Evaluation of bottlenecks in proinsulin secretion by Escherichia coli

• Type: Article in International Scientific Journal
• Date: 2004

Authors

Mergulhao, FJM (Author) Other; Taipa, MA (Author) Other; Cabral, JMS (Author) Other; Monteiro, GA (Author) Other

Journal

Title: Journal of Biotechnology

Vol. 109

Pages: 31-43

ISSN: 0168-1656

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 23 Citations

ISI Web of Science

Scopus - 27 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

CORDIS: Technological sciences > Technology > Biotecnology

Other information

Authenticus ID: P-000-AV1

DOI: 10.1016/j.jbiotec.2003.10.024

Abstract (EN): This work evaluates three potential bottlenecks in recombinant human proinsulin secretion by Escherichia coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. A maximum secretion level of 7.2 mg g(-1) dry cell weight was obtained in the periplasm of E. coli JM109(DE3) host cells. This value probably represents an upper limit in the transport capacity of E. coli cells secreting ZZ-proinsulin and similar proteins with the protein A signal peptide. A selective deletion study was performed in the fusion partner and no effect of the molecular size (17-24 kDa) was detected on secretion efficiency. The protective effect against proteolysis provided by the ZZ domain was thoroughly demonstrated in the periplasm of E. coli and it was also shown that a single Z domain is able to provide the same protection level without compromising the downstream processing. The use of this shorter fusion partner enables a 1.6-fold increase in the recovery of the target protein after cleavage of the affinity handle.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 13