Protein Expression and Analysis. Vijay Yajnik, MD, PhD GI Unit MGH

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1 Protein Expression and Analysis Vijay Yajnik, MD, PhD GI Unit MGH

2 Identify your needs Antigen production Biochemical studies Cell Biology Protein interaction studies including proteomics Structural studies

3 Antigen Production n Express protein as a fusion with a Tag which facilitates rapid purification by affinity chromatography n Express protein in E coli as an insoluble inclusion body which can then be made soluble by exchanging buffer in vitro and subsequently purified to inject rabbits for antibody production.

4 Biochemical studies protein function n For kinases and other enzymes, the method should preserve function n Cleavage of tag sequence may be necessary for functional studies

5 Cell Biology visualizing protein within a cell n GFP fusion proteins for direct immunoflourescence. n Alternatively, use epitope tag antibody for in direct immunoflourescence.

6 Protein interaction studies Protein expression strategy with preserved modifications such as glycosylation and phosphorylation. Example SH2 domains Purification of multi-protein complexes by affinity chromatography for mass spec. Example, identification of cytoskeletal proteins in focal adhesions

7 Structural studies n Soluble protein expression strategy is necessary n Large quantities of protein are often needed

8 O f f e r d i s t i n c t In vitro Bacterial yeast Insect Mammalian Expression Systems

9 Anatomy of an expression vector Plasmid or virus based backbone Polylinker to clone cdna Selectable marker i.e. drug resistance or GFP transcriptional promoter, often inducible Translational controls such as Ribosomal binding site in bacteria and Kozak sequence in mammalian cells

10 Fusion Proteins Can be either N or C terminal fusion or both Fusion component helps either in protein detection or protein purification or both Fusion protein can be cleaved by site specific proteases Rarely, fusion may alter protein function

11 Protease recognition sites Thrombin Leu Val Pro Arg Gly Ser Factor Xa Ile Glu Gly Arg Enterokinase Asp Asp Asp Asp Lys

12 Fusion tag come in various lengths Tag Size (aa) His Tag 6,8 or 10 GST Tag 220 FLAG Tag 8 HA Tag 9 Myc Tag 9

13 Fusion tag have different properties Contrast His to GFP

14 The 6xHis tag-salient Features Six consecutive histidine residues Much smaller than most other affinity tags Allows for purification, detection and assay of almost any 6xHis-tagged protein from all expression systems

15 The 6xHis tag 2 Rarely alters or contributes to protein immunogenicity Rarely interferes with protein structure or function Does not interfere with secretion Compatible with denaturing buffer systems

16 GFP: A must have in Cell Biology Approimately 1 kb coding sequence GFP fusion proteins, ideal for IF GFP in vector, not as fusion, serves as a marker for DNA transfection Good antibody available for western blot detection

17 Cloning Strategy Choose appropriate restriction site Ligate insert to vector Transform Identify positive clones by PCR and/or mini prep Sequence, very important in troubleshooting

18 Expression Systems Bacterial Eukaryotes Yeast Insect cells Mammalian cells in culture Transfection Viral infection Adenovirus Retrovirus Vaccinia virus

19 Bacterial Expression-1 Grow rapidly and cost effective Suited for large scale production No posttranslational modifications Inclusion body prep ideal for insoluble proteins Special strains that are protease deficient are readily available

20 Cloning Strategy

21 Bacterial Expression-2 Phage promoters vectors, pet system At, non-permissive temp, promoter is off BL21pLysE, at permissive temperature, rapidly transcribes vector cdna and within hours >30% of cellular protein is your protein of interest Limitation: needs conventional purification steps

22 Bacterial Expression-3 E coli uses codons that are rarely used by mammalian DNA, therefore, trna pool for amino acids like arginine, isoleucine, leucine and proline are limiting. BL21 RIL, BL21 RP and BL21 Rosetta are genetically altered strains that optimize codon useage.

23 Four archaeal genes expressed in BL21 (lane 1) and codon optimized BL21-RL (lane 2)

24 Insect Cell Expression Systems As higher eukaryotes, insect cells offer many posttranslational modifications Proteins are targeted to appropriate subcellular destination Cells grow in suspension culture Produce high levels of soluble recombinant proteins Drawbacks - not widely used, difficult for beginner

25 Baculovirus vectors Sf9 insect cell line Baculovirus can accommodate large cdna insert Baculovirus are non-infectious to vertebrates and their promoters are inactive in most mammalian cells

26 Troubleshooting 1 Check sequence for stop codons If, truncated products observed there is proteolytic degradation Your protein is toxic Modify 2nd amino acid Aberrant translation initiation

27 Troubleshooting 2 Secondary structure in the mrna transcript Excessive rare codon usage Domains express well

28 Troubleshooting-3 Solubility of the fusion protein Grow bacteria at a lower temperature Lower the level of expression Vary the strain background, some strains carry mutations in lacy gene allowing better permeability to IPTG Change the fusion partner

29 Expression in Mammalian Cells CMV promoter vectors SV40 origin of DNA replication Plasmids are grown in bacteria Ideal starting cell lines are COS, 293T and CHO Standardize transfection in specific cell lines

30 Mammalian expression vector

31 Transient Transfection of Mammalian Cells Calcium phosphate is gold standard Newer liposomal agents are very popular Special fusion proteins can be made in bacteria which are then engulfed by mammalian cells, example VP22 Adherent cell lines are difficult to transfect

32 Stable Transfection of Mammalian Cells Valuable when transfection efficiency is low Co-transfection with drug resistant marker such as G418, puromycin and hygromycin Test multiple clones for protein expression Functional studies can be performed on these cell lines

33 Use of COS cells Rapid, transient expression of protein Relatively easy to transfect Transformed with an origin-defective SV40 virus SV40 containing plasmids can replicate to a high copy number ~100,000 copies/cell 48 h post transformation

34 Use of CHO cells Relatively easy to transfect Cotransfect target gene with a selectable marker Stable integration into host cell chromosome and subsequent amplification

35 Troubleshooting 4 Check and standardize transfection efficiency Make new maxi prep DNA Endo free plasmid kits Toxic genes need an inducible system such as the Tet on or off system

36 Detection of the Target Protein Coomassie blue staining of cell extracts Silver staining of gels, SPYRO stains Western blotting using tag specific antibody

37 Western blotting SDS-PAGE Electrotransfer Block First antibody Wash Second antibody Wash Detection

38 Western blotting Blocking is a critical step Standardize antibody dilutions Adhere to one system PVDF membranes are great for re-probing Use X ray film designed for chemiluminescence

39 Case Study

40 Example: Express Novel Protein Make bacterial GST-Protein fusion protein for antibody production and protein interaction studies Mammalian Flag-Protein constructs for biochemical and sub-cellular localization studies

41 Purifying the target protein Conventional methods Affinity purification - fusion tags enable purification of the target protein in ONE step

42 Conventional protein purification techniques Salt fractionation Differential solubility Ion-exchange chromatography Hydrophobic interaction chromatography Affinity and pseudoaffinity chromatography Gel filtration

43 Concept Glutathione Agarose Protein GST

44 GST Fusion Proteins N terminal fusion in pgex vector series GST binds to glutathione which can be coupled with agarose and easily purified Expression in E. coli is inducible by IPTG Binding of glutathione agarose to GST fusion protein is reversible GST portion can be easily cleaved to recover native protein

45 GST-Protein Pick several clones and verify GST- Protein induction to identify best expressing clone Scale up, make liter prep Purify GST-Protein with Glutathione agarose beads Elute with glutathione Cleave Protein from GST, send Protein for antibody production (not absolutely necessary)

46 GST-Protein Interacting Proteins Express GST-Protein, immobilize to glutathione agarose Make mammalian cell extracts, incubate GST-Protein-agarose for binding with cognitive proteins Wash, PAGE, silver stain Run controls Mass Spec on novel bands

47 Interplay-mammalian TAP system by Stratagene Tandem affinity purification Purify your protein of interest Identify its interactors by mass spectrometry Key is use of 2 tags instead of one SBP strepavidin binding peptide CBP calmodulin binding peptide

48 CONCEPT TAP system

49 Protein Cell Biology Express Flag-Protein in COS cells Immunoflourescence for Nuclear or Cytoplasmic localization GFP-Protein fusion for sub-cellular details in live cells Test Protein antibody Make inducible stable cell lines

50 Sub-cellular localization

51 Case Study with Gateway Technology

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64 Summary Identify your needs before embarking on a system Understand basic concepts Troubleshooting expression requires multiple small scale experiments Standardize detection Focus on biological assays