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Cloning and expression of controllable cell membrane
poreforming protein gene
SHI Weiqing, CHEN Qin, SUN Huaichang
(College of Veterinary Medicine, Yangzhou University, Yangzhou 225009,China) |
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Abstract: Alphahemolysin (αHL) gene 0.9kb in length was amplified by high fidelity PCR from Staphylococcus aureus strain 1800 genomic DNA. Sequence analysis showed that the αHL gene differed by
6bp from that of the previouslypublished αHL gene of strain Wood 46, which caused only one amino acid substitution (275 Thr→Ile). Through twofragment PCR cloning and sitespecific mutation, five amino acids at position 130-134 of the αHL gene were substituted with five consecutive histidines andouter membrane protein A (omp A) signal peptide sequence was fused with the mutated αHL gene for secretive expression in Escherichia coli . After induction with IPTG, an additional protein band of expected size was revealed by SDSPAGE. The expression product was purified from the supernatant of cell lysate using
Ni2+chelated HiTrap column and dialyzed against an acidic buffer. Hemolytic activity assay showed that the purified protein (called H5) remained the hemolytic ability of the wildtype αHL to rabbit red blood cells (rRBCs), which was inhibited in the presence of
Zn2+. After treatment with H5, mouse fibroblast cell PA317 was stainable by trypan blue dye and permeable to trehalose, which was inhibited in the presence of
Zn2+. The results indicated that the expressed H5 protein is the controllable poreforming protein and thus usable for intracellular vitrification preservation studies of mammalian cells.
Key words: Staphylococcus aureus; alphahemolysin gene; mutagensis; procaryotic expression; controllable poreforming protein |
