Custom Protein Expression

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1 Custom Protein Expression Introduction Cosmogenetech Inc., an industry leader in gene, peptide synthesis, is pleased to launch the new protein expression and purification service. Cosmogenetech Inc. has the necessary know- how to deliver the best possible esults to our valued customers. We have the capacity to take your project directly from gene synthesis to protein expression, refolding and purification. At Cosmogenetech Inc., we are proud of our ability to optimize protein expression, maximize product yield, and ensure purity to serve every aspect of your research needs. Gene to Protein Starting from de novo gene synthesis, cdna or from almost anywhere a client asks us to begin; our scientists can deliver high quality purified protein in a timely manner. About Protein Purification Recombinant proteins are used throughout the whole spectrum of biomedical research. Once you have large quantity of purified protein, you could conduct many downstream experiments, such as enzyme kinetics, functional and structural studies. There are two main systems for the expression of recombinant protein - prokaryotic and eukaryotic. Prokaryotic recombinant protein expression systems (E.coli ) have several advantages, including ease of culture, rapid cell growth and relatively simple purification procedures. However, most post-translational modifications are not added by bacteria, and most proteins become insoluble in inclusion bodies and are very difficult to recover as functional proteins. Eukaryotic systems for the expression of protein include yeast, baculovirus cells, and mammalian cells. Advantages of these systems include high levels of expression, no inclusion bodies, and intact post-translational modifications. Expression System: In order to decide which host is most suitable the amount and the degree of purity of the product as well as its biological integrity and potential toxicity should be considered. For example, bacterial expression systems are not suitable if post-translational modification is required to produce a fully functional recombinant product. COSMOGENETECH E.coil expression service Step Description Step 1 Pretest Optimization of expression condition 1 week 500,000 Generation and identification of bacterial colonies expressing target proteins - Small scale expression은 large scale expression을 하기 전에 반드시 해야 할 단계로, Expression optimizaion(induction Time, IPTG concentration, Growth temperature, Bacterial strains, Protein solubility) 하는 단계입니다. Step 2 Western blotting Expression protein test 1 week 200,000 Pretest의 SDS-PAGE 분석을 통하여 Expression 조건이 결정되면, 목적 단백질에 tagging된 antibody(his, GST, MBP 등)를 이용하여 발현된 단백질을 확인하는 단계입니다 (anti-his or GST 외의 antibody 확인 요청 시 Ab 제공 필요) Step 3 Purification Protein Purification 1 week 300, One liter of bacterial culture will be induced with IPTG and harvested for protein purification 2. Protein purification using - Ni-NTA (for 6xHIS tagged proteins) - Glutathione (for GST-tagged proteins) beads - MBP resin at either native or denatured conditions 3. Non-tagging protein purification Total Protein Expression 3 weeks 1,000,000 Protein Expression(10건) 5 weeks 20% DC Addional service Taq removal 1 week 300,000 Expression Vector: Once the host has been selected, many different vectors are available for consideration. However, as for the selection of a suitable host system, the final choice of vector should take into consideration the specific requirements of the application and will, of course,be influenced by the behaviour of the target protein. Tag: As for the selection of host and vectors, the decision to use which tag must be made according to the needs of the specific application. The two most commonly used tags are glutathione S-transferase (GST tag) and 6 x histidine residues ((His)6 tag).

2 BIOBASIC E.coil expression service Description An Exciting New Launch 5 mg Intact-Native Protein Expression Description d 5 mg purified soluble protein Tag removal, endotoxin removal service available d 5mg purified soluble protein from E.coli expression system 3,244,000 Intact Native Protein Expression Ultrasonication, Refolding, Purification, Tag Removal 5mg of purified protein in its native fron (Notaq, 90-95% purity,endotoxin<1eu/ug) 7,582,000 d 15 mg purified soluble protein Tag removal, endotoxin removal service available Traditional Service Tag removal, endotoxin removal service available d 15mg purified soluble protein from E.coli expression system Purified protein from E.coli expression system 4,325,000 a. The guaranteed Packaes (PUR-15MG-E.coli & PUR-5MG-E.coli ) have the following features b. Customer provides the plasmid or the expression strain c. Size of the target protein is usually < 100KDa d. In some rare cases, in order to facilitate expression, Bio Basic Inc. may try different rags and final protein product may contain such tag e. The final purified protein is tag-containing f. Cosmogenetech Inc., does not guarantee the endotoxin level of purified protein g. Cosmogenetech Ine., guarantees the protein to be soluble, but not the bioactivity or functionality If target protein expresses as inclusion body, refolding will be done to make it soluble h. If Cosmogenetech Inc., failed to deliver the guaranteed amount due to the nature of the protein or other unpredictable circumstance, only a minimum set up fee will apply Other E. coli Expression System Services Description Lyophilization services Functional preservation and longer shelf life 845,000 Fermentation services cgmp manufacturing services Expression pilot study Sale-up of fermentation and purification process up to 300 liters Fully equipped cgmp facility 1,183,000 Description - Constrution of expressing plasmid containing the cdna encoding the protein of interest with N-terminus Tag - Small scale transient expression of HEK293 cells - Expression pilot study, expression level determination by Westhern Blot Mammalian Expression System Mammalian cell lines (CHO, HEK 293 etc) are widely used for the production of recombinant glycoproteins(vaccines, enzymes) as well as hormones and immunobiologicals (antibodies, interleukins, lymphokines), as they have the advantage of full posttranslational modifications of the expressed protein Mammalian - Protein production Description Transient expression and purification Stable cell line establishment Tag removal service available Purified protein * FREE Western is available for tagged proteins that can be detected by the anti-his antibody. For other tagged proteins, additional charges may apply.

3 Featured Catalogue Products In addition to the outlined custom protein purification services, Bio Basic Inc. possesses an extensive list of ready made proteins. This following table includes a sampling of the catalogue protein library (We have a lot more!). If your protein of interest is not on the list, please send an to to. Interleukins Growth Factors Chemokines IFNs Other Specialty Products Recombinant Human IL-1α Recombinant Human GM-CSF Recombinant Human IL-2 Recombinant Human EPO Recombinant Human SDF- 1α/CXCL12 Recombinant Human Lymphotactin/XCL1 Recombinant Human IFN-α1b Recombinant Human IFN-α2b Recombinant Human TNF-α Recombinant Bovine Enterokinase Baculovirus Expression System Stage 1 Gene Synthesis Stage 2 - Obtain cdna sequence - Codon optimization if needed - Gene synthesis - Plasmid amplification and purification - Subclone into pfastbac donor vector - Sequence verification - Isolation of recombinant plasmid DNA Description 1 Synthesized gene - Purified plasmid - Plasmid containing bacteria strain Sequence and size dependent 1-2 weeks Visit Gene Synthesis page Recombinant Murine IL-3 Recombinant Human IGF-1 Recombinant Human RANTES/CCL5 Recombinant Human IFN-γ Recombinant ProteinG Stage 3 Generation of Bacmid DNA - Selection of recombinant Bacmid DNA containing host strain - Isolation of recombinant Bacmid DNA - Bacmid DNA containing strain - Bacmid DNA 2 weeks Phase 1 Recombinant Human IL-12 Recombinant Human IL-33 Recombinant Human TPO Recombinant Human EGF Recombinant Human MIP- 1α/CCL3 Recombinant Human GRO-α/CXCL1 Recombinant Human IL-29/ IFN-λ1 Recombinant Rat IFN-γ Recombinant Cys-Protein G Recombinant Protein A/G Stage 4 Transfection of Insect Cells Stage 5 Pilot Run Stage 6 Protein Expression and Purification - Multiple transfection of Sf-9 to amplify virus - SDS-PAGE, Western-Blot analysis - Pilot run for determination of production yield and other characteristics of the protein Multiple methods for purification Tag removal (optional) SDS-PAGE, Western-Blot analysis 2 - High titer virus stock 3 weeks - Progress update on pilot experiment 1-2 weeks 0.1-2mg of purified protein 3 >90% purity Final report 2-3 weeks Phase 2 Stage 7 Additional Services - Endotoxin removal - HPLC, MS analysis - Purified protein - QC report 2 weeks 1 Listed is the typical turnaround time at Bio Basic Inc. Actual time may vary depending on individual project. 2 FREE Western is available for tagged proteins that can be detected by the anti-his antibody. For oth er tagged proteins, additional charges may apply. 3 Scale up is available upon request. Addition charge will apply.

4 Custom Antibody service [Cosmogenetech] Cosmogenetech Inc. produces high quality polyclonal antibody according to peptides and proteins provided by customer or Cosmo itself. Moreover, Cosmo supports one-step service of cloning, protein expression or peptide synthesis based on accumulated skills through cloning business. [Biobasic] Introduction Bio Basic is excited to launch our new Custom Antibody service! We have assembled Custom Antibody (monoclonal and polyclonal) packages that exceed our exacting standards and are sure to meet yours. Whether you desire antibodies for Western Blot or ImmunoHistochemistry, ELISA or ambitious projects that are highly-specialized to your lab, Bio Basic can help you obtain the results that you need. Our packages are structured give you the freedom to synthesize (from cdna, peptide or protein) an antibody that will be successful in basic applications or guaranteed to work in your assay of choice. Our qualified sales staff and experienced scientists will help you choose the Custom Antibody package that suits your project goals. We will keep you informed throughout the generation of custom antibodies. Features ELISA titer guarantee > 1:100,000 (Monoclonal) and > 1:50,000 (Polyclonal) and Dot Blot guarantee Application-guaranteed monoclonal and polyclonal Ab packages that will always produce the Ab you need Rapid and constant communication to keep you up-to-date on the status of your project All of our shipped antibodies are lyophilized for ease of transport and prevention of degradation Industry-leading peptide design and synthesis protocols to ensure your antigens generate optimal immune responses Highly competitive pricing Polyclonal antibody Polyclonal antibodies (pabs) are immunoglobulins that are obtained from different B-lymphocytes. pabs exist as a heterogeneous mixture of antibodies with differing antigen affinities in serum, and recognize multiple epitopes on any one antigen. COSMOGENETECH Antigen manufacture Antigen Description Lead time Peptide Recombinant protein Antibody manufacture Peptide design 1 day free Peptide synthesis 2 weeks Peptide conjugation (BSA/KLH) 2 day 160,000 Protein expression 2 weeks Protein gel elution 1 week 120,000 Immune animals Antigen volume Provides Lead time Mouse (5 heads) Rat (2 heads) Rabbit (1 head) Rabbit (2 heads) Rabbit (3 heads) Peptide : 1.3 mg Protein : 0.8 mg Strain : 1.0 mg Peptide : 1.2 mg Protein : 0.5 mg Strain : 0.8 mg Peptide : 1.5 mg Protein : 1.0 mg Strain : 0.8 mg Peptide : 3.0 mg Protein : 2.0 mg Strain : 1.6 mg Peptide : 4.5 mg Protein : 3.0 mg Strain : 2.4 mg ELISA identification step-by-step serum whole serum 10~15 weeks 600, , ,000 1,000,000 1,300,000 Antibody purification Description Volume Lead time Protein A/G affinity purification 10ml 1 week 250,000 PBS buffer exchange 10ml 2 days 250,000

5 BIOBASIC 1) PolyBasic Standard Peptide (Poly-Pep-Stan) 2) PolyBasic Standard Protein (Poly-Pro-Stan) 3) PolyBasic Standard Phospho-Specific (Poly-Phos-Stan) 4) PolyBasic d Phospho-Specific (Poly-Phos-Guar) *모든 antibody제작 과정시 배송료와 peptide 합성 비용은 추가 비용으로 청구됨을 알려드립니다. PolyBasic Standard Peptide (Poly-Pep-Stan)* PolyBasic Standard Peptide Poly-Pep-Stan Bio Basic guarantees to generate antibodies that are Dot Blot-positive against peptide and have ELISA titre > 1:50,000 Name, species, sequence and NCBI Number for target protein 1. Peptide design, synthesis and conjugation (additional fees required) 2. Immunize 2 New Zealand Rabbits 4. Antigen affinity purification ELISA and Dot Blot data against peptide ml/rabbit antiserum All purified antibody (at least 2 mg) 1 mg peptide PolyBasic Standard Phospho-Specific (Poly-Phos-Stan)* PolyBasic Standard Phospho-Specific Poly-Phos-Stan Bio Basic guarantees to generate antibodies that are Dot Blot-positive against peptide and have ELISA titre > 1:50,000 and modifide/unmodifide titer ratio > 8 2. Information of modification site 1. Peptide design, synthesis and conjugation, including the modified peptide and unmodifide control peptide (additional fee req.) 2. Immunize 4 New Zealand Rabbits 4. Antigen affinity purification and cross-reactivity reduction ELISA and Dot Blot data against modified and unmodified peptides All purified antibody (at least 1 mg) 1mg modified peptide 1mg unmodified peptide weeks from antigen synthesis to sample delivery Client shall provide full payment before project initiation PolyBasic d Phospho-Specific (Poly-Phos-Guar)* 6-10 weeks from antigen synthesis to sample delivery Client shall provide full payment before project initiation PolyBasic Standard Protein (Poly-Pro-Stan)* PolyBasic Standard Protein Poly-Pep-Stan Bio Basic guarantees to generate antibodies that are Dot Blot-positive against immunogen and have ELISA titre > 1:50,000 Customer to Provide Protein antigen 1. Qc client's antigen 2. Immunize 2 New Zealand Rabbits 4. Protein A affinity purification ELISA and Dot Blot data against immunogen ml/rabbit antiserum All purified antibody (at least 20 mg) 4-6 weeks from immunization to sample delivery Client shall provide full payment before project initiation PolyBasic d Phospho-Specific Poly-Phos-Guar Bio Basic guarantees to generate antibodies that are Dot Blot-positive against peptide and have ELISA titre > 1:50,000 and modifide/unmodifide titer ratio > 8 Bio Basic also guarantees antibody will work in client's experimental system 2. Information of modification site 1. Peptide design, synthesis and conjugation, including the modified peptide and unmodifide control peptide (additional fee req.) 2. Immunize 4 New Zealand Rabbits 4. Antigen affinity purification and cross-reactivity reduction ELISA and Dot Blot data against modified and unmodified peptides First Shipment : Any samples of the client's interest (50ul each) 1mg modified peptide 1mg unmodified peptide Second Shipment : All purified antibody (at least 1 mg) weeks from antigen synthesis to sample delivery Client shall pay 50% of price before project initiation Client shall pay remaining balance upon confimation that shipped antibodies work in their experimental system

6 Monoclonal antibody Monoclonal antibody service allows us to produce large amounts of pure antibodies in the following way: We can obtain cells that produce antibodies naturally; we also have available a class of cells that can grow continually in cell culture. If we form a hybrid that combines the characteristic of "immortality" with the ability to produce the desired substance, we would have, in effect, a factory to produce antibodies that worked around the clock. The mab's were produced based on the ability of a cancer cell's (a myeloma) ability to multiply indefinitely. The stages of producing a mab are: " Immunization of the host (usually mice) " Separation of the immune cells (the plasma cells) " Fusion of the myeloma cell with the plasma cell " Separation of the hwybridoma cells " Culturing of the cells " Screening of the cells producing mab's BIOBASIC Monoclonal Antibody s MonoBasic Library (Mono-Lib-10) MonoBasic Western Blot (Mono-WB-1) MonoBasic Immunofluorescence/Immunocytochemistry (Mono-IF-1 / Mono-ICC-1) MonoBasic Immunohistochemistry (Mono-IHC-1) MonoBasic Immunoprecipitation (Mono-IP-1) MonoBasic Omni-Application (Mono-Omni-1) MonoBasic Phospho-Specific (Mono-Phos-1) MonoBasic Omni-App/ Phospho-Specific Monoclonal Antibody cell line package 많은 수의 (10개) Hybridoma cell line을 원하는 경우!! MonoBasic Library (Mono-Lib-10) ; peptide, protein 모두 가능!! Monoclonal Antibody cell line Mono-Lib-10 Bio Basic guarantees to generate at least 10 clones ELISA titre > 1:100,000 and positive Dot Blot results against the immunogen Bio Basic also guarantees that at least 1 clone will work in client's own experiment system OR 2. Protein antigen with < 60% homology to mouse isoform 1. Antigen disign and production or QC client's antigen 2. Immunize 3 Balb/c mice 3. Fusion and ELISA screening 4. (2-4 rounds) 5. Antibody production 6. Antibody validation For each cell line: 1. Epitope and affinity data (if available) 2. ELISA and Dot Blot data on immunogen 10 ELISA-positive hybridoma cell lines weeks from antigen synthesis/qc to sample delivery Client shall pay 50% of price before project initiation Client shall pay remaining balance upon project completion *Antigen이 Peptide인 경우 Peptide의 합성비용은 추가요금으로 청구되며, 배송료도 추가요금으로 부과됩니다. MonoBasic IP/IF/ICC/IHC MonoBasic Western Blot Application specificity MonoBasic Library (ELISA-guaranteed) MonoBasic Custom Monoclonal Antibody s

7 Monoclonal Antibody basic package ELISA와 Western blot결과까지 원하는 경우!! MonoBasic Western Blot (Mono-WB-1) ; peptide, protein 모두 가능!! MonoBasic Western Blot Mono-WB-1 Bio Basic guarantees to generate at least 1 clone that specifically detects the overexpressed antigen by WB Bio Basic also guarantees that at least 1 clone detects endogenous antigen in client's WB system 2. Full length cdna plasmid 1. Antigen disign and production 2. Immunize 3 Balb/c mice 3. Fusion and ELISA screening 4. (2-4 rounds) 5. Antibody production 6. Protein G affinity purification 7. Antibody validation For each cell line: 1. Comprehensive validation data on overxpressed antigen, including ELISA and WB data 2. Epitope and affinity data (if available) 3. ELISA and Dot Blot data on immunogen First Shipment : any sanples of client's interest (50ul each) Secound Shipment : 1 hybridoma cell line with 2 mg purified antibody weeks from antigen synthesis to sample delivery Client shall pay 30% of price before project initiation Client shall pay remaining balance upon confirmation that a qualified clone works in their WB system *Antigen이 Peptide인 경우 Peptide의 합성비용은 추가요금으로 청구되며, peptide 합성 소요 기간은 별도로 소요됩니다. 또한 2번의 배송료도 추가요금으로 부과됩니다. Monoclonal Antibody phospho package Phospho-specific monoclonal antibody제작을 원하는 경우!! MonoBasic Phospho-Specific (Mono-Phos-1) ; Peptide만 가능!! MonoBasic Phospho-Specific Mono-Phos-1 Bio Basic guarantees to generate at least 1 clone that is Dot Blot-positive against peptide and have ELISA titre > 1:50,000 and modified/unmodified titer ratio > 8 Bio Basic also guarantees at least 1 clone will work in client's experiment system 2. Information of modification site 1. Peptide disign, synthesis and conjugation, including the modified peptide and unmodifide control peptide (additional fee req.) 2. Immunize 3 Balb/c mice 3. Fusion and ELISA screening 4. (2-4 rounds) 5. Antibody production 6. Protein G affinity purification 7. Antibody validation ELISA and Dot Blot data against modified and unmodified peptides First Shipment : Any samples of the client's interest (50ul each) 1mg modified peptide 1mg unmodified peptide Second Shipment : 1 hybridoma cell line with 2 mg purified antibody weeks from antigen synthesis to sample delivery Client shall pay 50% of price before project initiation Client shall pay remaining balance upon confirmation that shipped antibodies work in their experimental system *Antigen으로 이용되는 Peptide의 합성 비용은 추가요금으로 청구되며, peptide 합성 소요 기간은 별도로 소요됩니다. 또한 2번의 배송비도 추가요금으로 부과됩니다. (Monoclonal basic과 phospho package의 경우 1개의 hybridoma cell line을 제공하나, 주문 시 추가로 요구하시면 2개 이하의 hybridoma cell line을 추가 제공 가능함을 알려드립니다 (단, 추가비용이 발생될 수도 있습니다.))

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