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☆

重组DNA 大肠杆菌 亲和层析 融合蛋白 分子生物学 生物化学 生物 标志标签 麦芽糖结合蛋白 表达式向量 分子质量 蛋白酶 基因
作者
Saurabh Agarwal,Shweta Jha,Indraneel Sanyal,D. V. Amla
出处
期刊:Journal of Biotechnology [Elsevier]
卷期号:147 (1): 64-72 被引量:14
标识
DOI:10.1016/j.jbiotec.2010.03.008
摘要

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.
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