protein expression and analysis - massachusetts … expression.pdfbiochemical studies “protein...

Protein Expression and Analysis

Vijay Yajnik, MD, PhDGI UnitMGH

Identify your needs

■ Antigen production

■ Biochemical studies

■ Cell Biology

■ Protein interaction studies including proteomics

■ Structural studies

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.

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

Cell Biology“visualizing protein within a cell”

n GFP fusion proteins for direct immunoflourescence.

n Alternatively, use epitope tag antibody for in direct immunoflourescence.

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

Structural studies

n Soluble protein expression strategy is necessary

n Large quantities of protein are often needed

Expression Systems

■ In vitro■ Bacterial■ yeast ■ Insect■ Mammalian

■ Offer distinct advantages

■ Concepts are similar

■ Vector design is similar

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

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

Protease recognition sites

■ Thrombin Leu Val Pro Arg Gly Ser■ Factor Xa Ile Glu Gly Arg■ Enterokinase Asp Asp Asp Asp Lys

Fusion “tag” come in various lengths

Tag Size (aa)His • Tag 6,8 or 10GST • Tag 220FLAG • Tag 8HA • Tag 9Myc • Tag 9

Fusion “tag” have different properties

Contrast His to GFP

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

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

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

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

■ Bacterial ■ Eukaryotes

Yeast Insect cells

■ Mammalian cells in cultureTransfectionViral infection

AdenovirusRetrovirusVaccinia virus

Expression Systems

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

Cloning Strategy


■ 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


■ 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.

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

Insect Cell Expression Systems

■ As higher eukaryotes, insect cells offer many posttranslational modifications

■ Proteins are targeted to appropriate sub-cellular destination

■ Cells grow in suspension culture■ Produce high levels of soluble

recombinant proteins■ Drawbacks - not widely used, difficult for

beginner

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

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

Troubleshooting 2

■ Secondary structure in the mRNA transcript

■ Excessive rare codon usage■ Domains express well

Troubleshooting-3Solubility 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

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

Mammalian expression vector

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

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

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

Use of CHO cells

■ Relatively easy to transfect■ Cotransfect target gene with a selectable

marker■ Stable integration into host cell

chromosome and subsequent amplification

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

Detection of the Target Protein

■ Coomassie blue staining of cell extracts■ Silver staining of gels, SPYRO stains■ Western blotting using tag specific

antibody

Western blotting

SDS-PAGE

Electrotransfer

Block

First antibody

Wash

Second antibody

Wash

Detection

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

Case Study

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

Purifying the target protein

■ Conventional methods■ Affinity purification - fusion tags enable

purification of the target protein in ONE step

Conventional protein purification techniques

■ Salt fractionation■ Differential solubility■ Ion-exchange chromatography■ Hydrophobic interaction chromatography■ Affinity and pseudoaffinity

chromatography■ Gel filtration

Protein GST

GlutathioneAgarose

Concept

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

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)

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

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

CONCEPTTAP system

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

Sub-cellular localization

Case Studywith

Gateway Technology

Gateway Technology

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

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