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Biotechnology and Applied Biochemistry (2003) 38, (87–93) (Printed in Great Britain)

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Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli

Filipe J. M. Mergulhão*, Gabriel A. Monteiro*, Gen Larsson†, Maria Bostrom†, Anne Farewell‡, Thomas Nyström‡, Joaquim M. S. Cabral* and M. Ângela Taipa*¹

*Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal, †The Swedish Centre for Bioprocess Technology, Stockholm Centre for Physics, Astronomy and Biotechnology, 106 91 Stockholm, Sweden, and ‡Department of General and Marine Microbiology, University of Gothenburg, 413 90 Gothenburg, Sweden

Key words: Escherichia coli, induction, malK, recombinant system, stress.

Abbreviations used: DCW, dry cell weight; IPTG, isopropyl b-D-thiogalactoside; ppGpp, guanosine 5´-diphosphate-3´-diphosphate; SpA, staphylococcal Protein A.

¹To whom correspondence should be addressed (e-mail pcataipa@popsrv.ist.utl.pt).

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5´-untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.

Received 6 March 2003/11 April 2003; accepted 9 May 2003

Published as Immediate Publication 9 May 2003, DOI 10.1042/BA20030043

© 2003 Portland Press Ltd