title: Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.
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 IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 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.
authors.affiliation: Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
authors.forename: Shailesh Kumar R
authors.initials: SKR
authors.lastname: Yadav
authors.affiliation: Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
authors.forename: Tejram
authors.initials: T
authors.lastname: Sahu
authors.affiliation: Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India. adixit7@gmail.com.
authors.forename: Aparna
authors.initials: A
authors.lastname: Dixit
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D008826:Microbial Sensitivity Tests; D029777:Momordica charantia / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014242:Trichoderma / Q000187:drug effects*
chemical_list: D000935:Antifungal Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; C511888:napin-like protein, Momordica charantia
keywords: 2S albumin; Antifungal activity; Momordica charantia; Napin; Pichia pastoris; Trypsin inhibitor activity