A combined approach to improving large-scale production of tobacco etch virus protease

Abstract

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in E. coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible.Here we use a quantitative, high throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove 4 of the 5 arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 °C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of ~400 mg/ L of expression culture (~15 mg pure TEV protease per g of E. coli cell paste). Methods for producing glutathione S-transferase tagged TEV with similar yields (~12 mg pure protease fusion per g of E. coli cell paste) are also reported.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

“…The eluted fractions that were found to contain homogeneous DFTR via SDS-PAGE were pooled and concentrated using an Amicon ultracentrifugal filter, 3-kDa molecular mass cutoff (EMD Millipore, Bedford, MA). The NH 2 -terminal His 6 tag of the purified protein was cleaved by incubation at 4°C with recombinant His 6 -TEV (recombinant TEV-protease carrying a His 6 tag) (78), at an Mj-DFTR to protease ratio of 20:1 under overnight dialysis against a solution containing Tris-Cl buffer, pH 8.0, NaCl, and DTT at final concentrations of 50, 200, and 5 mM, respectively. DTT was then removed by three more rounds of dialysis against the same solution but without DTT and containing a higher final concentration of NaCl (400 mM).…”

Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase

“…pNIC28-BSA4 was a generous gift from Professor Opher Gileadi, University of Oxford, and pMHT238⌬ was from Professor Brian G. Fox, University of Wisconsin. The His-tagged and C-terminally truncated TEV protease encoded in pMHT238⌬ was expressed and purified essentially as described before (31). Primers were from Thermo Fischer Scientific; Phusion High-Fidelity PCR master mix was from Finnzymes, and Exonuclease I was from Fermentas.…”

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

“…The column was washed with the lysis buffer, and the protein was subsequently eluted using a gradient of imidazole in the buffer. Appropriate protein fractions were pooled and dialyzed at 4°C in the presence of rTEV protease (25) to remove the N-terminal His 6 tag. The His 6 -tagged rTEV protease, encoded in a pProEX HTa expression vector, was produced earlier from E. coli Rosetta TM (DE3)pLysS cells (Novagen), as described previously (25).…”

“…Appropriate protein fractions were pooled and dialyzed at 4°C in the presence of rTEV protease (25) to remove the N-terminal His 6 tag. The His 6 -tagged rTEV protease, encoded in a pProEX HTa expression vector, was produced earlier from E. coli Rosetta TM (DE3)pLysS cells (Novagen), as described previously (25). After overnight incubation of the purified FbiB proteins with rTEV protease, a subtractive immobilized metal affinity chromatography step was performed to remove the cleaved protein from uncut protein and rTEV protease.…”