Cloning, Expression, and Purification of a Synthetic Human Growth Hormone in Escherichia coli Using Response Surface Methodology

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• 作者：Mozhdeh Zamani ; Aydin Berenjian ; Shiva Hemmati ; Navid Nezafat…
• 关键词：Growth hormone ; Recombinant E. coli ; Production ; Media optimization ; Response surface methodology ; T7 promoter
• 刊名：Molecular Biotechnology
• 出版年：2015
• 出版时间：March 2015
• 年：2015
• 卷：57
• 期：3
• 页码：241-250
• 全文大小：3,507 KB
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• 刊物主题：Biotechnology; Biochemistry, general; Cell Biology; Protein Science; Biological Techniques; Human Genetics;
• 出版者：Springer US
• ISSN：1559-0305

文摘

The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10?g/L), tryptone (10?g/L), and K2HPO4 (2?g/L). The optimum hGH concentration was 391?mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119?mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.