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β-1,4-Galactosyltransferase-1 , β-l,4-GT I ,catalyzes the transfer of galactose (Gal) from UDP-Gal to the N-acetylglucosamine (GlcNAc) residue present at the nonreducing terminal end of glycans of glycoproteins and glycolipids, producing β-1,4 linked galactosylated glycan. In addition to GlcNAc as an acceptor, the enzyme can also use other sugars, such as N-acyl substituted glucosamines and N-acetyl-D-mannos-amine, as acceptors.Pichia pastoris hererologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins. It will be employed more widely for its many advantages, including fast culture, cheap medium, easy operating, active products by secreted expression, protein processing, protein folding, post translational modification,easy industrial production and so on.In this paper, the eukaryotic expression of the β-1,4-GT gene by Pichia pastoris hererologous gene expression system was studied. The β-1,4-GT gene was got from the plasmid pGEX-4T-2 through DT-PCR techniques. β-1,4-GT gene was inserted into a Pichia pastoris vector pPIC9K(with a-factor signal) constructing a recombinant expression plasmid pPIC9K-GT by getting a tandem of multiple copies. The recombinant plasmid was transformed into Pichia pastoris cell line GS115 by electroporation. The transformants were screened, cultured and induced by the addition of 0.5%(v/v) methanol. The SDS-PAGE analysis showed that the weight of the fusion protein was correct. The expression of β-1,4-GT reached up to 35% of the total proteins in medium supematant as shown by SDS-PAGE. β-1,4-GT was purified by gel filtration using Sephadex G-75 column. We applied the pH-indicator-based assay to detect activity. The active unit is 98.6, specific activity is 21.43U/mg.β-l,4-GT with biological activity was expressed, purified and indentified for the futher study or pharmacology application.
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