Abstract
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.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / metabolism
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Culture Media
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Escherichia coli / genetics*
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Escherichia coli / metabolism
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Gene Dosage
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Gene Expression Regulation, Bacterial
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Genetic Vectors
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Heat-Shock Proteins / genetics
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Heat-Shock Proteins / metabolism
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Proinsulin / biosynthesis*
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Proinsulin / genetics*
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Promoter Regions, Genetic
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
Substances
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ATP-Binding Cassette Transporters
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Bacterial Proteins
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Culture Media
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Escherichia coli Proteins
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Heat-Shock Proteins
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MalK protein, Bacteria
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MalK protein, E coli
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Membrane Proteins
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Recombinant Fusion Proteins
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UspB protein, E coli
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universal stress protein A, Bacteria
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Proinsulin