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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• 作者：Saurabh Agarwal ; Shweta Jha ; Indraneel Sanyal ; D.V. Amla
• 关键词：Recombinant α ; ₁-proteinase inhibitor ; MBP fusion protein ; Modified synthetic gene ; E. coli expression ; Thermostability ; Site-specific mutation
• 刊名：Journal of Biotechnology
• 出版年：2010
• 出版时间：3 May 2010
• 年：2010
• 卷：147
• 期：1
• 页码：64-72
• 全文大小：1007 K

文摘

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.