Construction Of New Plasmids For Surface Display On Cells Of Yarrowia Lipolytica And Preliminary Study On The Applications

In this study, two surface display plasmids (pINA1317-ylcwp131 and pINA1317-ylcwp110) were constructed in Yarrowia lipolytica using C-terminals of YlCwp1 with different lengths from Y. lipolytica based on plasmid pINA1317, a pre-existing auto-cloning system for heterologous protein production in Y. lipolytica. When the gene encoding enhanced green fluorescent protein (EGFP) was cloned into the newly constructed surface display plasmids and expressed in cells of Y. lipolytica, respectively, we found that the target protein was successfully displayed on the yeast cells and 100% of the yeast cells had found anchoring target protein. The target gene cloned on the plasmids can be expressed when the cell growth is at stationary phase without adding any inducer to the medium. To our knowledge, this work constitutes the first report of surface display expression systems in Y. lipolytica.The genes encoding phytase derived from a marine yeast Kodamaea ohmeri BG3 and haemolysin from Vibrio harveyi SF-1 were expressed in the yeast. Localization of the expressed phytase and haemolysin on the cell surface were confirmed by immunofluorescence microscopy. The phytase displayed on the yeast surface exhibited activity toward sodium phytate. The yeast cells displaying haemolysin had haemolytic activity on erythrocytes from Atlantic flounder.The gene encoding mature phytase was also cloned into pINA1317 expression vector and expressed in Y. lipolytica. The recombinant phytase secreted into the medium was purified by Ni2+ affinity chromatography. The purified phytase was analyzed by SDS-PAGE and western blotting. A specific band with molecular mass of approximatly 65.1 kDa was found. The found molecular mass was less than that of native phytase and more than that of recombinant enzyme expression in E. coli BL21 (DE3). The recombinant phytase maintained the general properties of the native enzyme. A maximum activity of 114.7 mU/mg dry cell weight was obtained from yeasts harboring phytase. Optimal pH and temperature of the displayed phytase were 5.0 and 60.0℃, respectively. The phytase displayed on the cell surface was found little more sensitive to temperature as compared with the native enzyme and more stable than recombinant phytase secreted into the medium. Surface displayed phytase was stable at pH3.0～8.0, and exhibited high and similar activity at pH 4.0～6.0. The effect of cations on displayed phytase was similar to that on secreted recombinant phytase expression in Y. lipolytica and native phytase of K. ohmeri BG3.The serum immunoglobulins of Atlantic flounder (Paralichthys dentatus) were purified by means of HiTrap rProtein A Sepharose affinity chromatography. SDS-PAGE analysis of the immunoglobulins showed that the heavy chain (H chain) and the light chain (L chain) of the purified serum IgM had molecular mass of the purified serum IgM were 74.5 kDa and 26.3 kDa, respectively. A mouse polyclonal antibody against Atlantic flounder immunoglobulin was produced.The anti-serum titer evaluated by immunodotting assay was 1:3200.The recombinant haemolysin with 6×His tag expression in E. coli JM109 was purified by Ni2+ affinity chromatography. The purified haemolysin with a molecular mass of about 45.0 kDa according to SDS-PAGE had haemolytic activity on the erythrocytes collected from Atlantic flounder.The yeasts displaying haemolysin were suspended in PBS and cell density of the suspension was adjusted to 5.0×108 cells/ml. 0.2 ml of the yeast cell suspension was administered i.p. to healthy Atlantic flounder. The immunization was carried out three times. After prime, each fish was immunized at both the second and the fourth week. No dead fish was found among all the fish vaccinated with the yeast cells displaying haemolysin, although the yeast cells had high haemolytic activity. This means that the yeast cells displaying haemolysin were safe to the marine animal tested. Indirect ELISA analysis indicated that the Atlantic flounders immunized with the yeast cells having the haemolytic activity had produced specific antibody against haemolysin in the serum.This demonstrates that the yeast cells displaying haemolysin may be used as the live vaccine in marine fish. Because the surface display system has many unique characteristics, increasing the amount of the target proteins and reducing the number of native cell wall proteins, the yeasts displaying target proteins will be useful in different fields such as immobilized biocatalyst, bioconversion, bioremediation, live vaccine development and ultra-high-throughput screening for the identification of novel biocatalysts.