Physicochemical Characterization and In Vitro Digestibility Study of an In Silico Designed Recombinant Protein Enriched with Large Neutral Amino Acids and Lacking Phenylalanine for Phenylketonuria.

Prakruthi Appaiah, L Sunil, Asha Martin, Prasanna Vasu

The protein journal 2022 Feb

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In our previous study, a 3D structure of LNAA66 model protein containing 4-5 α-helices, high large neutral amino acids (LNAA) and lacking phenylalanine was designed, refined, expressed in Pichia pastoris and confirmed by Western blotting. Here the study is focused on the characterization of the expressed and purified recombinant LNAA66 protein. The results revealed that the expressed protein had 68.59% of LNAA enrichment, containing 41.6% of α-helix, 50.4% turns and 8% β-sheet, which are as per the in silico designed protein. The LC-ESI-MS/MS results confirmed the recombinant protein by identifying the first 30 N-terminal amino acids with a sequence coverage of ~ 29%. The protein was digested entirely into smaller molecular weight fragments when treated with digestive enzymes mimicking the human GI tract digestion, which indicated complete digestibility of the protein. These results suggest that the protein can be utilized for the envisioned application of dietary treatment for phenylketonuria. © 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Prakruthi Appaiah, L Sunil, Asha Martin, Prasanna Vasu. Physicochemical Characterization and In Vitro Digestibility Study of an In Silico Designed Recombinant Protein Enriched with Large Neutral Amino Acids and Lacking Phenylalanine for Phenylketonuria. The protein journal. 2022 Feb;41(1):79-87