Molecular cloning and expression of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from human fetal liver

Based on the sequences of the highly conserved segments in the previously c loned sialyltransferases, a cDNA encoding Gal beta 1,3GalNAc alpha 2,3-sial yltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surroundin g tissue of hepatoma. The cDNA encoding the putative active domain was expr essed in COS-1, Escherichia coli, and Pichia pastoris. The recombinant prot ein expressed in COS-1 could catalyse the transfer of NeuAc from CMP-NeuAc to asialo-fetuin. No enzyme activity was detected with a 32-kDa protein in E. coli and both 32-kDa and 41-kDa proteins in P. pastoris. These results s uggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.