Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris

Shailesh Kumar R. Yadav, Tejram Sahu, Aparna Dixit

Research output: Contribution to journalArticle

Abstract

Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source.

Original languageEnglish (US)
Pages (from-to)6703-6713
Number of pages11
JournalApplied Microbiology and Biotechnology
Volume100
Issue number15
DOIs
StatePublished - Aug 1 2016
Externally publishedYes

Keywords

  • 2S albumin
  • Antifungal activity
  • Momordica charantia
  • Napin
  • Pichia pastoris
  • Trypsin inhibitor activity

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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