In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Aruna Vashishta, Tejram Sahu, Anshu Sharma, Shailesh Kumar Choudhary, Aparna Dixit

Research output: Contribution to journalArticlepeer-review

Abstract

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% α-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 μg/ml. Refolded His-rMcnapin exhibited ∼ 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.

Original languageEnglish (US)
Pages (from-to)847-855
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1764
Issue number5
DOIs
StatePublished - May 1 2006
Externally publishedYes

Keywords

  • Momordica charantia
  • Napin-like protein cDNA
  • antifungal activity
  • in vitro refolding
  • recombinant napin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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