Open AccessMolecular cloning and expression of Galβ1,3GalNAc α2,3‐sialyltransferase from human fetal liver
Author(s) -
Shang Jie,
Qiu Ruolun,
Wang Junqi,
Liu Junjian,
Zhou Rouli,
Ding Huiping,
Yang Shoujun,
Zhang Shuzheng,
Jin Cheng
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00733.x
Subject(s) - complementary dna , pichia pastoris , sialyltransferase , biology , microbiology and biotechnology , recombinant dna , molecular cloning , escherichia coli , gene , cloning (programming) , cdna library , gene expression , biochemistry , glycoprotein , computer science , programming language
Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galβ1,3GalNAc α2,3‐sialyltransferase (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 surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS‐1, Escherichia coli , and Pichia pastoris . The recombinant protein 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 suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.