Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic
space of the cells. To date, there is no information regarding DegP from halophilic bacteria.
Chromohalobacter
salexigens BKL5 is a moderately halophilic bacterium that has the 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
C. salexigens BKL5 and characterize its biochemical properties.
Results: DegP-encoding gene was 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 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 residue ratio of recombinant DegP was
0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly
lower than that of DegP from nonhalophiles (average, 0.94).
Conclusions: Recombinant DegP from
C. salexigens BKL5 showed proteolytic activity when β-casein was used as a
substrate.
In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic
proteins depending on its amino acid composition.
