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Heterologous expression of a thermostable plant cysteine protease in Escherichia coli both in soluble and insoluble forms

Paper ID Volume ID Publish Year Pages File Format Full-Text
34996 45067 2010 6 PDF Available
Title
Heterologous expression of a thermostable plant cysteine protease in Escherichia coli both in soluble and insoluble forms
Abstract

Ervatamin-C is a stable papain-like cysteine protease from a tropical plant Ervatamia coronaria. Proteases in this family have numerous industrial applications. Thus protein engineering to create tailor-made variants of them for biotechnological and other applications will be highly desirable. A prerequisite for such an approach is a recombinant expression system. The cDNA encoding pro-ervatamin-C (mature protease domain together with the N-terminal prodomain) has therefore been cloned and expressed in Escherichia coli using two T7 based expression vectors pET-28a(+) and pET-39b(+). The recombinant pro-ervatamin-C was expressed as inclusion body using pET-28a(+) vector and the protease was solubilized, purified and successfully refolded to its functionally active form. To express the recombinant protease in a soluble form, a DsbA (disulphide oxidoreductase) tag was placed before pro-ervatamin-C using pET-39b(+) vector to obtain folded active ervatamin-C without going through any in vitro refolding step. A comparison of the two procedures has been presented. The recombinant enzyme shows a similar enzymatic activity, specificity and thermal stability pattern like its native counterpart.

Keywords
Cysteine protease; Ervatamin-C; Overexpression; Inclusion body; Refolding; Soluble expression
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Heterologous expression of a thermostable plant cysteine protease in Escherichia coli both in soluble and insoluble forms
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 8, August 2010, Pages 1307–1312
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us