Soluble expression and bioactivity characterization of hepatic immunoregulatory protein LSECtin-CRD mutants / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To construct a prokaryotic expression vector for liver and lymph node sinusoidal endothelial cell C-type lectin carbohydrate recognition domain (LSECtin-CRD) in which two amino acids were mutated to Cys and to evaluate the bioactivity of LSECtin-CRD mutants.Methods:Mutation sites were selected based on predicted 3D model of LSECtin-CRD. Site-directed mutagenesis was carried out using sequence overlapped extension PCR (SOE-PCR). The cDNA fragments of LSECtin-CRD mutants were subcloned into the prokaryotic expression vector pET28b-Tat. The expression of soluble LSECtin-CRD mutants was induced with IPTG and identified by SDS-PAGE. Ni-NTA affinity chromatography was used for purification. The bioactivities of LSECtin-CRD mutants were assessed by mannose and GlcNAc binding assays.Results:The prokaryotic expression vectors for LSECtin-CRD mutants G205C-A227C and G205C-D279C were successfully constructed as demonstrated by PCR and gene sequencing. The two mutants were efficiently expressed in soluble forms and had significant sugar-binding activities.Conclusions:Functional LSECtin-CRD mutants were successfully obtained, laying an experimental foundation for further study on the relationship between the function and structure of LSECtin-CRD.