Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

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 0.21­0.29 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 ß-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.

Detection of Rickettsia bellii and Rickettsia amblyommii in Amblyomma longirostre (Acari: Ixodidae) from Bahia state, Northeast Brazil

Studies investigating rickettsial infections in ticks parasitizing wild animals in the Northeast region of Brazil have been confined to the detection of Rickettsia amblyommii in immature stages of Amblyomma longirostre collected from birds in the state of Bahia, and in immatures and females of Amblyomma auriculariumcollected from the striped hog-nosed skunk (Conepatus semistriatus) and armadillos (Euphractus sexcinctus) in the state of Pernambuco. The current study extends the distribution of R. amblyommii (strain Aranha), which was detected in A. longirostre collected from the thin-spined porcupine Chaetomys subspinosus and the hairy dwarf porcupine Coendou insidiosus. In addition, we report the first detection of Rickettsia bellii in adults of A. longirostre collected from C. insidiosus in the state of Bahia.

Periplasmic expression of TNF related apoptosis inducing ligand [TRAIL] in E.coli

Tumor necrosis factor-related apoptosis-inducing ligand [TRAIL], a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 [DE3] and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space

Escherichia coli disulfide-forming related proteins: structures, functions and their application in gene engineering for expressing heterologous proteins in Escherichia coli / 生物工程学报

The formation of disulfide bonds in secreted proteins of E. coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG. DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it. Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates. DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo. It is regarded that DsbE has the similar function with DsbD. All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds. Furthermore, their chaperone activity can promote the formation of protein disulfide bonds. There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E. coli. So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG. Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E. coli, and DsbG has additive effects with DsbC.