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

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

PDF jfbc.13151.pdf - Accepted Version Restricted to Registered users only Download (1MB) | Request a copy

Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	bioinformatics tools, large neutral amino acids, Pichia pastoris (syn. Komagataella phaffii), phenylketonuria, recombinant protein, synthetic gene
Subjects:	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 Oct 2020 05:07
Last Modified:	01 Oct 2020 05:07
URI:	http://ir.cftri.res.in/id/eprint/14485

Actions (login required)

View Item