Cloning, Mutagenesis And Expression Of Staphylococcus Aureus Alpha-Hemolysin Gene And Its Application For Cell Vitrification Preservation

With the development of vitrification preservation technology, it is possible to preserve sera, enzymes and other biologies at room temperature for relatively long time. However, this technology is presently limited to cellular biomaterials and thus it is of importance to develop a new intracellular vitrification preservative method. To this end, alpha-hemolysin gene of 0.9kb was amplified by high fidelity PCR from Staphylococcus aureus (strain 1800) genomic DNA and showed to be over 99% identical to that of the previously published alpha-hemolysin gene by DNA sequencing. The five amino acid codons at position 130-134 were replaced by five consecutive histidine codons and the resulted mutant was called H5. To obtain a secretive expression of the H5 in periplastic space of Escherichia coli (E.coli) cells, outer membrane protein A signal peptide sequence was fused with H5 and subcloned into prokaryotic expression vector pET-30a. The recombinant plasmid was named pET-H5 and transformed to BL21 (DE3) pLysS E.coli cells. After induction with IPTG a protein of expected size of 35kDa was revealed by SDS-PAGE analysis. The expression product was purified by using the Ni2+-chelated HiTrap column from supernatant of cell lysate and dialyzed against a buffer (pH 5.2) containing l0mM NaAC and 20mM NaCl. The purification of the protein was confirmed to be a single band by SDS-PAGE. Hemolytic activity assay showed that H5 remained the original hemolytic ability for rabbit red blood cells (rRBCs), with an HC50 of 180ng/ml at 37℃. Pore-forming ability of the H5 was controllable in the absence and presence of Zn2+. The abilities of the poration and intracellular trehalose loading were demonstrated by incubating mouse fibroblast cell PA317 in the solution containing 25 μ g/ml H5 and 0.2% of trypan blue dye or 0.5M trehalose for 1 h and observed under microscope. These data indicate that the H5 protein could be used to facilitate loading of trehalose into eukaryotic cells for intracellular vitrification preservation.