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Abstract
Background: DegP is serine protease that specifically cleaves and refolds unfolding proteins in peripalsmic space of the cells. So far, there is no information regarding the DegP from halophilic bacterium. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has an ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP encoding gene from Chromohalobacter salexigens BKL5 and characterize its biochemical properties.
Results: DegP-encoding gene could be overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 kDa and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21-0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic ratio of recombinant DegP was 0.56, which was slightly higher than that of the average basic/acidic ratio of extreme halophilic (0.45) but significantly lower than that of non halophilic (0.94).
Conclusions: Recombinant DegP from Chromohalobacter salexigens BKL5 showed proteolytic activity when β-casein was used as a substrate. In silico analysis indicated that recombinant DegP had similar characteristics to halophilic proteins based on its amino acid composition.
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