Heterologous Expression Of Chimeric Lysin And Comparison Of Enzymatic Properties

In recent years,phage lysin has been widely used in biological control in place of antibiotics,It plays a major role in the prevention and treatment of infectious diseases in humans,animals and other farmed animals.Lysins are usually expressed in prokaryotic E.coli systems,but have disadvantages such as low yield,instability,poor solubility,and high cytotoxicity.Therefore,this study aims to construct a highly efficient and safe yeast expression system to produce a broad-spectrum chimeric lysin ClyV,which will further lay the foundation for clinical application.In this study,the amplified ClyV gene was digested with yeast expression vectors pPIC3.5k and pPIC9 k to obtain pPIC3.5k-ClyV and pPIC9k-ClyV,which were electroporated into Pichia pastoris.A yeast-positive multi-copy strain was obtained by screening the geneticin plates.The pPIC3.5k-ClyV and pPIC9k-ClyV engineering bacteria obtained by the above fermentation were induced by methanol,and then the lysin ClyVp1 and ClyVp2 were purified by HisTrap FF columns.At the same time,the E.coli BL21/pET 28a-ClyV engineering bacteria deposited in our laboratory were also induced by prokaryotic expression,and the lysin ClyVe was obtained by HisTrap FF columns.ClyV was correctly expressed in both E.coli and Pichia pastoris.The yeast system is relatively better in terms of ClyV expression and solubility.To further compare the enzymatic differences between Pichia yeast-derived ClyVp and E.coli-derived ClyVe,we compared bacteria turbidity decrease assay of prokaryotic E.coli and eukaryotic yeast system by using Synergy H1 microplate reader,in different NaCl,EDTA,and pH buffers.The stability under EDTA and NaCl conditions showed that ClyV derived from E.coli and P.pastoris had higher lytic activity against S.agalactiae S12,and 100 ?g/ml could reduce S12 by about 3 orders of magnitude in 60 min.The activity of yeast ClyVp was slightly higher than that of Escherichia coli ClyVe at 45 °C;the optimum pH of ClyVp and ClyVe were 6.0 and 9.0,respectively;ClyVp was more stable than the lytic activity of ClyVe,even about 40% even at 1000 mM NaCl concentration.The activity retained approximately 60% activity at a concentration of 800 ?M EDTA.The cytotoxicity of ClyV on CHO-K1 in hamster ovary cells was tested by MTT assay.The results showed that ClyVe was incubated with CHO-K1 cells for 24 h after 500 ?g/ml,and some cytotoxicity could be detected,while yeast ClyVp's toxicity is still low at 2000 ?g/ml.On the whole,yeast ClyVp has more advantages than E.coli ClyVe,and has better application potential in industrial production and biomedical.