title: Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives.
abstract: Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.
authors.affiliation: College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Changsheng
authors.initials: C
authors.lastname: Sun
authors.forename: Jiongqiu
authors.initials: J
authors.lastname: Liang
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.forename: Xuna
authors.initials: X
authors.lastname: Gao
authors.forename: Ruijuan
authors.initials: R
authors.lastname: Zhang
authors.forename: Fulin
authors.initials: F
authors.lastname: Hong
authors.forename: Qihang
authors.initials: Q
authors.lastname: Yuan
authors.forename: Shengbin
authors.initials: S
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D002021:Buffers; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D002021:Buffers; D011994:Recombinant Proteins; D012492:Salts; D010450:Endopeptidases; C099488:TEV protease