PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach

Page Page 1 of 7

Title: Protocol #25 TEV protease expression & purification protocol

Protocol #25 TEV protease expression & purification protocol

Aim Tobacco Etch Virus NIa protease (TEV protease) is a valuable protease in the service of molecular protein engineering as various protein expressing plasmids contain the specific TEV site through which it is possible to remove a fusion tag. TEV protease recognizes the amino acid sequence [E-X-X-Y-X-Q-(G/S)] but it is best recognizes the sequence ENLYFQS. The aim of this protocol is to supply the researcher with the necessary steps required to express and purify the 27kDa catalytic domain of TEV at high amounts and level of purity (>20mg at >85% purity). This protocol utilizes the Auto-induction media for higher yield of bacteria expressing cells but the protein can be expressed also in the classical method of IPTG induction (1mM final concentration). Note that TEV protease was shown to be unstable in the presence of high concentration of Imidazole, thus it is important to remove it as soon as possible (Fang et al, 2006). This protocol requires a net work time of approximately 2 days and is based both on the references detailed below and on the experimental experience detailed in Chen Guttmans lab notebook dated 19-01-11. Buffers and media are detailed at the end of the protocol in appendix A. Materials & Equipment Refrigerated large volume capacity shaker. Sorval refrigerated centrifuge. 4x250ml buckets. Refrigerated ultracentrifuge. 2-4 Ultracentrifuge 40ml tubes Thermo French Press. TEV catalytic domain cloned into pET expression system with an hexahistidine BL21 (DE3) competent cells (heat shock or electroporation compatible 2x 5L flasks containing each1.8L sterile TY media (see appendix A). 200ml of sterile 20xNPS (see appendix A).

tag fused to its N-terminal portion. preparation)

PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach Page Page 2 of 7

Title: Protocol #25 TEV protease expression & purification protocol

80ml of sterile 50x5052 (see appendix A). 4ml of sterile 0.5M MgSO4 (see appendix A). 4ml of 50mg/ml Ampicilin antibiotic. 100ml Binding buffer1. 50ml Wash buffer2. 25ml Elution buffer. 4L Dialysis buffer. Ultrafiltration apparatus (MWCO >10,000). Protease inhibitor (for example, Sigma Cat# P8849)

Experiment procedure 1. plates. 2. flask). 3. 4. 5. 6. 7. mass3. 8. Protocol can be stopped at this point by placing pellets at -80C Thoroughly resuspend bacterial pellet in approximately Binding buffer
1 2

Transform BL21 competent cells with TEV plasmid and plate on Ampicilin agar Next day, pick single colony and inoculate overnight in a 25ml of sterile LB

within 250ml supplemented with 25l of Ampicilin 50mg/ml (starter per 2L of growing Next day, prepare 2x2L of AI media within 2x5L flasks and supplement it with Rotate flasks at 180-210 rpm for 3hr at 37C. Lower temperature to 20C and continue shaking overnight. Next day collect cells by centrifugation at 6000rpm for 7. Using spatula, transfer pellets to a 50ml falcon tube and weigh the bacterial

2ml of 50mg/ml ampicilin and add 25ml for each 2L of media.

(50ml/20gr pellet) till no large clumps are visible. Make sure you add: DNAse to remove DNA fragments from the sample (200l of 10mg/ml DNAse I for every 50ml of binding buffer)
Depends on pellet weight; prepare 50ml buffer/20gr pellet Depends on dripping column used. 3 Bacterial mass can give a good indication in regard to bacterial lysis (<10gr) or formation of inclusion bodies (>30gr)

PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach Page Page 3 of 7

Title: Protocol #25 TEV protease expression & purification protocol

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
4 5

Protease inhibitors according to manufacturer recommendations

Apply French press lysis X2 (see protocol #5). Transfer lysed cultures into two 37ml ultracentrifuge tubes balance tubes Centrifuge at 45Krpm, 40' at 4C (Note: while centrifugation, clean-dry French Prepare 10-20 numbered eppendorf tubes. Load dripping column (Biorad) with the supernatant of each tube (only one tube Collect flowthrough ("FT") and keep on ice. Repeat steps 15 and 16 with the 2nd tube. Wash X1 with wash buffer I and collect flowthrough ("Wash I" or "WI"). Wash X2 with wash buffer II and collect flowthrough ("Wash II" or "WII"). Wash X1 with wash buffer III and collect flowthrough ("Wash III" or "WIII"). Close Valve and then load 10ml of elution buffer onto the column; incubate for 3'. Collect elution at 1ml and close valve; incubate another 3' at RT and elute Repeat step 20 for the 3rd elution. Elute rest of suspension at 1ml volume. Measure fractions O.D at 280nm (1mg/ml of pure TEV absorbs 1.19). a. Remove 10l of each sample for 15% SDS-PAGE analysis. Dilute all fractions 1:2 (v/v) with storage buffer (w/o glycerol!). Pool selected fractions and concentrate via ultrafiltration apparatus (10,000 Dilute concentrated sample 1:1 (v/v) with the addition of 100% Glycerol. Aliquot 200l into eppendorf tubes and flash freeze in liquid nitrogen. Store at -80C.

precisely (50mg) with binding buffer till the sample reach the tubes neck line. press components)4,5.

volume per column volume!6) Tightly seal column and mix by inverting at RT for 5'.

additional 1ml.

MWCO7) to ~5mg/ml while exchanging the buffer solution8.

If there is a suspicion of inclusion bodies formation, centrifuge @ 8-10Krpm via Sorval for 1hr. Clean tubes with MQ and brush NOT with soap! 6 Narrow column; fat dripping column can accommodate supernatant of two ultracentrifuge tubes 7 Usage of 3,000 MWCO filter is recommended for sample yield but will require much more time to exchange the buffer. 8 Additional option is to use disposable destalting columns (GE healthcare)

PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach Page Page 4 of 7

Title: Protocol #25 TEV protease expression & purification protocol

PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach Page Page 5 of 7

Title: Protocol #25 TEV protease expression & purification protocol

Appendix A Buffer recipes Use only MiliQ 18.5M water! Autoinduction (AI) media (see Studier, FW) Solution (For 2L AI media) add ingredients in order of appearance TY media 1M MgSO4-7H2O 50x5052 20xNPS Antibiotics (depend on concentration) Overnight BL21 starter Volume (ml) 1800 2 40 100 25

TY (1.84L)

20 g bacto-tryptone 10 g yeast extract Glycerol 250gr Glucose 25gr 100gr -Lactose Add in sequence in beaker, stir until dissolve. Filter (do NOT autoclave) 0.5M (NH4)2SO4 1M KH2PO4 1M Na2HPO4 Autoclave 66gr 136gr 142gr

Fill to 1.84L Fill to 1L

50x5052

20xNPS

Fill to 1L

Base stock salts and buffers Buffer/salt 5M NaCl Molecular weight (gr/ml) 58.44 Amount (gr) 292.2 MQ (L) 1

PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach Page Page 6 of 7

Title: Protocol #25 TEV protease expression & purification protocol

1M Tris-HCl pH=8 121.13 3M Imidazole 68.08 6M Guanidine 95.5 0.5M EDTA 292.24 14.3M Stock -Mercaptoethanol (ME) All stock buffers should be filtered! Buffers (prepare fresh)9 Buffer Binding buffer (100ml) Ingredients 100mM Tris pH=8 300mM NaCl 20mM Imidazole 0.02% Triton X-100 5mM ME 20mM Tris pH=8 300mM NaCl 20mM Imidazole 5mM ME Wash buffer II (50ml) 20mM Tris pH=8 600mM NaCl 30mM Imidazole 5mM ME Ingredients 20mM Tris pH=8 300mM NaCl 40mM Imidazole 5mM ME Elution buffer (25ml) 50mM Tris pH=8 150mM NaCl 300mM Imidazole 5mM ME 50mM Tris pH=8

60.6 204.2 286.5 73.06

0.5 1 0.5 0.5L

Wash buffer I (25ml)

Volume10 10ml 6ml 666l 20l 35l 0.5ml/1ml11 1.5ml/3ml 166.5l/333 l 8.2l/17.5l 1ml/2ml 6ml/12ml 0.5ml/1ml 17.5l/35l Volume 0.5ml/1ml 1.5ml/3ml 333l/666 l 8.2l/17.5 l 1.2ml 750l 2.5ml 8.2l 0.5ml

MQ Fill to 100ml

Fill to 25ml/50ml

Fill to 50ml/100ml

Buffer Wash buffer III (25ml)

MQ Fill to 25ml/50ml

Fill to 25ml Fill to

Stripping buffer (25ml)

9

These are suggested buffers that can serve as a starting point; refinement or selection of specific buffers should be conducted to each protein according to it's physio-chemical properties. 10 Base stock buffer/salts 11 Bold indicates 60ml fat dripping column; Normal font indicate 30ml narrow dripping column

PROTOCOL Date created: 20-01-11 Last update: 12092011 17:51 Written by: Chen Guttman Laboratory: Raz Zarivach Page Page 7 of 7

Title: Protocol #25 TEV protease expression & purification protocol

500mM NaCl 2.5ml 25ml 50mM EDTA 2.5ml Storage buffer (25ml) 50mM Tris pH=8 1.2ml Fill to 150mM NaCl 750l 25ml 5mM ME 8.2l 50% Glycerol* * Add 1:1 (v/v) of 100% Glycerol to the sample before freezing the sample! References 1. 2. 3. 4. Waugh DS. TEV protease FAQ. Protein Engineering Section, MCL, NCI-Frederick, USA. Fang L. et al. An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization, 2006. Berg, S et al. Improved solubility of TEV protease by directed evolution, 2006. Studier, FW. Protein production by auto-induction in high-density shaking cultures, 2005.