Characterization of in silico modeled synthetic protein enriched with branched-chain amino acids expressed in Pichia pastoris.

Sunil, L. and Prakruthi, Appaiah and Asha, Martin and Prasanna, Vasu (2021) Characterization of in silico modeled synthetic protein enriched with branched-chain amino acids expressed in Pichia pastoris. International Journal of Biological Macromolecules, 168. pp. 518-525. ISSN 0141-8130

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Abstract

We have designed earlier the 3-dimensional structure of protein enriched with 56% branched-chain amino acids (BCAA) based on an α-helical coiled-coil structure. The chemically synthesized DNA (BCAA51 gene) was expressed in Pichia pastoris and confirmed by SDS-PAGE and western blot analysis. In the present study, the purified recombinant protein was characterized using circular dichroism and data revealed that the secondary structure contained 53.5% α-helix, 3.2% β-strand, and 43.3% turns, which is in concurrence with the overall structure predicted by in silico modeling. The LC-ESI-MS/MS spectra revealed that three peptide masses showed similarity to peptides like EQLTK, LEIVIR, and ILDK, of the modeled BCAA51 protein with the sequence coverage of ~16% from N-terminal region. The N-terminal sequence of the first seven amino acid residues (EQLTKLE) was exactly matching with the in silico designed protein. In vitro digestibility of the protein using SGF and SIF showed the disappearance of ~11 kDa band and appearance of low molecular weight peptides, which indicated that the protein was easily digestible and non-allergenic, which is the overall objective of this study. Further in vivo digestibility and toxicology studies are required to conclusively utilize this protein as a supplement for the treatment of chronic liver diseases.

Item Type:	Article
Uncontrolled Keywords:	Synthetic BCAA protein Protein identification In vitro digestion
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 16 Protein Biochemistry 600 Technology > 08 Food technology > 16 Nutritive value > 02 Amino acids
Divisions:	Food Safety Analytical Quality Control Lab
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Jan 2021 04:49
Last Modified:	01 Jan 2021 04:49
URI:	http://ir.cftri.res.in/id/eprint/14735

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