Recombinant expression of Streptomyces griseus genes and their variants in Bacillus subtilis and Escherichia coli
Year: 1994
Type: article
Abstract: The impetus for this study was a desire to produce inactive variants of Streptomyces griseus protease B (SGPB).Studies involving other catalytically altered serine proteases indicated a potential for a proteolytically inactive SGPB to be used in protein semi-synthesis and to be of value due to its specificity and its stability under various conditions.B. subtilis was initially chosen as the host for recombinant expression of this S. griseus gene due to the similarities in gene structure between SGPB and the extracellular Bacillus protease subtilisin and the ability of Bacillus to secrete large quantities of protein.The gene for SGPB had previously been found to code for a signal peptide and propeptide joined to the amino terminus of the mature protease.This suggested that SGPB (and the related protease, SGPA) is initially secreted as a precursor which is subsequently self-processed to remove the pro-region in a mechanism common to other bacterial extracellular proteases.Attempts at expressing SGPB without its pro-region in either Bacillus or E. coli or by substituting the pro-region of Bacillus cimyloliquefaciens subtilisin BPN' in Bacillus did not result in any SGPB protease activity.Properly folded active enzyme was produced only when the protease-specific pro-region was present, although the pro-region is cleaved and does not form part of the active protease.Expression of inactive variants of SGPB in either Bacillus or E. coli did not result in overproduction of the mutant protein.Although transcribed in Bacillus, the inactive SGPB precursor was not detectable and substitution of the active site serine with glycine resulted in specific targeting for degradation in E. coli due to a possible decrease in protease stability.Further study is necessary to determine the fate of the inactive precursor in Bacillus and ultimately overexpress the inactive SGPB variant. DedicationThis thesis is dedicated to my parents.
Source: Summit (Simon Fraser University)
Author: D. W. A. Carson
Language: English
Subfield: Biotechnology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2140730999