Expression And Activity Analysis Of Human Recombinant LYG2 And LYC5 Lysozyme In Pichia Pastoris

lysozyme is a natural basic protein. It specifically acts on the microbial cell wall through cutting off the ?-1,4-glycosidic bond between N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG) in peptidoglycan, and damages the peptidoglycan bracket. And this leads to cell lyse under the action of internal osmotic pressure. In recent years, more and more bacteria acquire resistance to antibiotics, due to overuse of antibiotics. And clinical treatment of infectious diseases becomes more difficult. Therefore, looking for an efficient and safe drug without resistance among bacteria becomes an important work. Lysozyme is an antibacterial protein which can naturally kill most Gram-positive bacteria and some Gram-negative bacteria. In the earlier studies by our laboratory, we have found and cloned a series of lysozyme proteins. Here, we expressed the LYG2 and LYC5 lysozyme proteins and analyzed their activities.Human g-type lysozyme includes LYG1 and LYG2, with LYG2 sequence being firstly discovered by our laboratory. And we had been granted with both domestic and foreign patents for it. Human C-type lysozyme gene family contains LYC1, LYC2, LYC3, LYC4, LYC5, and LYC6 (hLYZ). Though LYC1, LYC2, LYC3, LYC4 and LYC6 had been successfully expressed in Pichia yeast or E. coli expression system, LYC5 has not been expressed yet. Here, we designed mature protein coding sequences of LYG2 and LYC5 without the signal peptide sequences based on the preference of Pichia yeast codon, and added the Xho I and Not I restriction sites into its two ends. We then cloned the encoding gene into the MCS of expression plasmid pPIC9K in the correct reading frame, and the resulting recombinant plasmids were named as pPIC9K-LYG2 and pPIC9K-LYC5. The two plasmids were sequenced for confirmation. Then they were linearized by the restriction enzyme Sal I, and electro-transformed into the Pichia pastorios GS115 express bacterium. We have gained 28 Pichia pastoris recombinant strains with high copy LYG2 lysozyme gene and 6 Pichia pastoris recombinant strains with LYC5 lysozyme gene. The recombinant strains were screened through culturing and induction under methanol in baffled flask. The results of SDS-PAGE indicated that the LYG2 and LYC5 target proteins were secreted into the supernatant, about 20kD and 17kD in sizes. The results from mass spectral analysis confirmed that these proteins were LYG2 and LYC5 lysozymes, with expression levels being about 67mg/L and lOmg/L, respectively. The antibacterial activity was detected with the fermentation supernatant of the LYG2 Pichia pastoris recombinant strains and the 6 LYC5 Pichia pastoris recombinant strains. And the activities are about 301U/ml and 434U/ml, while the specific activities are 3,836U/mg and 25,700U/mg.In this study, we have successfully expressed LYG2 lysozyme protein and LYC5 lysozyme protein in Pichia pastoris, and their antibacterial activities were detected in the fermentation supernatant. These results will contribute to the mass production of LYG2 and LYC5 lysozymes.