The MacroLab provides TEV protease for our in-house users. We put considerable effort into comparing various TEV expression clones and purification methods so that we can provide you with affordable, yet high quality TEV. We test each batch with our own TEV assay and we are confident it will provide you with robust and reproducible results.
We express a double mutant (L56V / S135G ) pRK793 plasmid in Rosetta2(DE3)pLysS cells. 2L of cells are purified on a HisTrap Ni column, and the product is desalted with a 26/10 column. Final purification is performed on a HiTrap Capto-S column. Typical purity is shown below:
Your TEV Protease
*Keep your TEV frozen at -80C*
Your box of TEV contains 40mg of protein. We standardize the TEV concentration to 2mg/mL and aliquot each batch into 80 tubes. Each tube contains 250uL (0.5mg). Remember, unless you requested otherwise, the TEV is His6 tagged (so you can perform a subtractive nickel column with it).
Tev is frozen in the following:
25mM HEPES pH 7.5
We recommend cleaving your protein for at least 2 hr at room temperature or overnight at 4oC with a 1:20 (weight/weight) TEV/substrate ratio, or a 1:50 molar ratio. Some substrates may require more TEV. TEV is not inhibited by PMSF (1mM) , pepstatin A (1mM) , or complete protease inhibitor cocktail (Roche).
Each batch of TEV we produce is tested and known to cleave >90% of a substrate protein at a molar ratio of 1:50 in 2 hr at room temperature.
Special Handling Instructions
*Keep your TEV frozen at -80C*
TEV is prone to aggregation, so use it as soon as possible after thawing.
This is the protein expressed by TEV-DM-Prk793
MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDI IFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIK DVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTNSSSNNNNNNNNNNLGIEGRGENLYFQ^GHHHHHHHGESLFKGPRDYNPISST ICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLVVQSLHGVFKVKNTTTLQQHLI DGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSGDG IFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQ WVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNRRRRR^GHHHHHHHGESLFKG PRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLVVQSLHGVFKVK NTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSD TSCTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFM ELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNRRRRR
While it is expressing, self cleavage removes the N-terminal MBP and you are left with:
GHHHHHHHGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNN GTLVVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLV TTNFQTKSMSSMVSDTSCTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNF TNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNRRRRR
L56V and S135G improve protein solubility and thermal stability S. van den Berg, J. Biotechnol., 121 (2006), pp. 291-298
S219V – makes protein stable against autolysis. R.B. Kapust, Protein Eng., 14 (2001), pp. 993-1000
Download the TEV Info Sheet