Introduction of the hypocholesterolemic peptide, LPYPR, to the major storage protein of mung bean [Vigna radiata (L.) Wilczek] through site-directed mutagenesis

Abstract

The hypocholesterolemic peptide, LPYPR, was successfully introduced into the VR-1, VR-2, and VR-5 regions of the mung bean 8Sα globulin. The mutant protein (MP) has 96.69% structural homology and 97% sequence homology compared to the wild type (WT). Expression of the mutant protein in E. coli HMS174(DE3) was 40.66%, which was 144.42% higher than that of the WT. The WT protein and MP had MWs of about 48.4 and 48.7 kDa, respectively. These were purified using HIC and digested with trypsin. UPLC analysis of the tryptic digests of the MP revealed the successful release of the LPYPR peptide. Unlike the WT protein, cholesterol-binding capacity (mg/g sample) of the MP increased over time of tryptic digestion (average growth rate of 9.5% for crude MP and 12.5% for HIC-purified MP) for its undigested form (crude: 220.96 ± 8.65, purified: 214.71 ± 11.91), with maximum values of 380.76 ± 6.61 and 434.44 ± 10.88 were obtained for the 24-h digests of the crude and purified proteins, respectively. Similarly, the sodium taurocholate binding capacity (%) was also found to increase over time of tryptic digestion (average growth rate of 4% for crude MP and 5.67% for HIC-purified MP) for the tryptic digests of the MP. Minimum values for % bound sodium taurocholate was obtained with the undigested samples (crude: 46.71 ± 0.42, purified: 44.49 ± 0.13), while maximum values thereof were obtained with the 24-h digest samples (crude: 59.75 ± 0.30, purified 61.95 ± 0.51).