Single-Site Cys-Substituting Mutation of Human Lectin Galectin-2: Modulating Solubility in Recombinant Production, Reducing Long-Term Aggregation, and Enabling Site-Specific MonoPEGylation

The effector capacity of endogenous lectins on cell adhesion/growth prompts studies to turn them into pharmaceutically stable forms. Using human galectin-2 as a proof-of-principle model, we first introduced mutations at the site of one of the two Cys residues, that is, C57A, C57M, and C57S. Only the C57M variant was expressed in bacteria in soluble form in high yield. No notable aggregation of the modified homodimeric lectin occurred during 3 weeks of storage. This mutational process also facilitated the site-directed introduction of poly(ethylene glycol) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one chain per subunit in the homodimer. We note that neither the secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results thus document the feasibility of tailoring a human galectin for enhanced stability to aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as growth regulator and the rate of serum clearance.