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Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity ☆

Paper ID Volume ID Publish Year Pages File Format Full-Text
24402 43512 2010 9 PDF Available
Title
Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity ☆
Abstract

Human α1-proteinase inhibitor (α1-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant α1-PI has great potential in therapeutic applications. We report here the expression of a synthetic α1-PI gene and its variants in Escherichia coli. Modified α1-PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant α1-PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant α1-PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7–9 mg l−1 of bacterial culture (∼5.2 g wet cell mass). E. coli expressed recombinant α1-PI showed specific anti-elastase activity and appeared as a single band of ∼45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant α1-PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified α1-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active α1-PI and its variants in E. coli for further therapeutic applications.

Keywords
Recombinant α1-proteinase inhibitor; MBP fusion protein; Modified synthetic gene; E. coli expression; Thermostability; Site-specific mutation
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Expression and purification of recombinant human α1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity ☆
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 147, Issue 1, 3 May 2010, Pages 64–72
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