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 Escherichia coli (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 selfassociation
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.