Part:BBa_K5131002
SARS-Cov-2 nsp5_T21I
Information of SARS-Cov-2 nsp5 can be seen in BBa_K5131000.Based on structure prediction, we rational design the nsp5 to increase its activity. This part is a mutant of nsp5(nsp5-T21I) and incresed the kcat/Km of nsp5 from 27,691 s⁻¹M⁻¹(WT) to 35,069 s⁻¹M⁻¹ . We found kcat/Km of this mutant is much higher than TEV protease(kcat/Km =2,600 s⁻¹M⁻¹)[1] and HRV 3C protease(kcat/Km =840 s⁻¹M⁻¹)[2] which is commonly used for removal of recombinant tags during protein purification. We hope that this part can be a low-cost and efficient tag removal tool compared to directly purchasing commercial proteases for recombinant tag removal. We utilises pGEX-6P-1 to express this part. Rational design, expression, purification and characterisation of this part can be found in BBa_K5131009
Reference:
1. Parks TD, Howard ED, Wolpert TJ, Arp DJ, Dougherty WG. Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form. Virology. 1995 Jun 20;210(1):194-201. doi: 10.1006/viro.1995.1331. PMID: 7793070.
2. Wang QM, Johnson RB. Activation of human rhinovirus-14 3C protease. Virology. 2001 Feb 1;280(1):80-6. doi: 10.1006/viro.2000.0760. PMID: 11162821. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 402
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