Large-scale purification of a stable form of recombinant tobacco etch virus protease.
Tobacco etch virus NIa proteinase (NIa-Pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. We have designed a mutant NIa-Pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time. Histidine-tagged forms of both wild-type and mutant NIa-Pro were overexpressed in E. coli under conditions in which greater than 95% of the protease was in the insoluble fraction after cell lysis. An inclusion body preparation followed by denaturing purification over a single affinity column and protein renaturation yields greater than 12.5 mg enzyme per liter of bacterial cell culture. NIa-Pro purified according to this protocol has been used for quantitative removal of fusion domains from a variety of proteins prepared for crystallization and biochemical analysis.
Lucast
L J
LJ
Yale University, New Haven, CT, USA.
Batey
R T
RT
Doudna
J A
JA
D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014764:Viral Proteins / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C099488:TEV protease; C059992:nuclear inclusion protein a, mosaic viruses