Functional expression of horsegram (Dolichos biflorus) Bowman–Birk inhibitor and its self-association

Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, native to Southeast Asia and tropical Africa, contains multiple forms of Bowman–Birk inhibitors. The major Bowman–Birk inhibitor from horsegram (HGI-III) was cloned and functionally expressed in Escherichiacoli (rHGI), which moved as a dimer in solution similar to the natural inhibitor. The biochemical characterization of rHGI also points to its close resemblance with HGI-III not only in its structure but also in its inhibitory characteristics. To explore the electrostatic interactions involved in the dimerization, a site-directed mutagenesis approach was used. The role of reactive site residue K24 and the C-terminal Asp in the structure and stability of the dimer was accomplished by mutating K24 and D75/76. The mutants produced in this study confirm that the self-association of HGI-III is indeed due to the electrostatic interaction between K24 of one monomer and D75/76 of the second monomer, in agreement with our previous data. The functional expression of a Bowman–Birk inhibitor minus a fusion tag serves as a platform to study the structural and functional effects of the special pattern of seven conserved disulphide bridges.