Cloning, expression and site-directed mutagenesis of the bowman-birk inhibitor of horsegram (dolichos biflorus)

Abstract

Bowman Birk inhibitors (BBI) are small protease inhibitors found in the seeds of legumes in particular. Their molecular masses are in the range of 6- 9 kDa. They comprise of a binary arrangement of two sub domains with a conserved array of seven disulphide bridges, which play a pivotal role in the stability of the inhibitors. These inhibitors interact simultaneously and independently with two molecules of proteinases. In addition to the protease inhibitor activity, BBI is reported to have anticarcinogenic and newlineradio protective activity and immune stimulating properties. BBIs have also been implicated to play a vital role in plant defense mechanism. Horse gram (Dolichos biflorus) is a pulse crop native to South East Asia and Tropical Africa. Four isoforms of BBIs, from horsegram seeds have been isolated. The inhibitors of horsegram (HGIs) are single polypeptides with a molecular mass of 8.5 kDa. However SDS-PAGE and analytical gel filtration indicate the molecular mass to be 16 kDa, suggesting that they exist as dimers in solution. In contrast, inhibitors of germinated horsegram seeds, HGGIs exist as monomers. The role of active site residue Lys24 and C-terminal end in the dimeric status of the major inhibitor (HGI-III) was previously established by in vitro and homology modeling. To delineate their role in vivo the HGI-III gene was cloned in E. coli and expressed. HGI-III specific gene was isolated from the genomic DNA of horsegram by PCR based method. The gene was cloned in pRSET C vector such that the extra residues from the vector are avoided. pRSET-rHGI was functionally expressed in E.coli cells and was purified to homogeneity.The characterization of rHGI was carried out and was comparable to the HGI-III already reported. rHGI also existed as a dimer and the kinetic constants of the inhibitor towards trypsin and chymotrypsin were comparable to the HGI-III.