求助Biotechnol Appl Biochem.文献1篇。
【题名】：Production of thymosin alpha1 via non-enzymatic acetylation of the recombinant precursor.
【作者】：Esipov RS, Stepanenko VN, Beyrakhova KA, Muravjeva TI, Miroshnikov AI.
【杂志名全称】：Biotechnol Appl Biochem.
【年, 卷(期), 起止页码】：2010 Apr 21. [Epub ahead of print]
【DOI】：PMID: 20408810
【全文链接】：
http://www.ncbi.nlm.nih.gov/pubmed?term=Production%20of%20thymosin%20alpha1%20via%20non-enzymatic%20acetylation%20of%20the%20recombinant%20precursor 【摘要信息】：Human thymosin alpha1 is an effective immune system enhancer for the treatment of cancer and viral diseases. Therefore, the development of new methods of its synthesis is an urgent problem. In this work we propose an efficient scalable scheme for the production of recombinant thymosin alpha1. We used an expression system based on pET32b+ plasmid and E.coli strain ER2566 to obtain a fusion protein consisting of thymosin alpha1 and thioredoxin separated by a TEV protease cleavage site. The fusion protein was overexpressed in a soluble form and purified by ion-exchange chromatography. After proteolytic cleavage of the fusion protein with TEV protease, recombinant desacetyltimosin-alpha1 was isolated by ultrafiltration. Acetic anhydride was used for selective N-terminal acetylation of the obtained peptide (yield, 62%). The resultant thymosin alpha1 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The distinctive feature of this technology is a combination of different approaches: the biotechnological production of recombinant fusion protein, its enzymatic cleavage, and chemical acetylation of desacetylthymosin alpha1. Each stage of the process was optimized to increase the yield of the target peptide which averaged 29 mg per 1 litre of bacterial culture. The proposed method is simple, cost-effective and is suitable for large-scale production of recombinant thymosin alpha1
[ 此帖被zhenglei8501在2010-05-03 19:39重新编辑 ]
