Synthesis of mini-proinsulin precursors using N-termini of human TNF α as fusion partners in recombinant Escherichia coli

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Title
Synthesis of mini-proinsulin precursors using N-termini of human TNF α as fusion partners in recombinant Escherichia coli
Author(s)
C S Shin; M S Hong; D Y Kim; Jee Won Lee; Young Hoon Park
Bibliographic Citation
Journal of Industrial Microbiology & Biotechnology, vol. 22, pp. 176-180
Publication Year
1999
Abstract
Synthesis of human mini-proinsulin precursors was investigated in controlled fed-batch cultures at high cell concentrations of recombinant Escherichia coli. Transcription of the recombinant gene was controlled by a T7 promoter system. The human mini-proinsulin was prepared by substituting a C-chain peptide of natural proinsulin with a peptide sequence of only nine amino acids. The reduced size of fusion proinsulin and hence the increased purity of human insulin in the recombinant product may contribute to increasing the fermentation yield of human insulin. Three precursors (T1-, T2-, and T3-M2PI) were constructed by utilizing the N-terminus residues of human tumor necrosis factor α as fusion partners. The T2 precursor was most soluble in the cytoplasm, and exerted the most inhibitory effect on recombinant cell growth. In the production of T2-M2PI, significant amounts of undesirable metabolic by-products (acetate and ammonia) accumulated in the culture broth even at very low specific cell growth rate. The major portion of all synthesized precursors aggregated to insoluble inclusion bodies but the protein aggregates were easily converted to monomers in the presence of the anionic detergent (SDS) without using any reducing agent. With the expression of T1-M2PI, growth inhibition was minimal, and the maximum volumetric yield of mini-proinsulin (M2PI in fermentation cultures was at the highest level among the synthesized precursors.
Keyword
Escherichia colimini-proinsulinN-terminus residueshuman tumor necrosis factor αfusion partners
ISSN
0169-4146
Publisher
Springer
DOI
http://dx.doi.org/10.1038/sj.jim.2900619
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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