title: Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: a human Lymphatic Filarial parasite.
abstract: Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.
authors: CAS in Crystallography and Biophysics, University of Madras, Chennai 600 025, India.
authors: Nagampalli Raghavendra Sashi
authors: NR
authors: Krishna
authors: N S A
authors: NS
authors: Krushna
authors: R B
authors: RB
authors: Narayanan
authors: S S
authors: SS
authors: Rajan
authors: K
authors: K
authors: Gunasekaran
mesh_terms: D000818:Animals; D015153:Blotting, Western; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004605:Elephantiasis, Filarial / Q000378:metabolism / Q000469:parasitology; D004926:Escherichia coli; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010434:Pepsin A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010957:Plasmids; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D014169:Transformation, Bacterial
chemical_list: D002364:Caseins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D010434:Pepsin A; D005976:Glutaral