August 22, 2011 Biochemistry 201 D. Worthylake, 7152MEB, x5176

1. Expression & Purification of Recombinant Proteins August 22, 2011 Biochemistry 201 D. Worthylake, 7152MEB, x5176

2. Why express/purify protein(s)? • If you don’t have the gene that encodes the protein but you have a source, you may want to purify the protein to: • determine the amino acid sequence • make antibodies • Identification by mass spectroscopy • If you have the gene that encodes the protein, you may want to express/purify the protein for other reasons: • structural analysis (x-ray crystallography & NMR spectroscopy) • enzyme function • Interaction partners • biochemistry/biophysics (phosphorylation, regulation, etc.) • Functional studies (cellular localization by confocal microscopy, etc) • Pharmaceutical intervention

3. TOP 10 Things to consider Which protein construct to express Expression host (bacterial, insect cell, yeast, or mammalian) Cell line for expression Promoter for induction of protein production Codon optimization (for mammalian proteins expressed in bacteria) Cloning method May require expression as a fusion protein May require co-expression with molecular chaperones Affinity tag(s) for purification & protease cleavage site to remove the tag Purification protocol & buffer to keep protein happy and active

4. Protein construct and expression host Engineering of protein construct An entire protein ? If yes, don’t need to worry about limits A domain from a mosaic protein ? Need to worry about limits Which organism to express the protein in If protein is of bacterial origin, express in bacteria If protein is of non-bacterial origin, because of post-translational modification in non-bacterial cells, may need to express in higher organisms: Bacteria  Yeast  insect cells (SF9 or Hi5)  Mammalian cell lines (least expensive & time consuming) (most expensive & time consuming) May need to express as a fusion protein or require codon-optimization

5. Choice of expression host Bacterial expression system Advantages – Easy, great over-expression, low protease activity, no post-translational modifications Disadvantages – Protein solubility, lack of post-translational modifications Eukaryotic expression system Advantages – Protein solubility, post-translational modifications Disadvantages – Expensive, low yield, proteases, time consuming Isolate protein from native source Advantages – Protein solubility, authenticity Disadvantages – Expense/effort, yield, slaughter-houses Waring blenders Hierarchy: Bacteria, Yeast, SF9, Hela, native tissue

6. Bacterial host BL21(DE3) Hosts for protein expression DE3 Host-encoded lac Repressor represses host RNAPol transcription of T7 RNAPol from lac promoter. IPTG induction knocks lac Repressor off and allow host RNAPol to transcribe T7RNAPol. T7 RNAPol transcribe gene from T7 promoter on plasmid. Lon/OmpT protease deficient. DE3_pLysS DE3 strains have leaky expression, which leads to problems if expressed protein is toxic. Plasmid-encoded T7 lysozyme inhibits T7 RNAPol and decrease leaky expression. Host for cloning These hosts lack T7 RNAPol , and so are suitable for plasmid amplification and not protein expression. Isopropyl β-D-1-thiogalactopyranoside (IPTG) = allolactose mimic Figures from www.novagen.com

7. Specialized bacterial cell lines for protein expression Company Cell Genotype Phenotype Invitrogen BL21Star RNaseE(131) more stable mRNA BL21-AI ara- arabinose induction p15A p15A p15A F/p15A+F p15A Novagen Rosetta2 7 rare tRNA overcome codon usage deficiency Origami 2/B Thrx-/GluthRed- enhance formation of disulfide bonds Clontech BL21Pro TetR tetracycline induction Lucigen C41/C43 ?? Expression of toxic proteins p15A p15A all companies pLysS T7 lysozyme decreases leaky expression You cannot just mix and match, as plasmids with the same origin of replication cannot be transformed into the same cells. Most expression plasmids have pBR322 ori, which is compatible with p15A.

8. Consideration of codon usage

9. Basic elements of a plasmid/vector pET developed by WF Studier & BA Moffatt in 1986 Ap = ampicillin resistance ori = ColE1/pBR322 origin of replication lacI = lac repressor; bind lacO until IPTG induction T7P = T7 Polymerase promoter lacO = lac operator where lac repressor binds =multiple cloning site

10. Selection of cloning method is critical Restriction digestion-based methods are inefficient and require that your gene of interest does not have the same internal restriction site(s) as present in MCS. Gateway-based methods are powerful. Ligation-independent cloning is much more effective than ligation reaction. The green/red parts of the primers are not complementary with the gene!

11. Gateway technology overview (discussed for completeness) See file invitrogen_Gatewaymanual2003.pdf (optional reading)

12. Gateway recombination reactions Entry clone Expression clone See file invitrogen_Gatewaymanual2003.pdf (optional reading)

13. Generating an entry clone TAGG ATCC * Don’t put in stop codon (*) if to have a C-ter tag (Original site) (Original site) (Int + IHF) TAGG ATCC * See file invitrogen_Gatewaymanual2003.pdf (optional reading)

14. Recombining entry and destination clones to get an expression clone TAGG ATCC * (Int + IHF + Xis) TAGG ATCC * (Original site restored) (Original site restored) See file invitrogen_Gatewaymanual2003.pdf (optional reading)

15. PCR and ProcessiveDNAPol newcumulative Cycle 1: 2o = 11 = 21 - 1 Cycle 2: 21 = 2 2 + 1 = 3 = 22 - 1 Cycle 3: 22 = 4 4 + 3 = 7 = 23 - 1 www.neb.com Cycle n: 2n-1 2n - 1 … 15 cycles = 32,767 copies 20 cycles = 1,048,575 copies 30 cycles = 1,073,741,823 copies decreases lower-molecular weight fragments decreases extension time from 1’/kb to 10-20”/kb, so a typical amplification takes ~1hr instead of 3-4 hours. NEB Phusion (dbd) & Takara Speedstar (antibody-based)

16. LIC cloning, reminder Ligation-independent cloning is much more effective than ligation reaction.

17. LIC-Subcloning of a gene, from beginning to end DAY 1 (~6.5 hr) PCR gene from SOURCE vector (50l) (80’) Verify on agarose gel that gene was amplified (10l) (40’) Digest remaining sample with DpnI and do PCR clean-up (75’) Digest DESTINATION vector and PCR fragment with T4 DNAPol to generate single-stranded overhangs (60’) Add 100ng PCR fragment to 15ng DESTINATION vector and let anneal on ice (30’) Transform into XL10Gold supercompetent cells (100’) and plate overnight DAY 2 (~2.5 hr) Do colony PCR on 2-4 colonies to verify gene was inserted into vector: a) pick colony with pipet tip b) resuspend in 50l steril water and vortex to mix c) take 1l for PCR to verify insert (120’) d) inoculate remaining sample in TB for overnight growth e) if gene is inserted, do miniprep(30’) f) Verify construct by DNA sequencing

18. Bacterial transformation Frederick Griffith (1928) first demonstrated by showing that non-virulent Streptococcus pneumonia could be made virulent by exposing it to a virulent strain which has been heat-shocked. Oswald Avery (1944) demonstrated uptake of DNA into bacteria and coined the term bacterial transformation. To make cell competent: Grow cells to mid-log phase and treat/wash with CaCl2 solution. The bacterial cell wall is permeabilized by Chloride ion and swells up with the uptake of water. Bacterial transformation: Addition of plasmid DNA to cells, followed by cold- and heat-shock allows plasmid to enter through the small holes in the cell wall. (Can also use electroporation to create pores in cell wall). Amplify the number of cells in SOC media. Plate on LB agar (+ antibiotic) to select for transformed cells only (antibiotic resistance is conferred by gene encoded by on plasmid).

19. Selection of expression vector & fusion partner • Expression in insect and mammalian cells are expensive and time consuming. • Therefore, a feasible method is required for expression in bacterial cells as a • first choice. • Expression difficulty in bacterial cells may be overcome by expression as a • fusion protein. Expression at lower temperature improves solubility. Laila Niiranen, … Nils P. Willassen, Protein Expression & Purification 52 (2007) 210-218. • The apparent solubilizing effect of the fusion partner may be misleading, • as the purified protein can precipitate when cleaved from its fusion partner.

20. LIC vector for in-vivo cleavage from fusion protein In-vivo cleavage helps remove false-positive expression (protein once purified and cleaved from its fusion partner precipitates!). Co-express TVMV protease with fusion protein, with TVMV under control of a different promoter than the one used for the fusion protein.

21. In-vivo cleavage improves protein solubility Soluble insoluble

22. In-vivo cleavage helps protein purification Intact fusion proteinin-vivo cleaved protein M L FT W E +tev L FT W E +tev L FT W L FT W

23. Delayed In-vivo cleavage improves protein solubility 0hr delay 2hr delay

24. Co-expression with molecular chaperones • Trigger factor (Tf) • binds 50S subunit; • peptidyl-prolyl cis-trans isomerase • DnaJ/K • binds nascent polypeptides; • shield exposed hydrophobic patches • from folding unfavorably • GrpE • binds polypeptides released from • DnaK/J • releases polypeptides into folded form • or shuttles to GroEL/ES • GroEL/ES • helps fold/refold proteins already in • compact state but are not yet folded

25. Learn all you can before beginning MSA can often give you ideas for deciding on construct limits Even better if there’s some structural information!

26. If multiple sequence alignments do not help and there isn’t any structural info, try secondary structure prediction ..but try several starts and stops (primers are cheap!) http://www.compbio.dundee.ac.uk/www-jpred//

27. Do you have the gene?

29. Lots of output! Here’s what you want

33. buy

34. Shopping cart – price varies with order size

35. Before starting, confirm that you can make a significant quantity of soluble protein. Small scale solubility experiments are very important and typically will involve varying inducer concentration, expression temperature, expression construct, etc. Each protein is unique – must exploit differences Particular affinities GST, 6xHis, antibodies Solubility (NH4)2SO4, PEG precip. Charge ion exchange Hydrophobicity hydrophobic chromatography Size gel exclusion Iso-electric point iso-electric focusing Thermal stability alter temp.

36. Nickel-affinity chromatography (Histrap) Express protein in frame with an affinity tag – often tag is removable with a protease. Common tags: 6xHis, GST, CaM, MBP. Use affinity chromatography for first step! electron coordination bonds Imidazole Nitrilotriacetic acid pH  7.4 Kirkegaard & Perry Laboratories, Inc

37. If the affinity tag is removable, go back over column and collect flow-through (or digest on the column). Ion exchange chromatography (what is the theoretical pI of your protein?) DiEthylAminoEthane (DEAE), CarboxyMethyl (CM), Quaternary amine, Sulfonic acid. http://www.proteinchemist.com/tutorial/iec.html These functional groups are charged over a broad pH range. Why would that be desirable?

38. Anion exchange chromatography Anion #2 ( Cl- ) Anion #1 ( protein ) Na+ + + - - pH=6 Na+ Cl- + + - - + Na+ + Cl- + + YFP YFP - - + Na+ + + Cl- + - - + + Na+ Cl- + + Bind (Low salt) Elute (High salt)

39. Run a 20 x (column volume) linear gradient and collect fractions 500mM NaCl Linear gradient (also step) Trp, Tyr, Phe, disulfides 50mM NaCl example chromatogram Run SDS-PAGE of fractions to decide which to pool (sacrifice yield for purity?)

40. Stronger and higher resolution ion exchange media (Q, SP) may be employed to separate proteins that were not baseline separated with weak ion exchange step. Some proteins, usually larger proteins, can bind to both anion and cation exchange matrices – change pH to enhance interaction. Electrostatic potential mapped onto a molecular surface Q column SP column

41. Size exclusion chromatography Separates proteins by size. Your protein should elute at the proper volume for its expected MW. Want a nice, symmetric peak in the chromatogram. Small proteins “see” a bigger volume than do large proteins

42. Some other chromatographic techniques Salting out – Proteins precipitate differentially in the presence of (NH4)2SO4 or polyethylene glycol - It’s probably worth trying Hydrophobic – Load proteins onto phenyl sepharose in presence of ~1.5M (NH4)2SO4 and run decreasing [(NH4)2SO4] gradient. More hydrophobic elutes later. Isoelectric focusing – Electrophorese protein in matrix containing pH gradient. When the protein reaches that pH where it has no net charge it ceases to migrate. Retrieve protein from matrix.

43. Expression of TVMV protease Day 1 Transform Rosetta 2 cells with plasmid containing tvmv gene and plate overnight Day 2 (or -1) In the evening, pick a colony and grown a 10ml overnight starter culture @ 37C/200RPM. (you can save a day by inoculating from a glycerol stock.) Day 3 (or 1) a) In morning, inoculate 2L media with overnight starter and grow @37C/200PRM. b) After 2-4 hours (OD600 ~0.6-1), add IPTG to 0.5mM final concentration and induce for 4hrs @30C/200RPM. Expression optimization: at mid-log phase, lower temperature to 12-20C, add lower amount of IPTG and induce overnight, or for slow leaky expression, no IPTG for 2 days (membrane proteins) c) Harvest cells (4000 RPM/20’), resuspend in 50mL lysis buffer (+ protease inhibitors) and store in -80C. Day 4 (or 2) a) thaw cells from -80C and lyse by sonication or with Emulsiflex (cells are squeeze through a small pin-hole by high pressure). b) Start purification

44. Purification of TVMV protease • Load sample in 10mM Imi (pH8) • Wash 5CV of 10mM Imi in 1M NaCl (helps remove DNA bound to DNA-binding proteins) • Start gradient: • a) 1030mM / 5CV (initial wash) • b) 3060mM / 30CV (more stringent wash) • c) 60500mM / 1CV (start elution) • d) 500mM / 5CV (complete elution)

45. Purification of TVMV protease 1) Nickel column 2) Superdex 75 26/60 Pool for S75 Vo (~110ml) save Injection

46. Confirming expressed protein by Western Anti-His6 western

47. Western blotting • 30-45’/210V: run SDS-PAGE gel • [SDS binds protein tightly (1 SDS/2 aa) to give equivalent q/m ratio for all proteins; hence proteins are separated based on MW] • 2x 15’: rinse gel in TB • 45’/35V: electroblot (shown to the left) • 30’: wash NC in 25ml PBST + 5% w/v fat-free milk • 3x 5’: wash NC in 25ml PBST • 30’: soak NC in 20ml PBST + antibody_HRP (1:20000) • 3x 5’: wash NC in 25ml PBST each • 5’: expose NC to substrate, 0.75 + 0.75 ml • (Pierce SuperSignal West Dura) • 9) Develop/image NC • ~3.5 hours total • TB: 25mM Tris[8.3], 192mM Glycine) • PBS: 1.54mM KH2PO4, 2.71mM Na2HPO4, 167mM NaCl • PBST: PBS + 0.05% v/v Tween20)

48. 10 things you should know • How BL21(DE3) cells work (host-encoded T7RNAPol and lacI repressor) General idea about different bacterial cell lines for optimizing expression: a) different promoters b) increasing mRNA stability c) Lon/OmpT protease deficiency d) Rosetta2 cells and codon-optimization Basic idea of plasmid and antibiotic selection. Different cloning method (ligation, LIC, Gateway) How PCR works – use of processive DNAPol to save time Bacterial transformation Improving protein solubility by expression as fusion protein, and which partner works best. in-vivo cleavage of protein from fusion partner co-expression with molecular chaperones Basic idea of protein purification a) nickel-affinity (Histrap) b) Size-exclusion c) ion-exchange d) western blot e) hydrophobic