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Study of a bacillus subtilis promoter, vegI, for efficient secretory production of heterologous proteins

Authors Sze, Kin Fai
Issue Date 2011
Summary Extracellular expression is a viable approach to obtaining different kinds of valuable proteins, and logically, a strong promoter is preferred for protein expression. A strong promoter of Bacillus subtilis (B. subtilis), vegI (vegIP), has been employed for the expression of heterologous proteins in both Escherichia coli (E. coli) and B. subtilis in previous studies. Dramatic cell death of E. coli transformants and low levels of secretion of the heterologous products are obtained from the vegIP expression cassette. One approach to obtaining an optimized extracellular expression system is by down tuning the promoter strength in order to arrive at a balance between the optimal level of expression and the highest degree of cell viability. It has been shown that the upstream (UP) element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase α-subunit. By modifying the UP element of the vegIP, the heterologous protein secretion level could be increased to a level that is 4.5 times higher than the highest quantity of the same protein previously obtained in E. coli. The improvement is possibly due to the alteration of the bending ability of the UP element. When employing vegIP for heterologous gene expression in B. subtilis, a spontaneous point mutation of A to G at the -11 position of the -10 region of the promoter is obtained. The A to G point mutation results in a weaker promoter, designated vegGP. In this study, the results reveal that endonuclease activity and high expression level are not the main causes for the incapable transforming of vegIP. Through the use of competitive EMSA to study the binding affinity of vegGP and vegIP mutants with sigma factor A (σA), it is found that strong binding affinity to σA may account for the unsuccessful transformation of B. subtilis with plasmids harboring vegIP.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2011
Language English
Format Thesis
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