Glycosylation circuit enables improved catalytic properties for recombinant alkaline phosphatase

Abstract

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins. Here, we engineered a Campylobacter jejuni N-linked glycosylation machinery by overexpressing one of the core glycosylation-related enzymes, PgIB, to increase the glycosylation rate. It has been previously shown that by utilizing N-linked glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. We utilized N-linked glycosylation using an engineered glycosylation pathway to glycosylate a model enzyme, the alkaline phosphatase (ALP) enzyme in Escherichia coli. We have investigated the effects of glycosylation on enzyme properties. Considering the glycosylation mechanism is highly dependent on accessibility of the glycosylation tag, ALP constructs carrying the glycosylation tag at different locations of the gene have been constructed, and glycosylation rates have been calculated. Our results showed that, upon glycosylation, ALP features in terms of thermostability, proteolytic stability, tolerance to suboptimal pH, and denaturing conditions are dramatically improved. The results indicated that the N-linked glycosylation mechanism can be employed for protein manipulation for industrial applications.