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Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes.

Abstract：

:We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

journal_name

FEMS Microbiol Lett

journal_title

FEMS microbiology letters

authors

Thompson VC,McGuire BE,Frier MS,Legg MSG,Dyer TW,Gudavicius G,Potter S,Nano FE

doi

10.1093/femsle/fnaa162

subject

Has Abstract

pub_date

2020-10-16 00:00:00

issue

19

eissn

0378-1097

issn

1574-6968

pii

5917979

journal_volume

367

pub_type

杂志文章

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