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.
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Acknowledgements
F.J.M. Mergulhão acknowledges a PhD fellowship from the PRAXIS XXI Programme, Ministério da Ciência e Tecnologia, Portugal. Dr. Per-Ake Nygren (KTH, Stockholm) is acknowledged for his guidance in molecular cloning and Dr. Ioannis Tubulekas (Biovitrum AB) for his PCR work and cloning strategy. Eric Forsén, Kristopher Sandstrom and Olof Nord (KTH, Stockholm) are acknowledged for their help in vector construction and sequencing.
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Mergulhão, F.J.M., Monteiro, G.A., Larsson, G. et al. Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB . Appl Microbiol Biotechnol 61, 495–501 (2003). https://doi.org/10.1007/s00253-003-1232-8
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DOI: https://doi.org/10.1007/s00253-003-1232-8