Cloning And Expression Of A Lipase Gene From Sataphylococcus Hominis GIMT1.079

Many Staphylococci are clinical conditional pathogens of human beings and animals. To date,13different lipases from10staphylococci species have been identified, three from Staphylococcus epidermidis, two from S. aureus, one each from S. hyicus, S. haemolyticus, S. saprophyticus, S. xylosus, S. warneri, S. simulans, S. capitis and S. carnosus, respectively. The DNA and protein sequence comparison revealed high homology of these known lipases.In this dissertation, after confirming that S. hominis GIMT1.079secreted lipase (SHoL),we designed degenerate primers to amplify the conservative sequence of the S. hominis lipase gene, then nest primers near the5’and3’ends were designed based on the newly obtained conserved SHoL squence. Finally, the two flanking sequences of the conserved SHoL were obtained using the improved hiTAIL-PCR (Thermal Asymmetric Interlaced PCR) method.The two flanking sequences were combined with the conserved sequence to form the entire lipase sequence (GenBank accession no. GU207403). By sequence alignment analysis, the deduced SHoL precursor contains754amino acids with a signal peptide of35amino acids and a pro-peptide of332amino acids, which is processed to the mature lipase of387amino acids. The ATG start codon is preceded by a potential Shine-Dalgamo sequence (AGAGGTG) at position-7. The first35amino acids includes a typical signal peptide that contains the conserved residues Ser, He, Arg and Lys, which is known as the SIRK motif. The mature protein contains the conserved Ser483, Asp673and His712residues that form the catalytic triad of the lipase active site. As in other staphylococcal lipases, in the vicinity of the serine residue there are two glycines (Gly-Xl-Ser-X2-Gly). The amino acid sequence of the S. hominis mature lipase (mSHoL) exhibits high levels of homology ranging from53%to88%to those of several other staphylococcal species.The SHoL DNA sequence encoding the mature peptide was subcloned into the pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21host. The expressed protein was analyzed by SDS-PAGE and a specific band with molecular mass of about42kDa was detected. Enzymatic activity assay verified that the recombinant protein has a lipase activity. A maximum activity of0.35U/ml was obtained from cellular extract of E. coli BL21harboring the pET-28a(+)-mSHoL plasmid.