Enhanced soluble expression and characterization of human N-acetylglucosaminyltransferase IVa in Escherichia coli.

N-Glycosylation is one of the most important posttranslational modifications of proteins. Nearly the entire surface of cells and almost all secreted proteins in humans are modified with complex-type N-glycans, whose functions are affected by the number of N-glycan branches. N-Acetylglucosaminyltransferase-IVa (GnT-IVa) is a Golgi glycosyltransferase that transfers a GlcNAc to the α-1,3 mannose arm of the biantennary N-glycan GlcNAc2Man3GlcNAc2 to form a β-1,4 GlcNAc branched structure. The soluble expression of mammalian glycosyltransferases in heterologous hosts is often challenging. In the present study, human GnT-IVa (HsGnT-IVa) was cloned as an N-terminal truncated form that was fused with solubility-enhancing tags or signal peptides and overexpressed in Escherichia coli (E. coli). Our results showed that recombinant HsGnT-IVa could be overexpressed in its highest soluble and active form when the first 87 amino acids were removed and was fused with maltose-binding protein (MBP). By optimizing the induction conditions, the expression level of the recombinant protein was increased to yield approximately 540 mg per liter of culture after affinity purification. The purified enzyme exhibited appropriate glycosyltransferase activity, and the K value of the acceptor substrate was calculated as 1.1 mM. Characterization of the enzyme revealed that it reached its maximum activity with 5 mM Mn at 37 °C in MES/NaOH (pH 7.0). In addition, the effects of key amino acids in the catalytic and lectin domains on enzyme activity were measured. This work offers an efficient approach for the large-scale production of bioactive HsGnT-IVa, which can be used for in vitro synthesis and functional studies of multiantennary complex-type N-glycans.