Recombinant HyC antigen
The recombinant HyC protein was derived from P. silenus expressed
in Escherichia coli (strain BL21 DE3 pLysS) transformed with the
pET-32a(+) expression vector as described in Yadav et
al.18 The rHyC was obtained as fusion protein with
thioredoxin tag and used as antigen in the present study. Briefly, a
confirmed positive recombinant clone was cultured in LB broth containing
ampicillin and chloramphenicol, incubated at 37oC till
it attains an OD of 0.6. Subsequently it was induced by adding IPTG to a
final concentration of 1 mM. The culture was allowed to grow at 37oC with shaking at 200 rpm for 6 h and purified under
denaturing condition using 8 M urea dialyzed against gradient urea
concentration (8 M to 0 M) and finally in Tris buffer pH 8.0. The
homogeneity of purified protein was analyzed by 12% SDS-PAGE and
quantified by Bradford reagent.