Cloning, Expression And Site-Directed Mutagenesis Of The Bowman-Birk Inhibitor Of Horsegram (Dolichos Biflorus).

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
radio 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.
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To evaluate the role of active site residue Lys24 and the C-terminal
end in dimerisation, site directed mutagenesis was performed.
Characterization of the inhibitory activities of the mutants revealed that the
K24A mutant inhibited elastase instead of trypsin. D75A mutant and Δ76
mutant retained the trypsin inhibitory activity. All the mutants and rHGI
exhibited similar chymotrypsin inhibitory activity. The oligomerisation status
of the mutants was studied using SDS-PAGE and size exclusion
chromatography. The studies pointed that K24A mutant existed as a
monomer. The C-terminal mutants, D75A and Δ76 also existed as
monomers. Thermal stability studies revealed that the monomers were less
stable than the dimers. Thus the cloning and heterologous expression of a
functional rHGI provides a platform to unveil the fine specificity of the
interactions involved in the dimeric status of HGI-III.
The BBIs have been extensively studied and known to prevent
malignant transformation in cancerous cells. The large size of BBIs hinders
the bioavailability of orally administered the BBIs. Smaller peptides
comprising the inhibitor domain are an attractive alternative. The trypsin
inhibitory (TID) domain of horsegram BBI was genetically engineered. The
expressed rTID was purified to homogeneity and was evaluated. The
apparent molecular mass of the expressed protein was ~4000 Da. The
purified inhibitor was stable thermally and also to proteases like pepsin and
pancreatin. Kinetic studies indicated that the expressed peptide (rTID) is a
non-competitive inhibitor of trypsin and also inhibited tryptase. Preliminary
studies revealed that rTID inhibits other trypsin like proteases. This smaller
peptide is a better prospect for drug design targeted at tryptase, the
enzyme implicated in inflammatory, allergic disorders and multiple sclerosis.