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Improvement, cloning, and expression of an in silico designed protein enriched with large neutral amino acids in Pichia pastoris for possible application in phenylketonuria.

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Relation http://ir.cftri.com/14485/
https://doi.org/10.1111/jfbc.13151
 
Title Improvement, cloning, and expression of an in silico designed
protein enriched with large neutral amino acids in Pichia
pastoris for possible application in phenylketonuria.
 
Creator Prakruthi, Appaiah
Prasanna, Vasu
 
Subject 02 Amino acids
 
Description Phenylketonuria (PKU) is an inborn disease caused by defective phenylalanine hydroxylase, which consequently results in the accumulation of phenylalanine in the
brain leading to further complications. One of the promising approaches in dietary
treatment is the supplementation of large neutral amino acid (LNAA). The LNAA
compete with phenylalanine for the common L-type LNAA transporter across the
blood–brain barrier, and decrease phenylalanine levels in the brain. In this study, the
earlier LNAA-enriched protein model was improved (Protein Model-66) and validated in silico. The reverse translated and codon-optimized synthetic LNAA66 gene
was cloned into pPICZαC and expressed in Pichia pastoris. The expressed protein
was purified by His Select affinity chromatography. SDS-PAGE and Western blotting
analysis showed a band at an expected molecular weight of 12 kDa, confirming the
expression of the modeled protein. To our knowledge, this is the first report showing
the cloning and expression of an in silico designed LNAA-enriched protein.
Practical applications
One of the promising dietary treatment of phenylketonuria (PKU) is the supplementation of large neutral amino acid (LNAA), wherein high levels of LNAA compete with
phenylalanine for the same L-type LNAA transporter, and consequently decrease
phenylalanine accumulation in the brain, thereby decreasing neurological complications. For the first time, here, we are showing that an in silico designed and validated
Protein Model-66, rich in LNAA, can be successfully cloned and expressed in Pichia
pastoris. The complete biochemical and structural characterization of this protein will
give a clear insight into its potential application for PKU treatment. The protein can
be potentially used as a supplement to treat PKU to those who are non-adherent to
the restricted, non-palatable, and expensive diet. Furthermore, this novel and effective strategy of in silico designing, cloning and expression can be exploited to develop
proteins for various applications of industrial, food, medical, and academic relevance.
 
Publisher Wiley
 
Date 2020
 
Type Article
PeerReviewed
 
Format pdf
 
Language en
 
Identifier http://ir.cftri.com/14485/1/jfbc.13151.pdf
Prakruthi, Appaiah and Prasanna, Vasu (2020) Improvement, cloning, and expression of an in silico designed protein enriched with large neutral amino acids in Pichia pastoris for possible application in phenylketonuria. Journal of Food Biochemistry. ISSN 1745-4514