Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: A human Lymphatic Filarial parasite

Nagampalli Raghavendra Sashi Krishna, N. S.A. Krushna, R. B. Narayanan, S. S. Rajan, K. Gunasekaran

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)245-250
Number of pages6
JournalProtein Expression and Purification
Volume79
Issue number2
DOIs
Publication statusPublished - 1 Oct 2011
Externally publishedYes

Keywords

  • Brugia malayi
  • Casein agar plate
  • Pepsin inhibition
  • Refolding
  • Zymogram

Cite this