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RISS 인기검색어
- 검색키워드
- 저자 : Cuihong Du (검색결과 2 건)
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Expression and Purification of Soluble Recombinant Human Endostatin in Escherichia coli
Cuihong Du,Xiaoping Yi,Yuanxing Zhang 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.2
Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.
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Synthesis of S-adenosyl-L-methionine in Escherichia coli
Xiao-Nan Wei,Minjie Cao,Jian Li,Huan Li,Yi Song,Cuihong Du 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.6
S-adenosyl-L-methionine (SAM) is an importantphysiological metabolite in vivo and may be useful inmedicines. SAM is produced from L-methionine and ATPcatalyzed by S-adenosyl-L-methionine synthetase (SAMS)in vivo. In this study, the gene encoding SAMS was clonedand a genetically engineered Escherichia coli (E. coli)BL21(pET-28a-SAMS) was constructed. The recombinantSAMS with a molecular mass of approximately 46 kDawas expressed by inducing the engineered E. coli usingisopropyl-β-D-1-thiogalactopyranoside (IPTG) as an inducer. To produce SAM using a low-cost, nontoxic and highperformanceexpression system, lactose was used as asubstitute for IPTG to induce BL21(pET-28a-SAMS). Byoptimizing the expression conditions, the concentration ofSAM produced by the engineered E. coli was 48 mg/L in theculture medium supernatant. To increase the concentrationof SAM produced, a coupled system was constructedconsisting of E. coli BL21(pET-28a-SAMS) and Saccharomycescerevisiae (S. cerevisiae) JM-310. In this coupled system,ATP generated from S. cerevisiae was provided to E. colifor producing a higher concentration of SAM. The SAMconcentration in the coupled system reached 1.7 g/L. SAMwas purified by a weak acid cationic exchange resin D113,and a simple and economical purification procedure forSAM isolation was achieved. SAM was confirmed byHigh Performance Liquid Chromatography-tandem MassSpectrometry analysis. Our study provides a feasible andconvenient approach to produce SAM.
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