Purification, refolding, and characterization of recombinant LHRH-T multimer

Abstract

To make the native LHRH immunogenic, a multimer of LHRH interspersed with T non-B peptides (r-LHRH-d2) was expressed as recombinant protein in Escherichia coli. The expression level of the recombinant protein was around 15% of the total cellular protein and it aggregated as inclusion bodies. Inclusion bodies from the bacterial cells were isolated and purified to homogeneity. Instead of high concentrations of chaotropic agents, r-LHRH- d2 was solubilized in 50 mM citrate buffer at pH 3 containing 2 M urea. The protein was refolded by 5-fold dilution (pulsatile) with cold 10 mM citrate buffer at pH 6 in presence of 0.3 M l-arginine. Purification of r-LHRH-d2 was carried out by successive passages on CM–Sepharose column at pH 6.0 which retained extraneous proteins and pH 4.8 at which r-LHRH-d2 bound to the resin. The elution was carried out by using linear salt gradient (0.1–1 M NaCl). The overall yield of the purified r-LHRH-d2 was 40% of the initial inclusion body proteins. The purity and homogeneity were confirmed by a single homogeneous peak on analytical HPLC eluting out at 29.51 min and by single band on SDS–PAGE reactive with polyvalent anti-LHRH antibodies. Mass spectroscopic analysis indicated the protein to be of 16.6 kDa which equals the theoretically expected mass. The N-terminal amino acid analysis of r-LHRH-d2 showed the sequence which corresponded to the designed protein. The CD spectrum of the refolded r-LHRH-d2 showed that the multimer has considerable β sheet structure like the monomeric LHRH protein.