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.
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The work has been funded by institute funding (MLP0191) to PV.
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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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DOI: https://doi.org/10.1007/s10930-021-10039-0