Purification, refolding, and characterization of recombinant LHRH-T multimer.
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 beta sheet structure like the monomeric LHRH protein.
Raina
Komal
K
The Talwar Research Foundation, E8, Neb Valley, New Delhi 110068, India.
Panda
Amulya K
AK
Ali
Mushir M
MM
Talwar
G P
GP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013601:T-Lymphocytes
D000911:Antibodies, Monoclonal; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone