Gaie Fixed Expression In Saccharomyces Cerevisiae Surface And Nodc In Escherichia Coli Expression And Activity Determination

UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli K-12 catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, we cultured DH5α(pET15b/Gαl E) which was conserved in our lab, extracted the its plasmid pET15b/Gαl E. After PCR and digestion with EcoR I,BamH I , Gal E gene was cloned into commercial plasmid pYD1 and then transformed into Saccharomyces cereviae EBY100 for the expression of Gal E on cell surface, using YNB selective culture. By conformations of enzyme digestion and DNA sequencing, We cultured the S. cerevisiae containing recombinant plamid pYD1/Gαl E in YNB-CAA (0.67%YNB; 0.5%Casamino acids) with 2% glucose in 30℃, when OD₆₀₀ reached 2.0, transferred them into YNB-CAA (0.67%YNB; 0.5%Casamino acids) with 2% galctose in 20℃for induction. Different proteins need different inducing time in cell surface display, when recombinant yeast cells were induced for 24h, we got less cells; when induced for 36h, the medium was not in good condition. We induced 30h for expression on surface display. The fact that expression of Gal E gene with its secretion signal peptide at the C-terminal of a-agglutinin in Saccharomyces cerevisiae led to the display of the protein on the extracellular surface of S. cerevisiae was confirmed by immunofluorescence microscopy. After immunofluorescence analysis, we prepared to test the activity of the displayed GalE protein. Reaction system was constructed with 0.6mM UDP-galactose or UDP-glucose as substrate and EBY100 cells which net weight was 10mg. Reaction temperature was 24℃and pH was 7.0. Enzyme activity analysed with EBY100 cells showed that the enzyme displayed on yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It was about 3 minutes to reach the equilibrium with UDP-Gal as substrate, much faster than the enzyme expressed in Escherichia coli. N-acetylglucosaminyl transferase(chitin synthase; NodC) belongs to glycosyltransferase familyⅡ. All Rhizobia use N-acetylglucosaminyl transferase to synthesize nod factor which are essential for the nitrogen fixation in Legumes. N-acetylglucosaminyl transferase are necessary for the synthesis of the lipooligosaccharide backbone. Its reaction condition are very mild. In the reaction, UDP- N-acetylglucosamine (UDP- GlcNAc )as donor and N-acetylglucosamine (GlcNAc) as acceptor, throughβ-1,4 linkage, polymerization of GlcNAc occurs,generally (GlcNAc)2.5. If we express much N-acetylglucosaminyl transferase in vitro, we could produce chitooligosaccharides backbones to get a lot of nod factors which could be sprayed to the roots of Legumes to promote nitrogen fixation. In addition, when coupled with other enzyme, N-acetylglucosaminyl transferase can synthesize some important oligosaccharides. Therefore this transferase is very significant in biological nitrogen fixation and sugar industry. However, NodC is a membrane protein,it is quite difficult to purify or reconstitute its activity for investigation of the reaction mechanism and greatly limits the applications of NodC.In our study, the interest gene NodC were from Sinorhizobium.loti and had been cloned into plasmid pET28a which was located in Escherichia coli DE3 and conserved in our lab. On the basis of software analysis, we deduced the protein sequence from the gene sequence, and then utilizing the specific primer and the whole DNA sequence of NodC as template for PCR. In this PCR,we deleted the hydrophobic amino acid 1-21 in N-terminus and tranmembrane domain in C-terminus and left segment from the 22nd anmio acid to the 290th amino acid which contained the catalytic domain. In this way, the truncated N-acetylglucosaminyl transferase(NodC-tru) could catalyze the reaction and may be in soluble form, at the same time improve the expression compared to the whole NodC. NodC-tru was cloned into plamid pET22b and transformed into DH5a for expression. By the exploration of culture and induction condition, we found no matter low temperature,lone time and low IPTG concentration or high temperature, short time and high IPTG concentration, the protein NodC-tru was mainly in the existence of inclusion bodies. We used 6 mol/L guanidine-HCl for denaturalization and then restored its activity. On the condition of 20℃and pH7.0, with substrates UDP-GlcNAc,GlcNAc, the truncated enzyme had new production and showed its activity.Experimental results showed mat we had got active truncated NodC which was deleted in hydrophobic N-terminus and tranmembrane domain in C-terminus. Its expression was higher protein and the difficulty is lower in purification and activity reconstitution compared to the whole transmembrane NodC, which can benefit the industrial production.