Expression of recombinant proteins in Escherichia coli under the transcriptional control of the cold-shock inducible cspA promoter

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Expression of recombinant proteins in Escherichia coli under the transcriptional control of the cold-shock inducible cspA promoter

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Title: Expression of recombinant proteins in Escherichia coli under the transcriptional control of the cold-shock inducible cspA promoter
Author: Vasina, Jess A., 1968-
Abstract: Aggregation and proteolytic degradation can significantly decrease the recovery yields of recombinant proteins overproduced in the gram-negative bacterium Escherichia coli. Expression at low growth temperatures holds great promise in alleviating these problems since proteolysis is reduced and proper folding is facilitated under these conditions. Unfortunately, the transcriptional efficiency of traditional promoters decreases with the cell growth temperature. When E. coli is transferred from a physiological temperature of 37$\sp\circ$C to the 10-15$\sp\circ$C temperature range, the production of most cellular proteins stops while a handful of cold-shock proteins, that are virtually undetectable under normal growth conditions, become synthesized at very high levels. Because the major E. coli cold shock protein, CspA, is primarily regulated at the transcriptional level, we examined the advantages and limitations of the cspA promoter in directing the expression of recombinant proteins at reduced growth temperatures. Transcriptional gene fusions between the cspA promoter and either the lacZ gene or a gene encoding the tripartite fusion protein preS2-S$\sp\prime$-$\beta$-galactosidase were constructed to characterize the strength, regulation and inducibility of the major cold shock promoter and to delineate its usefulness for the production of aggregation-prone recombinant proteins. The cspA promoter was well repressed at 37$\sp\circ$C and efficiently directed gene expression upon temperature downshift to the 30-10$\sp\circ$C temperature range for 60-90 min, although it became repressed at later time points. Using the cspA promoter at 10$\sp\circ$C, the aggregation-prone protein preS2-S$\sp\prime$-$\beta$-galactosidase could be produced in a completely soluble form at 5-fold higher levels than obtained with the chemically-inducible tac promoter at the same temperature. Fermentation, strain and genetic engineering approaches were also investigated to determine if promoter repression following prolonged incubation at low temperatures could be circumvented. While fermentation engineering provided modest improvements on the recovery yields of enzymatic activity from a plasmid-encoded cspA-lacZ gene fusion, sustained synthesis of $\beta$-galactosidase for about 7 h and 3-fold higher levels of activity were observed in a rbfA host strain. Deletions in the 5$\sp\prime$ untranslated region of the cspA RNA further showed that this region plays a fundamental role not only in the low-temperature repression but also in the high temperature repression and cold-shock induction of the promoter.
Description: Thesis (Ph. D.)--University of Washington, 1997
URI: http://hdl.handle.net/1773/9866

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