利用通用应激启动子uspA和uspB研究培养基及拷贝数对大肠杆菌中人胰岛素原分泌的影响。

Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB.

作者信息

Mergulhão F J M, Monteiro G A, Larsson G, Sandén A M, Farewell A, Nystrom T, Cabral J M S, Taipa M A

机构信息

Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.

出版信息

Appl Microbiol Biotechnol. 2003 Jun;61(5-6):495-501. doi: 10.1007/s00253-003-1232-8. Epub 2003 Feb 20.

DOI:10.1007/s00253-003-1232-8

PMID:12764564

Abstract

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

摘要

描述了在大肠杆菌中使用uspA和uspB启动子（通用应激启动子）进行异源蛋白生产的情况。使用携带uspA启动子的中等拷贝数载体（15 - 60个拷贝）可获得最佳结果，分泌到周质中的ZZ - 胰岛素原达到4.6 mg/g干细胞重量（DCW），分泌到培养基中的为1.9 mg/g DCW。这些值比之前使用相同的ZZ融合标签和SpA前导肽所报道的值高约1.7倍，表明这些应激启动子对于大肠杆菌中重组蛋白的分泌具有潜在价值。进一步证明，与富含LB的培养基相比，使用M9基本培养基有利于蛋白分泌。