In silico Designing of Protein Rich in Large Neutral Amino Acids Using Bovine αs1 Casein for Treatment of Phenylketonuria

Appaiah, Prakruthi; Vasu, Prasanna

doi:10.4172/jpb.1000417

Cited by 10 publications

(11 citation statements)

References 40 publications

(53 reference statements)

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“…SWISS-MODEL server was utilized to perform homology modeling which aligns an input target with pre-existing templates to produce a series of predicted models. The Protein Model-66 had properly folded structure containing four to five α-helices without β-sheets, which was similar in structure to Protein Model-54 (Appaiah & Vasu, 2016). The Protein Model-54 had a sequence similarity of 61.4% with bovine αS1 casein, while that of Protein Model-66 showed 60.4% of sequence similarity, when analyzed by aller genon line.com.…”

Section: Mkvlilaklvmreleelvnkmlhrywtvpqklvnvy Telvtelikiheivnlitmyswimentioning

confidence: 86%

“…SWISS‐MODEL, ExPASy ProtParam, and http://Allergenonline.com programs were used to screen the best target model. The tertiary structure of the protein was built using software like SWISS‐MODEL (http://swissmodel.expasy.org/interactive), RaptorX (http://raptorx.uchicago.edu/), I‐TASSER, and PROFUNC (http://www.ebi.ac.uk/thornton-srv/databases/ProFunc/), as described earlier (Appaiah & Vasu, ). The refined and improved protein model was compared with Protein Model‐54 using various software.…”

Section: Methodsmentioning

confidence: 99%

“…The tertiary structure of the protein was built using software like SWISS-MODEL (http://swiss model. expasy.org/inter active), RaptorX (http://rapto rx.uchic ago.edu/), I-TASSER, and PROFUNC (http://www.ebi.ac.uk/thorn ton-srv/ datab ases/ProFu nc/), as described earlier (Appaiah & Vasu, 2016).…”

Section: In Silico Designing Of An Improved Protein Modelmentioning

confidence: 99%

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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

Appaiah

Vasu

2020

J Food Biochem

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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.

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Section: Mkvlilaklvmreleelvnkmlhrywtvpqklvnvy Telvtelikiheivnlitmyswimentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

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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

Appaiah

Vasu

2020

J Food Biochem

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“…Appaiah and Vasu, 2016 reported that uGDT above 50 is a good indicator of any protein model with more than 100 residues. 47 Because PKS protein has 2146 amino acid residues, the overall uGDT of 1364 infers the selected structure as of the high-quality model.…”

Section: Transmission Electron Microscopymentioning

confidence: 99%

cis-9-Hexadecenal, a Natural Compound Targeting Cell Wall Organization, Critical Growth Factor, and Virulence of Aspergillus fumigatus

et al. 2020

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Aspergillus fumigatus causes several nosocomial pulmonary infections and accounts for high morbidity and mortality rate globally. Among various virulence factors, 1,8-dihydroxynaphthalene-melanin plays an important role in the survival during unfavorable conditions both in vivo and in vitro, masks various molecular patterns associated with A. fumigatus, and protects it from the host immune system. In the present study, we aim to understand the potential of cis-9-hexadecenal as an antimelanogenic compound and its role in modulating other associated virulence factors in A. fumigatus. cis-9-Hexadecenal is a bioactive compound that belongs to C 16 mono-unsaturated fatty-aldehyde groups. Minimum effective concentration of cis-9-hexadecenal affecting A. fumigatus melanin biosynthesis was determined using broth microdilution method. The spectrophotometric analysis revealed reduced melanin content (91%) and hydrophobicity (59%) at 0.293 mM of cis-9-hexadecenal. Cell surface organizational changes using electron microscopy showed altered demelanized smooth A. fumigatus conidial surface without any protrusions after cis-9-hexadecenal treatment. The transcript analysis of polyketide synthase (PKS) pksP/alb1 gene was quantified through qRT-PCR which revealed an upregulated expression. Total proteome profiling conducted through LC-MS-MS showed upregulated PKS enzyme but other downstream proteins involved in the 1,8-dihydroxynaphthalene-melanin biosynthesis pathway were absent. The homology modeling of PKS using Expasy's web server predicted that PKS is stable at varied conditions and is hydrophilic in nature. The Ramachandran plot by PROCHECK confirmed the 3-D structure of PKS to be reliable. Docking analysis using AutoDock-4.2.6 predicted the binding of cis-9-hexadecenal and PKS at Thr-264 and Ser-171 residue via hydrogen bonding at a low binding energy of −4.95 kcal/mol.

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“…ERRAT determines the statistics of non-bonded interactions between different types of atoms (https://servicesn.mbi. ucla.edu/ERRAT/) (Appalah & Vasu, 2016). VERIFY 3D evaluates the sequence position and structural environment of the model to compare them with the databases of well-known structures of high quality (https://servicesn.mbi.ucla.edu /Verify3D/) (Monie et al, 2016).…”

Section: Validation Toolsmentioning

confidence: 99%

In silico molecular modelling and docking of allicin, epigallocatechin-3-gallate and gingerol against colon cancer cell proteins

Elengoe

Sebestian

2020

APJMBB

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Globally, colon cancer is the second most common cancer among men and women. There is an urgent need to search for a cure for colon cancer. Phytocomponents have shown to exhibit chemoprevention and chemotherapeutic effects related to colon cancer. Thus, phytocomponents can be used as the lead for new drug discovery. Computational biology approaches such as protein modelling and docking have helped in designing substrate-based drugs. In this study, three dimensional (3-D) models of tumour protein (p53), adenomatous polyposis coli (APC) and epidermal growth factor receptor (EGFR) were built using SWISS-MODEL; and their interaction with allicin, epigallocatechin-3-gallate and gingerol through blind docking were evaluated using BSP-SLIM server. These three target proteins are from colon cancer. Physiochemical characters of protein models were assessed through ExPASy’s ProtParam tool. Moreover, the protein structures were validated using PROCHECK, ProQ, ERRAT and VERIFY 3D servers. The protein models’ scores were within normal range. It also showed that the protein models were stable to proceed with the docking approach. Finally, the protein structures (target proteins) were docked successfully with allicin, epigallocatechin-3-gallate and gingerol (phytocomponent). The protein models had a strong interaction with the phytocomponents due to their good binding scores. The best docking scores of the protein-phytocomponent complexes (p53-allicin, APC-Epigallocatechin-3-Gallate and EGFR-gingerol) were 4.968, 6.490, and 6.034, respectively. Protein p53 had the strongest interaction with allicin due to its lowest binding score among all the protein-plant compound complexes. Thus, the results of this study can be used to design and develop a more powerful structure-based drug.

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In silico Designing of Protein Rich in Large Neutral Amino Acids Using Bovine αs1 Casein for Treatment of Phenylketonuria

Cited by 10 publications

References 40 publications

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

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

cis-9-Hexadecenal, a Natural Compound Targeting Cell Wall Organization, Critical Growth Factor, and Virulence of Aspergillus fumigatus

In silico molecular modelling and docking of allicin, epigallocatechin-3-gallate and gingerol against colon cancer cell proteins

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