Instruction Manual. Baculovirus. 6th Edition, May Expression Vector System

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1 Baculovirus Expression Vector System Instruction Manual 6th Edition, May 1999

2 Baculovirus Expression Vector System Manual 6th Edition May 1999 Instruction Manual General Methods 6xHis and GST Purification Direct Cloning For information or to place an order, please call: MABS (6227) For Technical Assistance call: TALK-TEC ( ) Torreyana Road San Diego, CA USA Tel: (619) Fax: (619) URL:

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4 Table of Contents Baculovirus Memorandum of Agreement Opening Remarks The Baculovirus Expression Vector System Advantages of using the Baculovirus Expression Vector System AcNPV Baculovirus DNAs AcNPV C6 Wild-type Baculovirus DNA BaculoGold Linearized Baculovirus DNA Linearized AcRP23.lacZ Baculovirus DNA Linearized AcUW1.lacZ Baculovirus DNA General Methods Selecting an Appropriate Baculovirus Transfer Vector Optimizing Gene Expression Cloning your Gene into a Baculovirus Transfer Vector Preparing Vector and Insert Ligating Vector and Insert Propagating Vectors Purifying Vectors Insect Cell Lines General Handling Techniques Monolayer Cultures Suspension Cultures Freezing and Thawing Insect Cells Producing and Maintaining AcNPV-derived Baculoviruses Generating Recombinant Baculoviruses by Co-Transfection End-point Dilution Assay Plaque Assay Plaque Pickup Amplifying Virus Storing Virus Particles Isolating AcNPV Particles Isolating AcNPV DNA Expressing Recombinant Proteins Monolayer Cultures Suspension Cultures Purifying Recombinant Proteins Non-secreted Recombinant Proteins Cell Lysate Preparation Secreted Recombinant Proteins vi vii iii

5 5. Purification Systems xHis Expression and Purification Kit Batch Purification Column Purification GST Expression and Purification Kit Batch Purification Column Purification Dialyzing GST-Fusion Protein Checking Purity and Recovery of Recombinant Protein Cleaving Fusion Proteins using Site-specific Proteases Thrombin Cleavage Factor X a Cleavage Generating 32 P-Labeled GST or 6xHis Fusion Proteins Generating Recombinant Baculovirus by Direct Cloning Troubleshooting Cloning Inserts into Baculovirus Transfer Vectors Insect Cell Culture Co-transfection Plaque Assay Virus Amplification Recombinant Protein Production xHis Expression and Purification System GST Expression and Purification System Thrombin Cleavage References Appendix A: BaculoGold Starter Package and Transfection Kit Appendix B: 6xHis Kits Appendix C: GST Kits Appendix D: vehuni and vecuni Baculovirus Reagent Sets Appendix E: Baculovirus Transfer Vectors I. Polyhedrin Locus-based Vectors Fusion Vectors BioColors Baculovirus Vectors Multiple Promoter Transfer Vectors II. p10 Locus-based Vectors Multiple Promoter Transfer Vectors Index iv

6 Figures 1. The Baculovirus life cycle in vivo and in vitro Design of AcNPV BaculoGold DNA Design of AcRP23.lacZ DNA Design of AcUW1.lacZ DNA Experimental scheme using BEVS Monolayer and suspension Sf cultures Comparison of uninfected and infected Sf9 cell monolayers well End-point Dilution Assay Western blot analysis of Retinoblastoma protein (Rb) in plaques Examples of recombinant protein expression levels in Baculovirus-infected Sf9 cells Characterization of native and Baculovirus-expressed Retinoblastoma protein (Rb) Functional activity of Baculovirus-expressed recombinant protein Expression, purification and cleavage of fusion proteins Strategy for directly cloning EcoRI fragments into the AcMNPV genome Baculovirus vectors for direct cloning BioColors in Sf9 cells Separation of Baculovirus-expression GFP and BFP using fluorescence-activated cell sorting Tables 1. Comparison of BEVS and bacterial expression systems Analysis of recombination frequencies by plaque assays Vector selection Recommended cell numbers and approximate densities for various assays v

7 BACULOVIRUS MEMORANDUM OF AGREEMENT NON-EXCLUSIVE RIGHTS TO USE BACULOVIRUS EXPRESSION VECTOR SYSTEM TECHNOLOGY FOR RESEARCH PURPOSES I. BACKGROUND The Texas Agricultural Experiment Station (TAES) claims rights to technology developed by Dr. Max D. Summers of the Department of Entomology relating to a recombinant Baculovirus expression vector system (BEVS) and the use of such vectors in insect cell culture media for expression of cloned genetic material. TAES is making the system and its components available for noncommercial research purposes. This Baculovirus expression vector system and related subject matter are claimed in two United States Patents, Numbers 4,745,051 and 4,879,236. Commercial rights to BEVS or products thereof are subject to a non-exclusive license, terms of which will be made available upon written request. Information and materials received from TAES relating to BEVS must be taken with the understanding that it is subject to a restrictive license for research purposes only. II. TERMS AND CONDITIONS OF AGREEMENT vi (1) All information and material received under this Agreement shall be used for research purposes only. (2) Access and distribution of the vectors and information must be limited to Recipient and to those personnel who report to Recipient, hereinafter referred to as "Recipient." (3) Recipient agrees to supply TAES preprints of any publications resulting from the use of the BEVS material promptly upon receipt of notice of acceptance from the publishing journal. Preprints should be sent to the attention of the Coordinator of Research Development for Industrial Relations, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas (4) Recipient and those who report to Recipients are aware of the proprietary interest involved herein and commit to honoring the terms and conditions of this Agreement. (5) Recipient accepts the biological material with the knowledge that it is experimental biological material and that is provided by TAES without warranty of any sort, expressed or implied. Recipient agrees to comply with all applicable governmental regulations for the handling thereof. Recipients shall hold TAES harmless for any damages which may be alleged to result in connection with the use and possession of the requested materials as provided in this Agreement, subject to any relevant state or federal government limitations. (6) This Agreement and Recipient s right to use biological material become effective upon breaking the seal of the package containing biological material and automatically terminates if Recipient fails to comply with any provisions of this Agreement. (7) TAES retains ownership and all rights to biological material not expressly granted and nothing in this Agreement constitutes a waiver of TAES' rights under U.S. Federal, State, or Patent law. NOTE: THESE RESTRICTIONS DO NOT APPLY TO INFORMATION OR TECHNOLOGY WHICH RECIPIENT CAN SHOW ARE IN THE PUBLIC DOMAIN OR FOR WHICH HE/SHE HAD PREVIOUSLY RECEIVED OR DEVELOPED IN GOOD FAITH THROUGH CHANNELS INDEPENDENT OF THE TEXAS AGRICULTURAL EXPERIMENT STATION.

8 Opening Remarks All reagents and materials listed in this manual are for research use only. Safety Requirements These research products have not been approved for human or animal diagnostic or therapeutic use. We suggest that all purchasers follow the NIH guidelines that have been developed for recombinant DNA experiments. All PharMingen products should be handled only by qualified persons trained in laboratory safety procedures. The absence of a product warning is not to be construed as an indication that the product is safe. All possible hazards of many biological products may not be known at this time. Always use good laboratory procedures when handling any of these products. Warranty Information presented in this manual is accurate to the best of our knowledge. It is not, however, guaranteed as such. It is the user s responsibility to investigate and verify the suitability of the supplied materials and procedures for a particular purpose. PharMingen expressly disclaims all warranties of merchantability and fitness for a particular purpose with respect to the use or suitability of the reagents and materials. PharMingen shall in no event be responsible for damages of any nature, directly or indirectly resulting from the use of the products of these kits. Disclaimer This manual is a practical guide for researchers to become familiar with the Baculovirus expression technology as a tool to overexpress foreign genes. It is not intended as a replacement to a textbook about Baculoviruses but rather to serve as an introduction to Baculovirus nomenclature and cite key references to guide the interested reader to additional literature. The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. PharMingen will not be held responsible for patent infringement or other violations that may occur with the use of our products. For commercial use of the 6xHis/Ni-NTA system, licenses may be granted by Hoffmann-La Roche Ltd. (Basel, Switzerland). Please contact QIAGEN Inc., 9600 De Soto Avenue, Chatsworth, CA for further information. All Baculovirus and related products sold by PharMingen are for research use only. The Polymerase Chain Reaction (PCR) is a process patented by Hoffmann-La Roche, Inc. Triton is a trademark of Union Carbide Chemicals and Plastics Co. Technical Assistance and Ordering Information At your request, we will furnish technical assistance and information about our products. Call 800-TALK-TEC ( ) to talk to a Technical Service Specialist. Our specialists have the education and experience necessary to answer your technical questions regarding the reagents and materials listed in this manual. All technical assistance is provided gratis and you assume sole responsibility for results you obtain by relying on that assistance. We make no warranties of any kind with respect to technical assistance or information we provide. Call MABS ( ) to place an order. vii

9 Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus Amp Ampicillin β-gal β-galactosidase BEVS Baculovirus expression vector system BFP Blue fluorescent protein BSA Bovine serum albumin BV Baculovirus CIAP Calf intestinal alkaline phosphatase CsCl Cesium chloride DTE Dithioerythritol DTT Dithiothreitol ECV Extracellular virus EDTA Ethylenediamine tetraacetic acid EtBr Ethidium bromide FACS Fluorescent activated cell sorting FBS Fetal bovine serum GFP Green fluorescent protein GST Glutathione S-transferase h Hour kb Kilobases kd Kilodalton LB Luria-bertani (broth) MCS Multiple cloning site min Minute MOI Multiplicity of infection (plaque-forming units/cell number) NaPi Sodium phosphate NaPPi Sodium pyrophosphate ORF Open reading frame OV Occluded virus particles PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline pfu Plaque-forming unit(s) = virus Pi Inorganic phosphate pi Post infection PMSF Phenylmethylsulfonyl fluoride Rb Retinoblastoma protein RT Room temperature Sf Spodoptera frugiperda Sj Schistosoma japonicum SDS Sodium dodecyl sulfate TBE Tris borate/edta TE Tris/EDTA U Unit v/v Volume: volume ratio wt Wildtype X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside YP Yellow protein yr Year viii

10 The 6xHis vectors were developed by PharMingen and produced in collaboration with QIAGEN 1The Baculovirus Expression Vector System The Baculovirus Expression Vector System (BEVS) is one of the most powerful and versatile eukaryotic expression systems available. 1,2 The BEVS is a helper-independent viral system which has been used to express heterologous genes from many different sources, including fungi, plants, bacteria and viruses, in insect cells. The Baculovirus DNA used in PharMingen s BEVS is the Autographa californica nuclear polyhedrosis virus (AcNPV). In this system several Baculovirus genes nonessential in the tissue culture life cycle (polyhedrin, p10, basic) may be replaced by heterologous genes. Since the Baculovirus genome is generally too large to easily insert foreign genes, heterologous genes are cloned into transfer vectors. Co-transfection of the transfer vector and AcNPV DNA into Spodoptera frugiperda (Sf) cells allows recombination between homologous sites, transferring the heterologous gene from the vector to the AcNPV DNA. AcNPV infection of Sf cells results in the shut-off of host gene expression allowing for a high rate of recombinant mrna and protein production. Recombinant proteins can be produced at levels ranging between 0.1% and 50% of the total insect cell protein. Factors influencing foreign gene expression are discussed, although it is difficult to precisely predict how efficiently different genes will be expressed. Baculoviruses (family Baculoviridae) belong to a diverse group of large doublestranded DNA viruses that infect many different species of insects as their natural hosts. 3 Baculovirus strains are highly species-specific and are not known to propagate in any non-invertebrate host. The Baculovirus genome is replicated and transcribed in the nuclei of infected host cells where the large Baculovirus DNA (between 80 and 200 kb) is packaged into rod-shaped nucleocapsids. 4 Since the size of these nucleocapsids is flexible, recombinant Baculovirus particles can accommodate large amounts of foreign DNA. AcNPV is the most extensively studied Baculovirus strain. Its entire genome has been mapped and fully sequenced. 5-7 Infectious AcNPV particles enter susceptible insect cells by facilitated endocytosis or fusion, and viral DNA is uncoated in the nucleus (Fig. 1). DNA replication starts about 6 h post-infection (pi). In both in vivo and in vitro conditions, the Baculovirus infection cycle can be divided into two different phases, early and late. During the early phase, the infected insect cell releases extracellular virus particles (ECV) by budding off from the cell membrane of infected cells. During the late phase of the infection cycle, occluded virus particles (OV) are assembled inside the nucleus. The OV are embedded in a homogenous matrix made predominantly of a single protein, the polyhedrin protein. 8, 9 OV are released when the infected cells lyse during the last phase of the infection cycle. Whereas the first ECV are detectable 10 h pi, the first viral occlusion bodies of wild-type AcNPV virus develop 3 days pi but continue to accumulate and reach a maximum between 5-6 days pi. Occlusion bodies are visible under light microscopy where they appear as dark polygonal-shaped bodies filling up the nucleus of infected cells. Not all known Baculoviruses form occlusion bodies; AcNPV is representative of the group of occlusion body-positive Baculoviruses. The polyhedrin protein, the major component of occlusion bodies, has a molecular weight of 29 kd. 1 During late phases of infection, the polyhedrin protein accumulates to very high levels. Up to 1 mg of polyhedrin protein vehuni and vecuni Baculovirus DNAs allow for direct cloning of heterologous genes into the BV genome (Chapter 6). 1

11 may be synthesized per infected cells accounting for 30-50% of the total insect protein. Although the polyhedrin protein seems to be one of the most abundant proteins in infected insect cells, it is not essential for the Baculovirus life cycle in tissue culture. However, in vivo viral occlusion bodies are an important part of the Baculovirus life cycle, essential for its dissemination into the environment (Fig. 1). Deletional or insertional inactivation of the polyhedrin gene in AcNPV results in the production of occlusion body-negative viruses, a phenomenon which simplifies the identification of recombinant viruses. The plaques formed by occlusion body-negative viruses are distinctly different from those of occlusion body-positive wild-type viruses. Newer modified AcNPV allow either color selection to identify recombinants or permit positive survival selection for recombinants (BaculoGold Cat. No K), rendering the occlusion body-based visual screening method obsolete. A variety of Baculovirus Transfer Vectors have been constructed for use with AcNPV DNA (Appendix E). Each vector contains: 1) an E. coli origin of replication, 2) an ampicillin resistance marker, 3) a promoter from the polyhedrin, p10 or basic protein AcNPV gene, 10 4) a cloning region downstream from promoter in which to insert foreign genes and 5) a large tract of AcNPV sequence flanking the cloning region to facilitate homologous recombination. Purified recombinant vectors containing the gene of interest may be co-transfected with AcNPV Baculovirus DNA into insect cells. After several days, recombinant viruses, which arise by homologous recombination between the transfer vector and AcNPV DNA, are selected. A Endocytosis Secondary Infection of Insect Cells Budding Virus B Transfer Vector Your Gene GST/ 6xHIS Tag Co-transfection BaculoGold DNA Uncoating Replication Homologous Recombination BaculoGold DNA Your Gene Viral Occlusion Fusion (Midgut Cells) Recombinant Protein Secondary Infection of Insect Cells Budding Recombinant Virus Soluble in Gut Primary Infection of Host Insect Ingestion Figure 1. The Baculovirus life cycle in vivo and in vitro. A) In vivo. Two distinct viral populations are formed in infected insect cells, occluded and budded virions. Occluded virions are protected from desiccation in the environment, allowing primary infection in susceptible larva. Once ingested, the occlusion body is solubilized in the gut, releasing virions which fuse with midgut cells. The virion nucleocapsid migrates through the cytoplasm to the nucleus. The core is uncoated from the capsid structure in the nucleus. Here replication takes place. Secondary infection is mediated by the budded form of the virus entering adjacent cells via adsorptive endocytosis. B) In vitro. The Baculovirus genome is too large to directly insert foreign genes easily. Hence, the foreign gene is cloned into a transfer vector that contains flanking sequences which are homologous (5 and 3 to your insert) to the Baculovirus genome. BaculoGold DNA and the transfer vector containing your cloned gene are co-transfected into Sf9 insect cells. Recombination takes place within the insect cells between the homologous regions in the transfer vector and the BaculoGold DNA. Recombinant virus produces recombinant protein and also infects additional insect cells thereby resulting in additional recombinant virus. 2

12 2 Advantages of using the Baculovirus Expression Vector System Choosing the right system for foreign gene expression can be particularly important in obtaining biologically active recombinant protein. Several unique features of the BEVS have made it the system of choice for many applications (Table 1). This chapter highlights the advantages of using BEVS to express recombinant proteins. Often, recombinant proteins expressed in bacterial systems are insoluble, aggregated and incorrectly folded. 11 In contrast, proteins expressed in BEVS are, in most cases, soluble and functionally active. Features BEVS Bacterial Simple to use Protein size unlimited <100 kd Multiple gene expression Signal peptide cleavage Intron splicing Nuclear transport Functional protein sometimes Phosphorylation sometimes Glycosylation Acylation Table 1. Comparison of BEVS and bacterial expression systems. The insect cell, unlike bacterium, is capable of performing many of the processing events that are required for forming biologically active, foreign proteins. 1) Functional activity of the recombinant protein The BEVS typically produces overexpressed recombinant proteins containing proper folding, disulfide bond formation and oligomerization. 2 Additionally, this system is capable of performing several post-translational modifications. This leads to a protein that is similar to its native counterpart, both structurally and functionally. However, in cases where the authentic protein functions as a heterodimer or relies on tissue- or species-specific modifications, the recombinant Baculovirus-expressed protein may not be functionally active, unless its binding partner or modifying enzyme is co-expressed. 2) Post-translational modifications Several post-translational modifications have been reported to occur in the BV, including N- and O-linked glycosylation, phosphorylation, acylation, amidation, carboxymethylation, isoprenylation, signal peptide cleavage and proteolytic cleavage The sites where these modifications occur are often identical to those of the authentic protein in its native cellular environment. However, the BEVS can express the gene of interest at a high expression rate which may overwhelm the ability of the cell to mod- 3

13 ify the protein product. This often results in lower levels of glycosylation or phosphorylation of the target protein than in the native cell line. Also, tissue- or species-specific post-translational modifications will not be performed in the BV, unless the modifying enzyme is being co-expressed. 3) High expression levels Compared to other higher eukaryotic expression systems, the most distinguishing feature of the BEVS is its potential to achieve high levels of expression of a cloned gene. The BV system has proven particularly useful in the generation of large quantities of proteins for structural analysis The highest expression level reported is 50% of the total cellular protein of an infected insect cell corresponding to approximately 1g of recombinant protein per cells. However, many recombinant proteins are not produced at such high amounts and it is usually difficult to predict the amount of protein expression. There are some guidelines one can follow to optimize protein production. Of primary importance is optimizing the design of the recombinant Baculovirus Transfer Vector (Chapter 4.1). 4) Capacity for large inserts The expandability of the capsid structure of Baculoviruses allows the packaging and expression of very large genes. There is no known upper size limit for the insertion of foreign sequences into the BV genome. 5) Capacity to express unspliced genes Insect cells have the capability to perform intron/exon splicing. However, certain virus-, tissue- or species-specific splicing patterns will not be obtained if they require the presence of particular splicing factors which are not available in the infected insect cell environment. In general, for high protein expression levels, a cdna insert rather than a genomic DNA fragment is recommended. 6) Simplicity of technology BaculoGold technology has made expression of full-length proteins fast, easy and reliable. Recombinant Baculovirus can be obtained in two simple steps cloning and co-transfection in as little as 5 days. The ease of use now rivals that of bacterial expression systems and BEVS technology does not require that the recombinant protein be expressed as a fusion protein. With the addition of several vectors containing genes encoding the green fluorescent protein from the jellyfish Aquorea victoria (Appendix E), protein expression can easily be monitored. 7) Simultaneous expression of multiple genes BEVS has the capability to express two or more genes simultaneously within single infected insect cells. Protein complexes that depend on dimer or multidimer formation for activity can be assembled. A well known example is the formation of complete virus capsids from a variety of viruses which have been assembled in vitro, using BEVS, by coexpressing the capsid subunits simultaneously. To this end, several multiple promoter plasmids have been constructed and are described in Appendix E. 4

14 8) Localization of recombinant proteins Baculovirus-expressed recombinant proteins are usually localized in the same subcellular compartment as the authentic protein. Nuclear proteins will be transported to the insect nucleus, membrane proteins will be anchored into the cell membrane, and secreted protein will be secreted by infected insect cells. 9) Ease of purification PharMingen has developed the 6xHis and glutathione S-transferase (GST) Baculovirus Expression and Purification Kits, designed for easy and reliable single-step purification of recombinant proteins. The 6xHis purification system (Cat. No K) relies on the high specificity of the 6xHis tag for Ni-NTA Agarose. The GST purification system (Cat. No K) takes advantage of the high affinity of glutathione agarose beads for reduced glutathione. These kits combine the advantages of expressing functional and soluble recombinant proteins using BEVS technology with the convenience of a GST or 6xHis affinity purification system. Even under the highest expression levels, most GST and 6xHis fusion proteins expressed in insect cells remain predominantly soluble. An extensive line of vectors has been developed for use in these systems. When using the GHLT, HLT or G series of vectors, the inserted gene will be produced as a fusion protein with an affinity tag on the amino terminus. Vectors in the GHLT series produce a fusion protein composed of both a 6xHis and a GST tag. Vectors in the pacg and the pachlt series produce proteins with a GST or a 6xHis tag respectively. The GST and 6xHis tags can be removed by incubating the protein in the presence of a site-specific protease (Chapter 5). 10) Direct cloning Generally, heterologous genes are cloned into transfer vectors, which homologously recombine with the BV genome in insect cells. vehuni and vecuni Baculovirus DNA allow the direct cloning of heterologous genes into the BV genome (Chapter 6). 21 5

15 6

16 3 AcNPV Baculovirus DNAs Infection of susceptible insect cells AcNPV wild-type virus results in the production of occlusion bodies. These opaque, light-refractive particles can be easily visualized under the light microscope (Chapter 4.5). This phenomenon aids in the identification of recombinant Baculoviruses in which the polyhedrin gene has been replaced by a cloned gene of interest. Recombinant viruses expressing the protein of interest rather than the polyhedrin protein fail to produce occlusion bodies and can be visually identified as occlusion body-negative plaques. However, in the past, non-recombinants were the vast majority over recombinants, usually 1,000:1. Modified AcNPV DNA (BaculoGold, AcRP23.lacZ and AcUW1.lacZ DNA) revolutionized the BV technology and made the occlusion body-based visual screen method obsolete. To improve recombination efficiencies, a single restriction site was added behind the polyhedrin or p10 promoter (AcRP23.lacZ or AcUW1.lacZ, respectively) so that the modified Baculovirus DNA can be linearized. Co-transfecting the linearized AcNPV DNA with a Baculovirus Transfer Vector shows an improved recombination frequency of 30%. The addition of three restriction sites in the polyhedrin locus of BaculoGold allows for the deletion of essential portions of the virus genome. Co-transfecting BaculoGold with a Baculovirus Transfer Vector rescues the lethally deleted virus at recombination frequencies greater than 99% (Table 2). Volume Virus Used Number of Plaques Recombination Viral Stock 10 ml 1 ml 0.1 ml 0.01 ml Frequency A. AcNPV wt high titer stock solution >5,000 >5,000 >5,000 1,096 NA B. AcRP23.lacZ R 1, Baculovirus DNA ~34% XylE NR >5, C. BaculoGold R >5, Baculovirus DNA XylE NR R = Recombinant NR = Nonrecombinant ~99.9% Table 2. Analysis of recombination frequencies by plaque assays. Plaque assays were done using viral inoculum from wild-type high titer viral stock (A), and 5-day transfection supernatants from Sf9 cells co-transfected with either AcRP23.LacZ Baculovirus DNA and pvl1392-xyle plasmid DNA (B) or Baculo- Gold Baculovirus DNA and pvl1392-xyle plasmid DNA (C) on X-gal plates. After 7 days the plates were analyzed and the number of recombinant (R) (yellow in the presence of Catechol) versus non-recombinant (NR) (blue in the presence of β-gal) plaques were noted above. Recombination frequencies were determined by the number of R versus NR plaques. Each lot of BaculoGold Baculovirus DNA undergoes testing to insure that the recombination efficiency is greater than 99%. To improve selection and screening methods, a polyhedrin-driven lacz gene coding for β-galactosidase was inserted into the virus genome. Preparation of linearized BaculoGold DNA removes the lacz gene. Non-recombinant, lacz positive plaques stain blue and recombinant, lacz negative plaques are colorless. Recombinant virus are selected as colorless in a plaque assay overlayed with agar containing X-gal: (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). 7

17 AcNPV C6 Wild-type Baculovirus DNA The wild-type AcNPV DNA is a super-coiled, double-stranded, circular DNA molecule with a molecular weight of 130 kb BaculoGold DNA, AcRP23.lacZ DNA and AcUW1.lacZ DNA are all derivatives of the AcNPV wild-type DNA. Originally, AcNPV wild-type DNA was widely used for co-transfection with recombinant Baculovirus Transfer Vectors to obtain recombinant BV particles. However, the identification of recombinants is time-consuming, requires considerable skills, and the recombination frequency is only 0.1%. Wild-type AcNPV DNA has no advantages over BaculoGold, AcRP23 DNA or AcUW1.lacZ DNA. PharMingen sells purified ready-to-use AcNPV C6 Wild-type Baculovirus DNA (Cat. No D). BaculoGold Linearized Baculovirus DNA BaculoGold DNA 22, 23 is a modified AcNPV Baculovirus DNA which contains a lethal deletion and does not code for viable virus (Fig. 2). Co-transfection of the BaculoGold DNA with a complementing Baculovirus Transfer Vector rescues the lethal deletion by homologous recombination. Since only the recombinant BaculoGold produces viable virus, recombination frequencies exceed 99%. The flanking sequences of the complementing vector s promoter region must be derived from the polyhedrin locus of the AcNPV wild-type DNA. p10 locus derived vectors (pacuw1, pacuw41, pacuw42, pacuw43) will not recover the lethal deletion of BaculoGold. Furthermore, not all polyhedrin derived vectors are compatible with BaculoGold DNA. The lethal deletion in BaculoGold spans 1.7 kb downstream of the polyhedrin gene. Small streamlined vectors may not contain the entire region and will not rescue the lethal deletion. PharMingen sells purified ready-to-use linearized BaculoGold DNA (Cat. No D). AcNPV wt DNA polyhedrin gene p10 gene Bsu36I Bsu36I Bsu36I Uncut BaculoGold DNA ORF603 LacZ ORF1629 polyhedrin promoter Figure 2. Design of AcNPV BaculoGold DNA. The polyhedrin gene locus of AcNPV DNA has been altered in the following ways: (1) a lacz gene has replaced the viral polyhedrin gene; (2) three Bsu36I cutting sites have been added, in ORF 603, 1629 and in lacz, which do not alter the amino acid sequences of their coding regions. The modified AcNPV DNA is linearized at the Bsu36I cutting sites deleting essential portions of the ORF ORF603 LacZ ORF1629 Cleaved BaculoGold DNA containing lethal deletion

18 Linearized AcRP23.lacZ Baculovirus DNA Linearized AcRP23.lacZ DNA is a modified AcNPV Baculovirus DNA in which the viral polyhedrin gene was replaced by a lacz gene (Fig. 3). AcRP23.lacZ is linearized at a single Bsu36I site introduced downstream of the polyhedrin promoter. Homologous recombination occurs during co-transfection of polyhedrin locus derived Baculovirus Transfer Vectors with AcRP23.lacZ. Approximately 30% of the resulting virus will be homologously recombined Baculovirus DNA. Since recombination disables the lacz gene, recombinant Baculoviruses can be selected by plaque assay on X-gal plates. Nonrecombinant virus express the lacz gene and plaques will appear blue. Recombinant virus do not express lacz and plaques appear colorless. PharMingen sells purified readyto-use linearized AcRP23.lacZ DNA (Cat. No D). Note: When using AcRP23.lacZ DNA, a plaque assay is necessary to identify and isolate recombinants from non-recombinant Baculovirus. AcNPV wt DNA polyhedrin gene p10 gene ORF603 Bsu36I LacZ ORF1629 Uncut AcRP23.lacZ DNA polyhedrin promoter ORF603 LacZ ORF1629 Linearized AcRP23.lacZ DNA polyhedrin promoter Figure 3. Design of AcRP23.lacZ DNA. The polyhedrin gene locus of AcNPV DNA has been altered in the following ways: (1) a lacz gene has replaced the viral polyhedrin gene; (2) a single Bsu36I cutting site has been added downstream of the polyhedrin promoter. The modified AcNPV DNA is linearized at the Bsu36I site. 9

19 Linearized AcUW1.lacZ Baculovirus DNA Linearized AcUW1.lacZ DNA is a modified AcNPV Baculovirus DNA which contains a p10 promoter driven lacz gene (Fig. 4). AcUW1.lacZ DNA is linearized at a single single Bsu36I introduced downstream of the p10 promoter. Homologous recombination occurs during co-transfection of p10 locus derived Baculovirus Transfer Vectors with AcUW1.lacZ. Approximately 30% of the resulting virus will be homologously recombined Baculovirus DNA. Since recombination disables the lacz gene, recombinant AcUW1.lacZ DNA can be color-selected by plaque assay on X-gal plates. Non-recombinant virus express the lacz gene and plaques appear blue. Recombinant virus do not express the lacz gene and plaques appear colorless. Both non-recombinant and recombinant virus are occlusion body positive. PharMingen sells purified ready-to-use linearized AcUW1.lacZ DNA (Cat. No D). Note: When using AcUW1.lacZ DNA, a plaque assay is necessary to identify and isolate recombinants from non-recombinant Baculovirus. AcNPV wt DNA polyhedrin gene p10 gene Bsu36I Uncut AcUW1.lacZ DNA p26 LacZ p74 p10 promoter Linearized AcUW1.lacZ DNA p26 LacZ p74 p10 promoter Figure 4. Design of AcUW1.lacZ DNA. The p10 gene locus of AcNPV DNA has been altered in the following way: (1) a lacz gene has replaced the viral p10 gene; (2) a single Bsu36I cutting site was added downstream of the p10 promoter. The modified AcNPV DNA is linearized at the single Bsu36I cutting site. 10

20 4 General Methods The steps necessary to construct recombinant Baculoviruses using BaculoGold DNA (Cat. No D) are outlined in Figure 5. Protocols for each step are given within this chapter. BaculoGold DNA Clone foreign gene into transfer vector Co-transfect into insect cells Amplify recombinant virus Produce recombinant protein Purify recombinant protein Propagate and purify vector containing foreign genes Figure 5. Experimental scheme using BEVS. Choose the appropriate transfer vector and clone in the foreign gene. Propagate the transfer vector containing the foreign gene using competent cells and purify by suitable means. Co-transfect BaculoGold DNA and recombinant transfer vector into Sf9 insect cells. Amplify the resultant recombinant virus in Sf9 insect cells. Use the amplified viral stock to produce protein. Purify your protein using appropriate methods. 4.1 Selecting an Appropriate Baculovirus Transfer Vector The BaculoGold Starter Package and Transfection Kit (Cat. No K and No K) both contain the Transfer Vector Set pvl1392/1393 (Cat. No P) (Appendix A). The pvl1392 and pvl1393 vectors are based on the polyhedrin locus and contain an extended MCS downstream of a polyhedrin promoter (Appendix E). These vectors have been used extensively to express a variety of proteins and should be adequate in most cases (Chapter 4.6). However, your protein expression needs may require that you use a specialized vector. For this reason, a variety of different Baculovirus Transfer Vectors have been constructed. The choice of vector will be determined by the application of the purified recombinant protein and in some cases by the nature of the protein itself. This section is intended as a guide to help researchers choose a vector which best fits their needs. First, decide whether to clone the gene of interest into a Baculovirus Transfer Vector that will produce the authentic protein encoded by its own ATG, or into a fusion-protein vector providing an N-terminal tag. The BEVS allows the expression of full length authentic proteins and does not require the expression of an N-terminal fusion sequence. This is a major advantage over many other expression systems, although, for certain applications it may be desirable to express a fusion protein. The tag may provide a sequence which can be used to label or modify the protein in a desired way that may not be available with the 11

21 authentic protein. The pacgp67 and pacsecg2t vectors incorporate a secretion signal sequence fused to the desired protein to force the recombinant protein into the secretory pathway. A fusion tag may also ease purification of non-secreted proteins. The pacghlt and pachlt vectors contain GST and 6xHis tags which can be purified on glutathione and Ni-NTA Agarose beads, respectively. Secondly, a suitable promoter must also be chosen. Baculovirus encoded promoters can be divided into the following classes according to the time, the viral infection cycle and conditions under which they are activated. PharMingen s vectors contain either late or very late promoters. Immediate Early Promoters: Baculovirus promoters, activated due to the action of insect encoded transcription factors, control early viral transcription factors. Early Promoters: Baculovirus promoters, activated before viral DNA synthesis occurs, usually control genes necessary for the onset of viral replication (not usually used for foreign gene expression). Late Promoters: Baculovirus promoters, active during and after viral DNA synthesis, when the cell is producing Baculovirus components, control genes necessary to assemble the virus particles (e.g., 39K protein promoter, basic protein promoter). Very Late Promoters: Baculovirus promoters, activated very late during the infection cycle, well after virion assembly has been completed, control genes involved in the formation of occlusion bodies and cell lysis. Most genes controlled by very late promoters are non-essential under tissue culture conditions (e.g., p10 promoter, polyhedrin promoter). The early and immediate early promoters are generally very weak and are not routinely used in Baculovirus Transfer Vectors. The late promoters (the 39K and basic protein promoters) are moderately strong promoters which express their products late in the infection cycle when enzymes needed for post-translationally modified proteins are still present. The pacmp2 and pacmp3 transfer vectors (Cat. No P) contain the basic protein promoter and should be considered when the foreign protein is glycosylated, phosphorylated, etc. The polyhedrin and the p10 protein promoters are very strong promoters expressed during the very late phase of viral infection. They are essentially non-competitive and have been used together to construct multiple promoter vectors. The polyhedrin promoter is most commonly used and has been cloned into a variety of Baculovirus Transfer Vectors. Third, consider whether you want to use a single or multiple promoter vector. Multiple promoter vectors are useful for expressing subunits of heterodimers or for expressing a cell type- or tissue type-specific modifying enzyme along with your protein of interest. Table 3 is designed to help you to decide which Baculovirus Transfer Vector may be most appropriate for your work. 12

22 BaculoGold DNA AcRP23.lacZ DNA AcNPV wild-type DNA AcUW1.lacZ DNA Vector Compatibility Promoter Type Fusion Protein Features Cat. # Polyhedrin locus-based Single Promoter Plasmids pvl1392/3 (set) Polyhedrin very late no Standard polyhedrin locus vectors 21201P pacsg2 Polyhedrin very late site dependent Recommended for large inserts, has an ATG 21410P pacmp2/3 (set) Basic protein late no Facilitates post-translational modifications 21209P pacuw21 p10 very late no Allows for in-larval expression, F1 origin 21206P pacghlt-a, -B, -C (set) Polyhedrin very late yes GST-tag, 6xHis-tag thrombin cleavage site 21463P pachlt-a, -B, -C (set) Polyhedrin very late yes 6xHis-tag, thrombin cleavage site 21467P pacg1 Polyhedrin very late yes GST-tag 21413P pacg2t Polyhedrin very late yes GST-tag, thrombin cleavage site 21414P pacg3x Polyhedrin very late yes GST-tag, factor Xa cleavage site 21415P BioColors BV Control (set) Polyhedrin very late yes BioColors Genes 21518P BioColors His (set) Polyhedrin very late yes BioColors Genes, 6xHis tag, thrombin 21522P cleavage site Secretory pacgp67 A, B, C (set) Polyhedrin very late yes Signal sequence 21223P pacsecg2t Polyhedrin very late yes Signal sequence, GST-tag 21469P Multiple Promoter Plasmids pacuw51 Polyhedrin, p10 very late no Simultaneous expression of 2 foreign genes; 21205P F1 origin pacdb3 Polyhedrin, p10 very late no Simultaneous expression of 3 foreign genes; 21532P F1 origin pacab3 Polyhedrin, p10 very late no Simultaneous expression of 3 foreign genes 21216P pacab4 Polyhedrin, p10 very late no Simultaneous expression of 4 foreign genes 21412P p10 locus-based Single Promoter Plasmids pacuw1 p10 very late no Standard p10 locus vectors 21203P Multiple Promoter Plasmids pacuw42/43 (pair) Polyhedrin, p10 very late no Simultaneous expression of 2 foreign genes; 21208P F1 origin 13 Table 3. Vector Selection. The Vector Selection Chart gives a comprehensive overview of the vectors available for use with the BEVS. Please refer to Appendix E for vector maps and descriptions.

23 4.2 Optimizing Gene Expression Once the vector is chosen, the gene of interest is cloned into a restriction enzyme site downstream of the BV promoter. The efficiency of heterologous gene expression in the BV System can differ by approximately 1000 fold due to the intrinsic nature of the gene and the encoded protein. Modifying the heterologous gene will generally influence gene expression by only 2-5 fold. Researchers should not feel compelled to excessively modify their gene. However, there are some general rules for improving gene expression. Since translation will start at the first ATG initiation codon downstream of the chosen BV promoter, there should be no additional ATG codons upstream of the gene. Additionally, the 5 untranslated sequence between the promoter and the start ATG should be kept to a minimum. In some cases, genes have been efficiently expressed from constructs with around 150 nucleotides between the promoter and the start ATG. However, it is advisable to trim down 5 untranslated sequences to less than 50 nucleotides. The 3 untranslated region downstream of the stop codon is of minor importance. There have been conflicting results regarding the importance of the polyadenylation signal. We have found that the expression level is generally not affected by the sequence downstream of the stop codons. 4.3 Cloning your Gene into a Baculovirus Transfer Vector The techniques required for inserting a foreign sequence into a Baculovirus Transfer Vector and preparing high quality plasmid DNA for co-transfections are described in this chapter. Most of the techniques are not unique to BEVS and we suggest referring to 24, 25 molecular biology manuals for supplementary cloning information. Preparing Vector and Insert Examine the endonuclease restriction map for both the transfer vector and your gene of interest. Identify restriction site(s) common to the cloning site of the vector and to your gene of interest. The 5 cloning site of your insert should be as close as possible to the ATG start codon of your gene (not more than 100 bases upstream). A polyadenylation sequence for the 3 cloning site is optional and has not been shown in this system to improve stability or expression of recombinant protein. Both the insert and Baculovirus Transfer Vector DNA should be digested with appropriate restriction enzymes to generate compatible ends for cloning. If a single restriction enzyme is used to prepare the vector, the DNA must be treated with calf intestinal alkaline phosphatase (CIAP) to remove 5 phosphate groups and prevent recirculation of the vector during ligation. When preparing the insert DNA, the correct restriction fragment (gene of choice) should be purified from an agarose gel by electroelution or DNA purification using glass-milk beads. PCR products should be similarly purified. 14

24 Materials Needed Agarose minigel (agarose concentration depends on the size range of the fragments) 0.5 M EDTA TE-saturated phenol/chloroform Chloroform:isoamyl alcohol (24:1) 7.5 M ammonium acetate Ethanol (100% and 70%) TBE gel electrophoresis buffer CIAP [(0.01 U/pmol of ends) if vector has been digested w/single endonuclease] TE buffer 1. Prepare insert and Baculovirus Transfer Vector DNA by restriction endonuclease digestion. The following 20 µl reaction is provided as an example: 5 µl plasmid DNA (1 µg/µl) 1 µl appropriate restriction enzyme (e.g. BamHI, 20 U/µl) 2 µl appropriate restriction buffer (10X) 12 µl sterile deionized water 20 µl final volume 2. Incubate sample(s) at the appropriate temperature (depending on the restriction endonuclease used, usually 37 C) for 2 4 h. 3. If the vector has been digested with a single restriction endonuclease, the DNA should be treated with CIAP. Thus, add the following components directly to the restriction endonuclease digest after the incubation time has been completed: 20 µl previous volume 3 µl CIAP 10X buffer 1 µl CIAP 6 µl sterile deionized water 30 µl new final volume 4. Incubate for 20 min at 37 C. 5. Add 1 volume of TE-saturated phenol/chloroform. Vortex each sample for 10 s and centrifuge samples for 5 min at 12,000 g in a microcentrifuge. 6. Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1). Vortex each sample for 10 s and centrifuge samples for 2 min at 12,000 g in a microcentrifuge. 7. Transfer the upper aqueous phase to a fresh tube and add 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of ice-cold 100% ethanol. Mix carefully by slowly inverting tubes several times by hand. Precipitate DNA by placing for 1 h at 20 C or 20 min on dry ice. 8. Collect the DNA pellets by centrifugation at 12,000 g for 5 min. 9. Carefully remove the supernatant, wash the pellet with 1 ml of 70% ethanol, dry briefly in a 37 C oven or in a vacuum desiccator. Resuspend pellet in 20 µl TE buffer. Determine the approximate DNA concentration by agarose gel electrophoresis with comparison to known amounts of DNA standards. 15

25 Ligating Vector and Insert An insert DNA:vector molar ratio of 1:3, 1:1 and 3:1 should be used to determine optimal insert:vector ratios. The total amount of DNA for recessive-end cloning per 10 µl volume should be 200 ng. assuming: s i is the size of the insert s v is the size of the vector r iv is the molar ratio of insert:vector t is the amount of total DNA (insert plus vector) i is the amount of insert needed in the DNA ligation reaction v is the amount of vector needed in the DNA ligation reaction the formula for this is as follows: (s i = i x t) t x s and v = v [(s v /r iv ) + s i ] s v + (s i x r iv ) Materials Needed T4 DNA ligase 10X ligase buffer containing 10 mm ATP Sterile deionized water 1. Set up a ligation reaction as described below. This example assumes an insert:vector ratio of 3:1. Therefore, r iv = 3. We define s v = 10 kb and s i = 3.3 kb. The total DNA for recessive end cloning should be 200 ng. Therefore, t = 200 ng. If we insert these values into the formula above we calculate that we need 100 ng of vector DNA and 100 ng of insert DNA. Thus, our sample ligation looks as follows: 1 µl vector DNA (100 ng/µl, 10 kb) 1 µl insert DNA (100 ng/µl, 3.3 kb) 1 µl T4 DNA ligase (1 Weiss unit) 1 µl 10X ligase buffer 6 µl sterile deionized water 10 µl final volume 2. Incubate the mixture at 16 C overnight. 3. Following the ligation reaction, transform the ligated plasmid DNA (usually 1 µl of the ligation mixture) into competent cells of an appropriate host strain (e.g., HB101, DH5α). Note: To monitor the efficiency of the ligation and transformation steps, competent cells should also be transformed with uncut nonrecombinant vector DNA as well as cut vector DNA which has been ligated in the absence of an insert. 16

26 Propagating Vectors There are many different E. coli strains available which are suitable for preparation of competent cells used in transformations, e.g., DH5α or HB101. Many of these strains are available as commercially prepared competent cells. Several comprehensive manuals containing procedures for preparation of competent cells are listed in the Reference section of this manual. PharMingen s transfer vectors are high copy number vectors and should generate yields of up to several milligrams per liter. Transforming bacterial strains Materials Needed SOB Medium (liter): 20 g Bactotryptone 5 g yeast extract 0.5 g NaCl Autoclave solution 2 M Mg solution Mix equal volumes of 2 M MgCl 2 and 2 M MgSO 4 Filter sterilize solution 2 M Glucose Filter sterilize solution LB/Amp (150 µg/ml) plates Competent cells β-mercaptoethanol Before starting: Place DNA and 5 ml culture tubes on ice. Place culture plates in 37 C incubator to dry. Make SOC medium: (1 ml for each transformation) to each ml of SOB medium, add: 10 µl 2M Glucose solution and 10 µl 2M Mg solution Place SOC medium in the 37 C water bath. Make β-mercaptoethanol dilution - 1:20 dilution in sterile water. 1. Thaw competent cells on ice (100 µl/ transformation). 2. Gently thaw cells by hand. Aliquot 100 µl into pre-chilled 15 ml polypropylene tubes (Falcon Cat. No. 2097). 3. Add 1.7 µl of the fresh β-mercaptoethanol dilution to the 100 µl of bacteria, resulting in a final concentration of 25 mm. Gently swirl. 4. Incubate on ice for 10 min, swirling every 2 min. 5. Add ng of recombinant plasmid DNA (1 µl) to cells and swirl gently. As a positive control, add 1 ng of pbr322 to another 100 µl of cells. 6. Incubate on ice for 30 min. Heat shock at 42 C for seconds (critical!). Return to ice for 2 min. 7. Add 0.9 ml of SOC medium preheated to 37 C. Incubate at 37 C for 1 h, shaking at 225 RPM on an orbital shaker. 17

27 8. Spin down bacteria using a table-top centrifuge at 10,000 g for 5 min. Remove all media except for 100 µl. Resuspend bacteria in remaining 100 µl and spread thin on an LB-Amp plate. 9. Incubate plates at 37 C overnight. 10. The next day, pick up several colonies for miniprep DNA isolation to confirm the presence of the recombinant plasmid. Perform restriction endonuclease analysis to confirm the presence and orientation of the insert. Note: After transformation of a suitable E. coli host strain (e.g., HB101, DH5α, etc.) by a Baculovirus Transfer Vector and plating the bacteria on selective medium, cells harboring recombinant plasmid DNA will grow into colonies. Since all current Baculovirus Transfer Vectors contain an ampicillin resistant gene, the selection should be done on LB plates containing 50 µg/ml ampicillin. Purifying Vectors The quality of both the vector and viral DNA is critical for successful co-transfections. Sf cells are sensitive to some contaminant s found in crude plasmid preparations, which cannot be removed by phenol/chloroform extraction or ethanol precipitation. Vector DNA purified by CsCl-EtBr density gradient centrifugation, anion exchange chromatography (QIAEX resin, QIAGEN Inc) or by extraction with glassmilk will be sufficiently pure for co-transfection. Refer to molecular biology manuals for comprehensive purification 24, 25 techniques. 4.4 Insect Cell Lines Several established insect cell lines are highly susceptible to AcNPV virus infection. The two most frequently used insect cell lines are Sf9 and Sf21 (Cat. No L and No L). Both cell lines were originally established from ovarian tissues of Spodoptera frugiperda larvae and are highly recommended for use in the BEVS. Healthy insect cells attach well to the bottom of the plate forming a monolayer and double every h. Infected cells become uniformly round, enlarged, develop enlarged nuclei, don t attach as well and stop dividing. Sf9 and Sf21 cells may also be grown in suspension. Antibiotics are not required, but gentamicin sulfate (50 µg/ml) and Amphotericin B ( Fungizone ) (2.5 µg/ml) are often added to the media. CO 2 supplementation is not required. We routinely use Sf9 cells and will refer to them from here on; however, Sf21 cells may be substituted. We commonly receive questions concerning cell confluency and BEVS assays. Table 4 was designed to help new users determine accurate cell densities per desired assay. Table 4. Recommended cell numbers and approximate densities for various assays. These numbers are routinely used for Sf9 insect cell cultures in PharMingen s laboratories. Individual users may want to further optimize these numbers for their own experimental systems. 18 Plate # Cells % # Cells/ Assay Size per Assay Confluent Confluent Plate Transfection 60 mm 2.0 x 10 6 ~60% 3.2 x 10 6 Dilution Assay 12 well 1.0 x 10 5 ~30% 3.0 x 10 5 Plaque Assay 100 mm 6.2 x 10 6 ~70% 8.8 x 10 6 Viral Amplification 150 mm 2.0 x 10 7 ~70% 2.9 x 10 7 Protein Production 150 mm 2.0 x 10 7 ~70% 2.9 x 10 7

28 General Handling Techniques The following information is helpful when handling insect cells. Healthy Sf9 cells generally double every h when grown in TNM-FH media (Cat. No M). To maintain healthy cultures, Sf9 cells should be subcultured 1:3 when they reach confluency on plates (three times a week). They will grow reasonably well at temperatures between C. However, after infection it is important to keep the temperature at 27 C ±0.5 C; otherwise, recombinant protein production may be poor, although cells will look infected. An adjustment period ranging from a few days to several weeks should be allowed when transferring Sf9 cells between monolayer and suspension cultures. Always equilibrate insect cell culture medium to RT before using. When removing liquid from a plate of cells, tip the flask at a angle so the liquid pools toward the bottom of the flask. Remove the liquid without touching the cell monolayer using a sterile pipette. When seeding cells into a tissue culture plate or flask, be sure the vessels are placed on a flat surface to ensure homogenous cell density. It is extremely important when doing a plaque assay to provide the proper cell density for plaque formation (Table 4). Rock the plate back and forth to evenly distribute the cells over the surface of the plate. To pellet cells, gently dislodge cells from monolayers and transfer the cell suspension to a sterile centrifuge tube of appropriate size. Spin the suspension for 2 5 min at 1,000 g. Carefully remove the supernatant without disrupting the cell pellet. To resuspend the cell pellet for culture, add the desired volume of fresh medium to the side of the tube and gently resuspend the pellet by pipetting the suspension up and down several times. Insect cells are sensitive to centrifugal forces. For resuspension, cells should be centrifuged for 2 5 min at 1,000 g in a GH-3.7 Beckman GPR horizontal rotor or equivalent. TNM-FH and Grace s medium do not contain ph color indicators. These media usually have a ph around 6.2. Cell viability may be checked using trypan blue. To 1 ml of cells add 0.1 ml of a 0.4% stock solution of trypan blue (in PBS or other isotonic salt solution). Non-viable cells will take up trypan blue. Healthy, log-phase cultures should contain more than 97% unstained viable cells. To minimize centrifugation cells may be transferred to a new tissue culture plate using the old medium. Once cells have adhered (10 min), change to fresh media. Insect cells grow well both in suspension and as monolayer cultures and can be transferred from one to the other with minimal adaptation (Fig. 6). Small-scale propagation of cells can be maintained on plates; however, for large scale it is time-consuming and costly to use plates. Spinner flasks are ideal for scaling up insect cell cultures. Both monolayer and suspension cultures should be evaluated for optimal levels of protein expression. 19

29 A B Figure 6. Monolayer and suspension Sf cultures. A) Monolayer cultures. 150 mm tissue culture plates (Falcon Cat. No. 3025) used at PharMingen for protein production. B) Suspension cultures. 2L, 1 L and 5 L (not shown) spinner flasks (Techne) used at PharMingen for cell propagation. Sf9 cells are suspended in Techne spinner flasks by a magnetic arm that spins at ~60 rpm. The culture volume should always remain less than half of the full volume of the flask. For example, a 1-liter flask should contain <500 ml suspended culture. Monolayer Cultures Materials Needed Sf9 cells (Cat. No L) per plate TNM-FH media (Cat. No M) 15 cm tissue culture plate (Falcon Cat. No. 3025) Sterile 10 ml pipets (Falcon Cat. No. 7551) Hemocytometer (Fisher Cat. No ) 27 C incubator In monolayer cultures, you may notice loosely attached cells or cells floating in the medium. These floaters are especially frequent in cultures that are overgrown. In healthy cultures, floaters should be less than 2% of the cell population. When resuspending attached cells, use a stream of medium from a 10 ml pipette or a Pasteur pipette and gently dislodge the cells from the surface. Strongly attached cells may require persistence and more forceful pipetting. Try to minimize foaming. Trypsin and other enzymes are not recommended to dislodge Sf9 cells. Cell scrapers should be used only if absolutely necessary, as scraping may damage cells. Initiate new plate or flask cultures by adding one volume of the cell suspension to two volumes of fresh medium. Confirm that initial cell density is approximately 30%. 20

30 Cells may be grown in BaculoGold Protein-free medium (Cat. No M). Sf9 and Sf21 cells may attach more firmly in Protein-free medium and doubling time may vary. Sf9 cells can be adapted to Protein-free medium by slowly decreasing the ratio of TNM-FH to Protein-free medium (Cat. No M and No M). Split a confluent plate of Sf9 cells, and allow them to attach to a fresh tissue culture plate. Remove the medium and replace it with a 1:2 ratio of Protein-free:TNM- FH media. Incubate cells until confluent. Split the cells and allow them to attach. Remove the media and replace with a 1:1 ratio of Protein-free:TNM-FH medium. Incubate cells until confluent. Split the cells and allow them to attach. Remove the media. and replace with a 2:1 ratio of Protein-free:TNM-FH medium. For the next medium change use pure Protein-free medium. Suspension Cultures Materials Needed TNM-FH media (Cat. No M) Spinner flask (Techne) Sf9 cells per ml of culture (Cat. No L) Hemocytometer (Fisher Cat. No ) 27 C Incubator Spinner apparatus Sf9 and Sf21 cells grow well in suspension cultures. A spinner culture should be started at an initial density of cells/ml. The cell density can be easily determined using a hemocytometer. Incubate spinner flasks at 27 C under constant stirring at rpm. Routine maintenance of spinner cultures requires subculturing when the cell density reaches approximately cells/ml (2-3 times a week). Remove 65-75% of the cell suspension and replace with fresh medium. Re-seed the culture in a clean sterile spinner flask at least every 2 weeks to prevent build-up of by-products or other contaminants. Aeration may be required for large cultures or during infections. Optimum conditions depend on the particular setup and should be determined empirically. 1 Sf9 cells grown in suspension culture may be adapted to serum-free medium by slowly decreasing the ratio of TNM-FH to Protein-free medium. Remove 2/3 of a healthy suspension culture containing cells/ml and replace with Protein-free media. When a density of cells/ml is reached (usually 3 4 days) replace 2/3 of the culture with Protein-free media. Repeat these steps until the culture is 100% Protein-free. 21

31 Generating Recombinant Baculoviruses by Co-Transfection Prepare at least 10 µg of highly purified plasmid DNA for co-transfection. Sf cells are sensitive to some contaminants found in crude plasmid preparations that are not removed during phenol/chloroform extraction or ethanol precipitation. Impure preparations of plasmid DNA are toxic to the cells, and many cells may lyse shortly after transfection. The result is a lower viral titer. At about 24 h post-transfection, Sf9/Sf21 cell viability should be greater than 97%. Materials Needed Sf9 cells (Cat. No L), cells per plate Three 60 mm tissue culture plates (Falcon Cat. No. 3802) 15 ml TNM-FH insect medium (Cat. No M) 1.0 µg BaculoGold DNA (Cat. No D) (0.5 µg per co-transfection) 2-5 µg Recombinant Baculovirus Transfer Vector DNA containing your insert Wild-type AcNPV Virus supernatant (Cat. No E) 2 µg pvl1392-xyle Control Vector (Cat. No P) Transfection Buffer A and B Set (Cat. No A) 100 µl catechol solution (500 µm catechol, 50 µm sodium bisulfate solution) 1. Prepare and label three tissue culture plates, one each for the experimental cotransfection, positive co-transfection control, and negative control. Seed Sf9 cells onto each 60 mm tissue culture plate. Initial cell density should be 50 70% confluent. Cell attachment should be done on a flat and even surface, allowing the cells to attach firmly, usually about 5 min. If cells don t attach after that time, they are either not healthy or the wrong plates have been used (e.g., non-coated petri dishes). Note: A fourth tissue culture plate may be seeded with 2 x 10 6 Sf9 cells for infection with wild-type AcNPV virus as a positive control for infection. 2. Experimental co-transfection: Combine 0.5 µg BaculoGold DNA and 2 5 µg recombinant Baculovirus Transfer Vector, containing your insert, in a microcentrifuge tube. Mix well by gentle vortexing or by flicking the tube. Let mixture sit for 5 min before adding 1 ml of Transfection Buffer B. 3. Positive control co-transfection: Combine 0.5 µg BaculoGold DNA and 2 µg pvl1392-xyle Control Transfer Vector DNA in a microcentrifuge tube. Mix well by gentle vortexing or by flicking the tube. Let mixture sit for 5 min before adding 1 ml of Transfection Buffer B. 4. Aspirate the old medium from the cells on the experimental co-transfection plate and replace with 1 ml of Transfection Buffer A. Make sure that the entire surface of plate is covered to prevent the cells from drying out. 5. Aspirate the old medium from the positive control co-transfection plate and replace it with 1 ml of Transfection Buffer A as in Step Aspirate the old medium from the negative control plate and replace it with 3 ml fresh TNM-FH medium. Nothing else will be added to this plate. 7. Add the 1 ml of Transfection Buffer B/DNA solution from Step 2, drop by drop to the experimental co-transfection plate. After every 3 5 drops, gently rock the plate 23

32 back and forth to mix the drops with the medium. During this procedure, a fine calcium phosphate/dna precipitate should form. This precipitate is characterized by a fine white milky color. 8. Add 1 ml of the Transfection Buffer B/XylE Positive Control DNA solution from Step 3 drop by drop to the positive control co-transfection control plate, as in Step Incubate all three plates at 27 C for 4 h. 10. After 4 h, remove the medium from the experimental and positive control cotransfection plates. Add 3 ml fresh TNM-FH medium and rock the plates back and forth several times before once again removing all the medium. Add 3 ml of fresh TNM-FH medium and incubate the plates at 27 C for 4 5 days. It is not necessary to change the medium of the negative control plate. 11. After 4 days, check the three plates for signs of infection. Compare the negative and positive controls to the experimental co-transfection plate. Infected cells will appear much larger than uninfected ones, will have enlarged nuclei, will stop dividing, and will often float in the medium. 12. After 5 days, collect the supernatant of the positive control and experimental cotransfection plates. Assess co-transfection efficiencies by end-point dilution assay or identify recombinant viruses by plaque assay. Transfection supernatants should be amplified to produce high titer virus stocks that are used for recombinant protein production. Alternatively, single recombinant viruses, obtained by plaque purification or end-point dilution assay, may be used for virus amplification. To check the expression of your protein of interest, lyse the transfected cells or use an aliquot of the supernatant (depending whether the recombinant protein is secreted or not) and spin down debris. Transfected cells expressing the XylE protein can be assayed by adding µl catechol solution to the cells after the cotransfection supernatant has been removed and replaced with fresh media. Infected cells will turn bright yellow in approximately 5 min. End-point Dilution Assay The end-point dilution assay (EPDA) is a versatile assay that is useful for a variety of screens. A 96-well plate EPDA may be used to replace the plaque assay and plaque purification as a method for either determining viral titer or identifying and purifying recombinant virus. 1 We use a modified 12-well plate EPDA on a routine basis. In the 12-well EPDA, individual wells containing equal amounts of insect cells are inoculated with 100, 10, 1 or 0 µl aliquots of the original transfection supernatant, wild-type virus, or recombinant XylE positive control viral (typically pvl1392-xyle) supernatant (Fig. 8). This modified EPDA is useful for determining the efficiency of the initial co-transfection, identifying infected cells, approximating viral titers, and amplifying viral titer. Cells are visually inspected for signs of infection following an initial co-transfection in tissue culture plates. However, it may be difficult to identify infected cells as signs of infection are not always visually apparent, particularly if the transfection efficiency is low. The EPDA is then used to amplify the viral titer, and a visual comparison between cells inoculated with 100, 10, 1 and 0 µl of the original transfection supernatant is used to ascertain whether or not the initial co-transfection was successful. For example, if cells receiving 100 µl of the initial co-transfection supernatant look infected in the EPDA, but cells receiving 10, 1 and 0 µl do not, then it is likely that the viral titer is low and should be amplified to produce a high titer stock. If the EPDA is used as an amplification step to generate a high titer stock, care should be taken to avoid cross-contamination between 24

33 wells containing different viruses. Wild-type virus is highly infectious and can contaminate wells containing recombinant virus. If cells receiving 100 µl of the original co-transfection supernatant look similar to those receiving 0 µl, it is likely that the original cotransfection did not result in a significant viral titer, and must be amplified. Two types of positive EPDA controls are recommended. The supernatant from a pvl1392 XylE transfection is a particularly useful positive control. Cells expressing the gene turn yellow in the presence of catechol and are easily identifiable (Fig. 8B). A small number of infected cells may not turn yellow. For example, cells which are newly infected will show signs of infection (stop dividing, become enlarged and float) but may not yet be producing protein. Additionally, cells near the 5th day of infection may have begun to lyse and much of their protein may be dispersed throughout the media. The wild-type virus, a viral stock of known titer, can be used as a standard against which to approximate viral titers. Cells infected with wild-type virus will, in addition to showing typical visual signs of infection, contain occlusion bodies. This criterion is particularly useful for first-time users who have not previously visualized infected cells. The wildtype virus can also be used to verify the health and infectivity of the cells. Viral titers may be approximated by performing the 12-well EPDA with your transfection supernatant and a viral stock of known titer. A high titer stock at 2 X 10 8 pfu/ml (wild type viral stock, Cat. No E) will show equal signs of infection in all three (100, 10 and 1 µl) infected wells, 3 days pi. Each well of the 12-well plate contains 3X10 5 cell and a high titer stock contains 2 X 10 5 virus/µl, resulting in nominal cell proliferation and total cell infection three days pi. If your transfection supernatant shows a 10-fold decrease in the number of infected cells between dilutions, you should amplify the virus once or twice more to generate a high titer stock for protein production. High titer virus stocks are used for infection of cells at optimal multiplicity of infection (MOI = No.virus/No.cells) resulting in maximum protein production. 100 µl 10 µl 1 µl 0 µl A. AcNPV wild-type viral stock B. Recombinant AcNPV-XylE C. Recombinant AcNPV-IL-2 Figure well End-point Dilution Assay. A twelve-well tissue culture plate was seeded at 30% confluency with Sf9 cells and infected with 100, 10, 1 and 0 µl aliquots of viral inoculum from AcNPV wild-type high titer stock (A), Recombinant AcNPV-XylE transfection supernatant (B) and recombinant AcNPV-IL2 transfection supernatant (C). Photographs of each well were taken 3 days pi. 25

34 Materials Needed Sf9 cells (Cat. No L) One 12-well tissue culture plate (Falcon Cat. No. 3043) 12 mls TNM-FH insect medium (Cat. No M) Baculovirus transfection supernatant 1. Seed Sf9 cells per well on a 12-well plate. Allow cells to attach firmly. Replace medium with fresh TNM-FH. 2. Add 100, 10, 1 and 0 µl of the recombinant virus supernatant (usually obtained 5 days after the start of transfection) to separate wells. Do the same for the positive control, e.g., pvl1392-xyle supernatant or wild-type viral stock. 3. Incubate the cells at 27 C for three days. Examine the cells for signs of infection. 4. A successful transfection should result in uniformly large infected cells in the 100, 10, and 1 µl experimental wells. The cells in the 0 µl control wells should not look infected because they were not inoculated with virus. 5. If only the 100 µl and 10 µl wells seem to have infected cells and the 1 µl well looks more like the control, then the titer of your virus supernatant is low. Amplify the virus an additional time before you proceed with protein production. 6. The cells from the 100 µl well can be harvested and lysed in lysis buffer (Chapter 5.6.1). The desired protein production may be checked by western blot analysis (if antibodies are available) or by Coomassie blue-stained SDS-PAGE gel. 7. It is recommended that the virus supernatant from the 100 µl well is kept as the first viral amplification stock, however care should be taken to avoid crosscontamination between wells containing different virus. 8. To further purify the virus population, a plaque assay purification may be performed. It is optional for BaculoGold DNA users, but required if any other AcNPV DNA was used for co-transfection. Plaque Assay The plaque assay can be used to plaque purify virus or to determine viral titer in plaque-forming units per ml (pfu/ml) so that known amounts of virus can be used to infect cells during subsequent experimental work. In this assay, cell monolayers are infected with a low ratio of virus, such that only isolated cells become infected. An overlay of agarose keeps the cells stable and limits the spread of virus. When each infected cell produces virus and eventually lyses, only the immediate neighboring cells become infected. Each group of infected cells is referred to as a plaque. Uninfected cells are dispersed throughout the culture, surrounding the plaques. After several infection cycles, the infected cells in the center of the plaques begin to lyse and the peripheral infected cells remain surrounded by uninfected cells. This phenomenon causes the light passing through the infected cells to refract differently than the surrounding uninfected cells, and the plaque can be visualized either by the naked eye or by light microscopy. Each plaque represents a single virus. Therefore, clonal virus populations may be purified by isolating individual plaques. Individual plaques obtained from varying dilutions of a viral stock can be counted to determine the viral titer (pfu/ml) 26

35 of a given transfection amplification supernatant. The condition of the cells and their even distribution over the surface of the tissue culture plate is important to the success of a plaque assay. Cells should be healthy and in log growth phase at the time of the assay and at least 90% viable. Clumpy cells, cells that are not evenly distributed at the correct density (~ 70%) over the plate, and cells that do not adhere to the tissue culture dishes within about 2 h after plating are detrimental to the assay. A kd B kd Figure 9. Western blot analysis of Retinoblastoma protein (Rb) in plaques. 10 randomly picked plaques were amplified from plates inoculated with pvl1392-rb rescued BaculoGold virus (A) and plates inoculated with BaculoGold alone (B). As expected, Rb expression was only detected in plaques obtained from cultures inoculated with pvl1392-rb (A), and not in the background plaques in non-rescued cultures (B). Rb was detected using an anti-human Rb monoclonal antibody (clone G3-245, Cat. No A). Materials Needed Sf9 cells (Cat. No L) Three 100 mm tissue culture plates (Falcon Cat. No. 3003) 130 ml TNM-FH insect medium (Cat. No M) Baculovirus transfection supernatant 1-2 g Agarplaque-Plus Agarose (Cat. No A) 100 ml protein-free insect medium (Cat. No M) 1. Seed Sf9 cells at cells on a 100 mm plate. Allow the cells to attach firmly to the plate (10 min). It is important that this is done on a level surface to allow the cells to spread evenly over the bottom of the plate. 2. Replace medium with 10 ml fresh TNM-FH. 3. Add virus inoculum to the plate. Commonly, serial dilutions of the viral transfection supernatant (10 3, 10 4, 10 5 ) are made and 100 µl of each dilution is added to the medium of each plate. Mix gently by rocking the plate. Larger dilutions will be necessary for high titer stock solutions. 4. Incubate the plates at 27 C for 1 h to allow virus particles to infect the cells. 27

36 5. While the cells are incubating, prepare a 2% agarose solution using Agarplaque- Plus Agarose (low melting point agarose) in protein-free medium. Heat the solution in a microwave until the agarose is dissolved; allowing the solution to reach boiling will help to ensure its sterility. Take care that all the agarose is melted but do not overheat. High heat will cause precipitation of certain nutrients. Cool to 45 C in waterbath. Prewarm 1X volume of TNM-FH to room temperature (RT). 6. Mix equal volumes of the agarose solution and pre-warmed TNM-FH medium. The final agarose solution should be between 0.8% and 1%. A final agarose concentration less than 0.8% will not solidify well, whereas concentrations over 1% will cause damage to the cells. Remove plates from incubator and remove medium. Overlay cells with 10 ml of the Agarplaque-Plus Agarose solution by carefully adding agarose to the side of the tilted plate. Allow plates to sit undisturbed on a level surface until agarose hardens (about 20 min). If color selection is required (e.g., AcRP23.lacZ or AcUW1.lacZ), add 100 µl of an X-gal stock solution (25 mg/ml X-gal in DMF) to 10 ml of agarose solution before pouring it onto the plates. 7. Plates should be kept in a humid atmosphere at 27 C until visible plaques develop (6-10 days). Plaques can be visualized by inverting the plates on a dark background and illuminating them with a strong light source from the side of the plate, or by holding them at a 45 angle into a light source. Plaques can be used to determine virus titer or for screening to identify recombinant virus. Plaque Pickup To ensure proper isolation, it is best that plaques are picked from plates containing fewer than 50 plaques. Plate several dilutions of the virus to ensure that a sufficiently low number of plaques are obtained. Plaques maybe picked up using sterile micropipette tips (1,000 µl) or microcapillary tubes. 1. Mark the plate under the plaque with a marker. Using a sterile pipette tip, remove an agarose plug directly over the plaque; pick up between 10 and 100 plaques in this manner. 2. Place each agarose plug in separate microcentrifuge tubes containing 1 ml tissue culture medium. Elute the virus particles out of the agarose by rotating the tube overnight at 4 C. 3. Add 200 µl of each plaque pickup to separate wells of a 12-well tissue culture plate seeded with cells per well in 1 ml fresh TNM-FH media. Incubate the plates for 3 days at 27 C. 4. The virus supernatant of this passage one stock can be collected and centrifuged for 5 min at 1,000 g at 4 C to remove debris. Store at 4 C. 28 Materials Needed Sf9 cells (Cat. No L), 12-well tissue culture plate (Falcon Cat. No. 3043) 100 mm tissue culture plate (Falcon Cat. No. 3003) TNM-FH media (Cat. No M) Sterile micropipette tips or capillary tubes Microcentrifuge tubes

37 5. Seed a 100 mm tissue culture plate with cells for each plaque pickup. Allow cells to attach and replace medium with 10 ml fresh TNM-FH media. 6. Add 200 µl of the passage one stock to the 100 mm plate and incubate at 27 C for 4 days. Save the remaining 800 µl passage one stock at 4 C as a backup. 7. Harvest the viral supernatant and centrifuge to remove debris. Determine the titer of this passage two stock. If the titer remains below pfu/ml, proceed to Amplifying Virus. Amplifying Virus Prepare large stocks of virus by infecting insect cells at a low MOI (<1) and harvesting supernatant 4 5 days pi. It is critical to use a low MOI because passaging the virus at high MOI increases the number of virus with extensive mutations in their genome. 1 The number of mutant virus is also increased by serial passage, and it may be advantageous to maintain a low passage seed stock from which larger working stocks are amplified. Eventually the titer in the seed stock will be reduced through storage, and it becomes necessary to generate a new passage seed stock. Since BaculoGold recombinants are greater than 99% of the total virus population, it is not generally necessary to initially prepare all stocks from a clonal viral population. However, if there is a reduction in protein production after multiple passages of a viral stock, it may be necessary to isolate clonal viral populations by EPDA or plaque purification. 1 After verification of protein production, the clonal virus population can be amplified to produce a high titer stock. The viral stock is then ready for large-scale protein production. Materials Needed Sf9 cells (Cat. No L) per plate 15 cm tissue culture plate (Falcon Cat. No. 3025) 100 mls TNM-FH insect medium (Cat. No M) Baculovirus low titer virus stock 1. Seed Sf9 cells on a 15 cm plate. Allow them to attach for 15 min and change to fresh TNM-FH. 2. Add 100 µl-1 ml of your low titer recombinant stock to the plate. If you know the virus titer of your stock solution, make sure that the MOI is below one. Repetitive infections with an MOI of substantially higher than one will select for deletion mutants which may no longer express your gene. 3. Incubate the cells at 27 C for 3 days. Check for signs of infection 2 days pi. 4. Harvest the supernatant from the plate, then spin down the cellular debris in a table-top centrifuge 10,000 g. 5. Store the virus supernatant in a sterile tube at 4 C for up to 6 months. For longer storage periods, virus supernatant should be frozen at 80 C. Store in a dark area; the viral titer appears to decrease when exposed to fluorescent light for prolonged periods of time. 6. Determine the viral titer of your amplification solution using the plaque assay procedure. Amplification typically is done 2 or 3 times to attain a high viral titer ( pfu/ml). 29

38 Storing Virus Particles Supernatants containing Baculovirus particles may be stored at 4 C for up to 6 months or frozen at 80 C for a longer period of time. If frozen, avoid multiple freeze and thaw cycles. Upon freezing, the viral titer may decrease and should be reamplified when thawed. Store viral stocks in the dark; titers appear to decrease when exposed to fluorescent light for prolonged periods of time. The best way to preserve a recombinant virus is to isolate its DNA and store it at 80 C. Isolating AcNPV Particles For long-term storage, you may want to isolate the recombinant Baculovirus particles and purify the viral DNA. 1. Produce several liters of high titer Baculovirus stock solution. Remove cell debris by spinning the stock solution at 10,000 g for 5 min. 2. Transfer the supernatant to ultracentrifuge tubes (Nalgene Cat. No ) and pellet the virus particles by spinning the supernatant at 40,000 g for 30 min (18,000 rpm in an SS34 rotor). A bluish-white pellet should be visible at the bottom of each tube. 3. Decant the supernatant and invert the centrifuge tubes on a paper towel for a few minutes. Wipe the residual medium from the inside of the tubes. Carefully avoid touching the virus pellet. 4. Resuspend virus pellet in 10 ml of TE buffer. 5. Prepare a 5%/40% sucrose step gradient in an ultracentrifuge tube: pipette several milliliters of a 40% sucrose cushion into the tube, and carefully layer 3 ml of a 5% sucrose cushion on top of it. 1 Finally, place a layer of the resuspended viral particles on top of the 5% layer. 6. Spin the tubes at 40,000 g for 30 min. During that time, the viral particles will move through the 5% sucrose layer and will be collected at the interface between the 5% and 40% sucrose solution layers. It will appear as a white band. Most contaminants will either float on top of the 5% sucrose layer or precipitate to the bottom of the tube. 7. Use a sterile 9-inch Pasteur pipette to harvest the virus particles located between the two layers. 8. Transfer the harvested virus to a new ultracentrifuge tube and fill up the tube with TE buffer. If necessary, this resuspension can be stored at 4 C for a few days. 9. Spin the tube at 40,000 g for 30 min to pellet the virus. 10. Decant the supernatant and invert the centrifuge tubes on a paper towel for a few minutes. Wipe the residual buffer from inside of the tubes. Avoid touching the virus pellet. 11. Resuspend the virus pellet in an appropriate volume of TE buffer (1 ml per viruses). 12. If necessary, store the virus resuspension for a few days at 4 C. Otherwise, proceed with DNA isolation. 30

39 Isolating AcNPV DNA 1. Digest the resuspended virus particles with RNase A (10 µg/ml final concentration) for 30 min at 37 C. 2. Add 10% SDS to the resuspended virus particles such that the final SDS concentration is 0.5%. 3. Digest with Proteinase K (10 µg/ml final concentration) for 30 min at 37 C. 4. Extract once with phenol/chloroform: Add one volume of phenol to the solution, mix well but avoid vortexing. Spin mixture in a table-top centrifuge to separate the organic and aqueous layers, and then transfer the upper layer to a new tube. Add one volume of phenol/chloroform (1:1 mixture) to the aqueous layer. Mix well and spin tubes in a table-top centrifuge to separate the organic and aqueous layer. Transfer the upper aqueous layer to a new tube. Add one volume of chloroform to the aqueous layer. Mix well and spin tubes in a table-top centrifuge to separate the organic and aqueous layers. Remove the top aqueous layer to a new tube, being careful not to remove any chloroform. 5. Dialyze the aqueous layer against TE (ph 8.0) to eliminate traces of chloroform. We recommend 3 dialysis changes: 2 2 h, 1 overnight. 6. Measure the A 260 of the obtained viral DNA solution to determine concentration and measure the 260/280 ratio to verify purity. The 260/280 for DNA ~1.8. Run a 0.5% agarose gel to verify that the purified DNA is intact and of high molecular weight. 7. The DNA should be aliquoted and stored at 4 C. Since the Baculovirus genome is large, avoid freezing which may shear the DNA. 4.6 Expressing Recombinant Proteins Recombinant proteins have been produced in the Baculovirus system at levels ranging between 0.1% and 50% of the total insect cell protein. For optimal protein production, the MOI should be between 3 and 10. Researchers should test different MOI to empirically determine optimum levels for protein production. The supernatant from protein production should not be used as a viral stock. Since the MOI used was much higher than one, a considerable portion of the virus population may contain deletion mutations. Several variables influence protein levels, functional activity and post-translational modifications of Baculovirus-expressed protein (refer to Chapter 4.2). An example of the variation of expression levels between four proteins cyclin A, cdk2, TR2 orphan receptor, and androgen receptor is seen in Fig. 10(A). The percent of protein expressed in the system is highly dependent on the intrinsic property of the protein. The cyclin A from Fig. 10(A) was not visible by Coomassie blue-staining, and was analyzed by western blot analysis Fig. 10(B). Figure 10(C) shows post-translationally modified IL 4. A comparative analysis of phosphorylated Baculovirus-expressed and native retinoblastoma protein (Rb) is shown in Fig. 11. Figure 12 shows assays used to measure the functional activity of Baculovirus-expressed granulocyte macrophage colony stimulated factors (GM-CSF) and Interleukin-4 (IL-4). We suggest that the user consult the literature pertinent to their recombinant protein to gain information regarding expected post-translational modifications and levels of functional activity. 31

40 A kd Cyclin A Cdk2 TR2 Androgen R B kd Anti-Cyclin A Control C kd IL IL SDS-PAGE 1 2 Western Blot 6 SDS-PAGE Figure 10. Examples of recombinant protein expression levels in Baculovirus-infected Sf9 cells. A) Protein expression levels. Amido black SDS-PAGE of total insect cell lysate (20 µg/lane) containing Baculovirus-expressed cyclin A (lane 1), Cdk2 (lane 2), TR2 (lane 3), or androgen receptor (lane 4). B) Western blot analysis of Baculovirus-expressed cyclin A. Anti-cyclin A monoclonal antibody (clone BF683, Cat. No A) (lane 1). Isotype (negative) IgE control (lane 2). C) SDS-PAGE analysis of Baculovirus-expressed, purified mouse IL-4. IL-4 was purified using an anti-mouse IL-4 monoclonal antibody (clone BVD6-24G2, Cat. No D). The gel was stained with coomassie blue. Note that although Baculovirus-expressed cyclin A was not visible by staining (A, lane 1) it was readily visible by western blot analysis (B, lane 1). IL-4 migrates as two bands due to differential glycosylation (C). For large-scale protein production, we have found that cell propagation in spinner flasks and protein production on tissue culture plates is optimal. Protein may be produced in suspension, but often the levels are lower than on plates. Monolayer Cultures 1. Seed several individual 15 cm tissue culture plates with Sf9 cells per plate. Add fresh TNM-FH medium to make up a total of 30 ml media per plate. 2. Calculate the amount of virus needed using the formula: ml of inoculum needed = MOI (pfu/cell) number of cells/titer of virus per ml. 3. Infect seeded cells with high titer recombinant Baculoviruses stock solution (virus titer should be pfu/ml). For optimal protein production, the MOI should be between 3 and 10. Often researchers will test different MOIs to empirically determine the optimum level of infection. 32 Materials Needed 15 cm tissue culture plate (Falcon Cat. No.3025) High titer viral stock ( pfu/ml) Sf9 cells (Cat. No L) per plate TNM-FH media (Cat. No M) 27 C Incubator

41 4. Incubate the cells for 3 days at 27 C. Check for signs of infection 2 3 days after inoculation. Cells should be enlarged in size (about 2 fold) and a large nucleus should be visible. 5. Harvest the cells and supernatant from the plates and spin down the cells at 10,000 g for 5 min using a table-top centrifuge. Non-secreted proteins will be found in the cell pellet, which can be stored at 80 C. Secreted proteins will be found in the supernatant, which can be stored at 80 C. When purifying secreted protein, the cell pellet should be tested to determine the amount of protein, if any, that remains in the cells. Suspension Cultures Materials Needed TNM-FH media (Cat. No M) Spinner flask (Techne) Sf9 cells per ml of culture (Cat. No L) Hemocytometer (Fisher Cat. No ) 27 C Incubator Spinner apparatus 1. Seed approximately Sf9 cells/ml in a spinner flask. The cells should be healthy (98% viable). 2. Calculate the amount of virus needed using the formula: ml of inoculum needed = MOI (pfu/cell) x number of cells/titer of virus per ml. The desired MOI for protein production is Add the inoculum to the flask. Incubate the flask at 27 C with stirring for 2-4 days. Check the progress of the infection by examining aliquots of the culture under the microscope. 4. To harvest, pellet cells by centrifugation. For secreted protein, store the supernatant in sterile tubes. For non-secreted proteins, store the cell pellet at 80 C and discard the supernatant. 33

42 A kd MOLT-4 Q G1 S M B Rb Sf9 + Rb Sf pprb prb C MOLT-4 Sf + Rb D Sf9 + Rb PAP + PAP Rb pprb prb 1a 2a Rb 1 2 1b 2b Figure 11. Characterization of native and Baculovirus-expressed Retinoblastoma protein (Rb). A) Western blot analysis of native Rb during different stages of the MOLT-4 (a human leukemia cell line) cell cycle. Native Rb migrates as multiple bands due to varying degrees of phosphorylation. Cell cycle stages are denoted as Q (quiescent), G1, S, and M. B) SDS-PAGE analysis of recombinant Rb. Rb is detected in Baculovirus-infected (lane 1) but not in mock-infected (lane 2) Sf9 cell lysates. The gel was stained with Coomassie blue. C) Comparative analysis of native and Baculovirus-expressed Rb by western blot. Rb expressed in MOLT-4 cells (lane 1) is more highly phosphorylated than Rb expressed in Baculovirus-infected Sf9 cells (lane 2). D) Analysis of phosphorylation in Baculovirus-expressed Rb. Baculovirus-infected Sf9 cells were labeled with 32 P orthophosphate and treated or not treated with placental alkaline phosphatase (PAP). Both untreated (lanes 1a and 1b) and treated (lanes 2a and 2b) lysates were immunoprecipitated with anti-rb antibody (clone G3-245, Cat. No A). Detection by autoradiography (top gel) shows that the radioactive label (lane 1a) is greatly reduced (lane 2a) following PAP-treatment. Western blot analysis of the autoradiographs (bottom gel) show that Rb in untreated lysates migrated at a higher molecular weight (lane 1b) than Rb in PAP-treated lysates (lane 2b). Collectively, the data indicate that Baculovirusexpressed Rb is phosphorylated, although at a lower level than native Rb. Abbreviations: prb, underphosphorylated Rb. pprb, phosphorylated and highly phosphorylated Rb species. 34

43 A CPM TdR Incorporated TF-1 Based GM-CSF Assay hgm-csf B kd Serial 3-Fold Dilutions C CPM TdR Incorporated CTLL-2 Based mil-4 Assay mil Serial 3-Fold Dilutions D kd Figure 12. Functional activity of Baculovirus-expressed recombinant protein. hgm-csf and mil-4 were cloned into pvl1393 and expressed in Sf9 cells. A) hgm-csf assay. hgm-csf activity was measured using the continuous cytokine dependent human cell line, TF hgm-csf, at 10 µg/ml, was serially diluted 3 fold in 12 wells across a 96-well flat-bottom microtiter plate in 50 µl. 50 µl of TF-1 cells at cells/ml were then added to each well for a final cell density of /ml. After a 44 h incubation at 37 C in the presence of 5% CO 2, the cultures were pulsed with 0.5 µci tritiated thymidine (20 Ci/mM) for an additional 4 h. The cultures were then harvested and the incorporated thymidine measured by scintillation counting. The data shown represent the cpm of thymidine incorporation versus 3 fold serial dilutions of hgm-csf. Each point represents the mean of three replicates. B) Western blot analysis of hgm-csf. Recombinant human GM-CSF (Cat. No V) loaded at 100 ng/lane and tested by Western blot analysis against purified anti-human GM-CSF (Cat. No D) at 1 µg/ml (lane 1) and normal rat serum at 1:500 dilution (lane 2). C) mil-4 assay. IL-4 activity was measured using the continuous IL-2 dependent murine cell line, CTLL-2. 27,28 mil-4 at 10 µg/ml was serially diluted 3 fold in 12 wells across a 96-well flat-bottom microtiter plate in 50 µl. 50 µl of CTLL-2 cells at cells/ml were then added to each well for a final cell density of /ml. After a 20-h incubation at 37 C in the presence of 5% CO 2, the cultures were pulsed with 0.5 µci tritiated thymidine (20 Ci/mM) for an additional 4 h. The cultures were then harvested and the incorporated thymidine measured by scintillation counting. The data shown represents the cpm of thymidine incorporated versus 3 fold serial dilutions of mil-4. Each point represents the mean of three replicates. D) Western blot analysis of mil-4. Recombinant mil- 4 lysate (Cat. No N) was loaded at 100 ng/lane and tested by Western blot analysis using purified anti-mouse IL-4 (Cat. No D) at 5 µg/ml (lane 1), and normal rat serum at 1:500 dilution (lane 2). 35

44 4.7 Purifying Recombinant Proteins Proteins expressed in the BEVS may be either secreted or non-secreted proteins. Proteins may be isolated by any conventional means including polyacrylamide gel electrophoresis and affinity columns. The purification of GST and 6xHis tagged proteins using affinity columns is described in Chapter 5. Additional protein purification methods are beyond the scope of this manual and are described in specialized manuals. 24 Non-secreted Recombinant Proteins Non-secreted proteins will remain in the cells. Cells should be pelleted and lysed to release the protein. Cell Lysate Preparation 1. Harvest cells infected with recombinant virus 3 days pi. 2. Spin down cells at 2,500 g for 5 min. 3. Resuspend cell pellet in ice-cold Insect Cell Lysis Buffer (Cat. No A) containing reconstituted Protease Inhibitor Cocktail (Cat. No Z). Use 1 ml of lysis buffer per cells. Lyse cells on ice for 45 min. 4. Clear lysate from cellular debris by centrifuging at 40,000 g for 45 min, or filter lysate through a 0.22 µm filter. 5. Harvest clear supernatant, which should contain your recombinant protein. 6. Run an SDS-PAGE gel to determine the amount of your recombinant protein in the total insect lysate. Note: Insect cells infected with either wild-type AcNPV or with XylE recombinant virus should be lysed as a negative control for western blot analysis. This lysate should lack the protein band derived from your cloned gene of interest. If occlusion body-positive virus particles are used for infection, an additional intense band of 29 kd should be visible, which represents the polyhedrin protein of the wild-type virus. If XylE infected cells are used, an additional band should be visible at 35 kd. 36

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