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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

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Abstract

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.

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Abbreviations

3D:

3-Dimensional

LNAA:

Large neutral amino acids

PKU:

Phenylketonuria

PAH:

Phenylalanine hydroxylase

CD:

Circular dichroism

Trp:

Tryptophan

Tyr:

Tyrosine

Thr:

Threonine

Leu:

Leucine

Ile:

Isoleucine

Val:

Valine

Met:

Methionine

His:

Histidine

Phe:

Phenylalanine

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

HR-LCMS:

High resolution liquid chromatography mass spectrometry

PITC:

Phenylisothiocyanate

SGF:

Simulated gastric fluid

SIF:

Simulated intestinal fluid

TOF:

Time-of-flight

RT:

Room temperature

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Acknowledgements

Authors are thankful to Dr. Sridevi Annapurna Singh, Director, CSIR-CFTRI, Mysuru, India, for providing infrastructural facilities and Institute funding (MLP0191), and the Indian Council of Medical Research (ICMR), Delhi, India, for providing the SRF fellowship to PA and SL. We also thank Dr. Sridevi Annapurna Singh, Director, CSIR-CFTRI, Mysuru, India, and Shri Govindaraju K for their kind support in carrying out the CD spectroscopy and the amino acid analysis, respectively.

Funding

The work has been funded by institute funding (MLP0191) to PV.

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Authors and Affiliations

  1. Department of Food Safety and Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, 570020, India

    Prakruthi Appaiah, L. Sunil, Asha Martin & Prasanna Vasu

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, 201002, India

    Prakruthi Appaiah, L. Sunil, Asha Martin & Prasanna Vasu

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  1. Prakruthi Appaiah
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  2. L. Sunil
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Correspondence to Prasanna Vasu.

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Appaiah, P., Sunil, L., Martin, A. et al. 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. Protein J 41, 79–87 (2022). https://doi.org/10.1007/s10930-021-10039-0

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