Heterologous Expression Of HuGALNT3 And Prepartation Of Anti-huGALNT3 Antibody

Glycans and glycoconjugates play an important role in the occurrence and development of diseases. Great changes can be seen in glycans between Cancer cell and normal cells. There are no templates for the glycans synthesis, but through a series of orderly arranged glycosyltransferases completed. Differences in glycosyltransferase expression will directly result in changes in the structure of glycans. Therefore, the research about the glycosyltransferase could essentially clarify the glycan changes and the regulation of glycans in the process of tumor occurrence and development. UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (ppGalNAc-T3, GALNT3) has been proposed to be one memeber of the GalNAc transferase family. This family catalyses the transference of GalNAc to Ser/Thr residues in peptide, which is the first step in the formation of O-glycan. Different members of the family have various enzyme activity and substrate selectivity. And the activity of GALNT3 is closely related with the formation of mucin-type O-glycans, whose abnormity is found universally in epithelium-derived tumors.Due to the initial study on the function of GalNAc transferases, firstly our research isolated cDNA of GALNT3 from MKN45 cell line, and then constructed expression vectors and expressed the protein in E. coli and Pichia Pastoris respectively.In E. coli the soluble region of GALNT3 without the hydrophobic transmembrane domain (GALNT3-sol) was expressed efficiently and purified as both soluble protein and inclusion body. The result of activity assay showed that GALNT3 obtained as both two forms had no activity, and the reason may lie in E. coli which lacks the protein modification mechanism of Eukaryotes (such as glycosylation, acetylation, phosphorylation, and disulfide bond formation). GALNT3 is a complex glycoprotein derived from Homo sapiens containing several disulfide bonds, so the GALNT3 expressed in E. coli cannot form natural and active conformation.Besides, Our research also used GALNT3-sol inclusion body as immunogen to produce anti-GALNT3 antibody. After protein denaturation, refolding and purification, we isolated GALNT3-sol trough SDS-PAGE and cut off the gel containing the protein to inject BALB/C mice. Then the serum after the second and forth immunization was collected respectively, and the antibody titer was tested by ELISA. The antibody titer after the second immunization was 1:3200, and the titer after the forth was 1:25600. Subsequently the antibody was tested by Western Blot, and the positive serum could combine and show the protein GALNT3-sol, while the negative serum couldn't. These results reveal that the anti-GALNT3-sol antibody has high titer and could be used in Western Blot, which may prove important in the development of commercial anti-GALNT3 monoclonal antibody used in clinical assay.To obtain active GALNT3, we tried to express it in Pichia pastoris. The gene of GALNT3-sol was cloned into the plasmid pPIC9K, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains were screened out after defect medium, yeast PCR and gradient antibiotic (G418) and so on. The expression of GALNT3-sol was confirmed through Western Blot after methanol induction. The expression of GALNT3-sol was best induced for 72 hours at 20℃in Pichia pastoris. After the concentration of culture medium, the enzyme activity was tested. The results show that GALNT3-sol expressed in Pichia pastoris has the activity of transferring GalNAc to Ser/Thr residues in peptide, which provide support for the further research on the substrate specificity (sugar nucleotide or acceptor peptide) of GALNT3.Our research expressed massive GALNT3-sol protein in microbes successfully. The efficient polyclonal antibody was prepared using GALNT3-sol expressed in E. coli as immunogen for the first time, and furthermore it is also the first active GALNT3 protein expressed in microbes which make the production of other human ppGalNAc-Ts and ever other various glycotransferases in Pichia pastoris possible. The expression of active GALNTs will provide new means for initiating O-glycosylation and new platform for attaching compounds with special biological function or activity to therapeutic proteins.