scholarly journals CRISPR-TAPE: protein-centric CRISPR guide design for targeted proteome engineering

2020 ◽  
Author(s):  
DP. Anderson ◽  
HJ. Benns ◽  
EW. Tate ◽  
MA Child
Keyword(s):  
Amino Acid ◽  
Molecular Engineering ◽  
Design Tools ◽  
Design Algorithm ◽  
Homology Directed Repair ◽  
Post Hoc

AbstractRational molecular engineering of proteins with CRISPR-based approaches is challenged by the gene-centric nature of gRNA design tools. To address this, we have developed CRISPR-TAPE, a protein-centric gRNA design algorithm that allows users to target specific residues, or amino acid types within proteins. gRNA outputs can be customized to support maximal efficacy of homology-directed repair for engineering purposes, removing time consuming post-hoc curation, simplifying gRNA outputs, and reducing CPU times.

scholarly journals Combining simulations and experiments for the molecular engineering of multifunctional collagen mimetic peptide-based materials

Soft Matter ◽  
2021 ◽  
Author(s):  
Amber Hilderbrand ◽  
Phillip Taylor ◽  
Francesca Stanzione ◽  
Mark LaRue ◽  
Guo Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Engineering ◽  
Complex Materials ◽  
Mimetic Peptide ◽  
Synergistic Approach ◽  
The Creation

Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of...

scholarly journals Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

2020 ◽  
Vol 8 (4) ◽  
pp. 519
Author(s):  
Lisheng Xu ◽  
Fangkai Han ◽  
Zeng Dong ◽  
Zhaojun Wei
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Residue ◽  
Rational Design ◽  
Molecular Engineering ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Mutant Library ◽  
Thermal Stability Of

To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

scholarly journals CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

2021 ◽  
Vol 3 ◽  
Author(s):  
Mehmet Tufan Oz ◽  
Angelika Altpeter ◽  
Ratna Karan ◽  
Aldo Merotto ◽  
Fredy Altpeter
Keyword(s):  
Amino Acid ◽  
Gene Targeting ◽  
Crop Improvement ◽  
Herbicide Tolerance ◽  
Acetolactate Synthase ◽  
Amino Acid Substitutions ◽  
Dna Double Strand Breaks ◽  
Nucleotide Substitutions ◽  
Multiple Alleles ◽  
Homology Directed Repair

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

A new concept for molecular engineering of artificial enzymes: a multiscale simulation

Soft Matter ◽  
2016 ◽  
Vol 12 (3) ◽  
pp. 689-704 ◽  
Author(s):  
Pavel V. Komarov ◽  
Pavel G. Khalatur ◽  
Alexei R. Khokhlov
Keyword(s):  
Amino Acid ◽  
Multiscale Simulation ◽  
Molecular Engineering ◽  
Artificial Enzymes ◽  
Amino Acid Residues ◽  
Protein Amino Acid ◽  
Functional Analog ◽  
First Time

We have designed, for the first time, a functional analog of chymotrypsin from synthetic monomers imitating protein amino acid residues.

scholarly journals A Post-hoc Study of D-Amino Acid Oxidase in Blood as an Indicator of Post-stroke Dementia

2019 ◽  
Vol 10 ◽  
Author(s):  
Yi-Chun Chen ◽  
Wen-Hai Chou ◽  
Hsiao-Hui Tsou ◽  
Chiu-Ping Fang ◽  
Tung-Hsia Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Post Stroke ◽  
Post Hoc

scholarly journals AutoDesigner, a De Novo Design Algorithm for Rapidly Exploring Large Chemical Space for Lead Optimization: Application to the Design and Synthesis of D-Amino Acid Oxidase Inhibitors

2021 ◽  
Author(s):  
Pieter H Bos ◽  
Evelyne M. Houang ◽  
Fabio Ranalli ◽  
Abba E. Leffler ◽  
Nicholas A. Boyles ◽  
...  
Keyword(s):  
Amino Acid ◽  
Drug Discovery ◽  
De Novo ◽  
Chemical Space ◽  
De Novo Design ◽  
Lead Optimization ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Design Algorithm ◽  
Starting Point

The lead optimization stage of a drug discovery program generally involves the design, synthesis and assaying of hundreds to thousands of compounds. The design phase is usually carried out via traditional medicinal chemistry approaches and/or structure based drug design (SBDD) when suitable structural information is available. Two of the major limitations of this approach are (1) difficulty in rapidly designing potent molecules that adhere to myriad project criteria, or the multiparameter optimization (MPO) problem, and (2) the relatively small number of molecules explored compared to the vast size of chemical space. To address these limitations we have developed AutoDesigner, a de novo design algorithm. AutoDesigner employs a cloud-native, multi-stage search algorithm to carry out successive rounds of chemical space exploration and filtering. Millions to billions of virtual molecules are explored and optimized while adhering to a customizable set of project criteria such as physicochemical properties and potency. Additionally, the algorithm only requires a single ligand with measurable affinity and a putative binding model as a starting point, making it amenable to the early stages of a SBDD project where limited data is available. To assess the effectiveness of AutoDesigner, we applied it to the design of novel inhibitors of D-amino acid oxidase (DAO), a target for the treatment of schizophrenia. AutoDesigner was able to generate and efficiently explore over 1 billion molecules to successfully address a variety of project goals. The compounds generated by AutoDesigner that were synthesized and assayed (1) simultaneously met not only physicochemical criteria, clearance and central nervous system (CNS) penetration (Kp,uu) cutoffs, but also potency thresholds; (2) fully utilize structural data to discover and explore novel interactions and a previously unexplored subpocket in the DAO active site. The reported data demonstrate that AutoDesigner can play a key role in accelerating the discovery of novel, potent chemical matter within the constraints of a given drug discovery lead optimization campaign.

Gp120 substitutions at positions associated with resistance to fostemsavir in treatment-naive HIV-1-positive individuals

2020 ◽  
Vol 75 (6) ◽  
pp. 1580-1587 ◽  
Author(s):  
Luciana Lepore ◽  
Claudia Fabrizio ◽  
Davide Fiore Bavaro ◽  
Eugenio Milano ◽  
Anna Volpe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Position ◽  
High Rate ◽  
B Virus ◽  
Treatment Naïve ◽  
Constant Domains ◽  
Hiv Gp120 ◽  
Post Hoc ◽  
Hiv 1

Abstract Objectives Fostemsavir, a novel attachment inhibitor targeting the HIV-1 gp120, has demonstrated wide in vitro activity. However, the high rate of HIV gp120 substitutions could jeopardize its efficacy. We investigated envelope (env) substitutions at positions associated with resistance to fostemsavir in patients with a new HIV-1 diagnosis according to HIV subtype and tropism. Methods Gp120 sequences from 409 subjects were retrospectively analysed and the presence of the L116P, A204D, S375H/M/T, M426L, M434I and M475I mutations was evaluated. Other amino acid changes at the same positions were also recorded. The variability at each amino acid position was evaluated using Shannon entropy. Results The frequency of mutations was: S375T (13.2%); M426L (6.8%); M434I (2.9%); M475I (2.7%); S375H (1.0%)/M (0.8%) and L116P (0.31%). Statistically significant differences were found at positions 375 (R5/non-R5 strains and B/non-B subtypes) and 426 (B/non-B subtypes); post hoc analysis revealed that significance for position 375 was steered by S375T while for position 426 significance was governed by unusual substitutions, in particular M426R (B/non-B, P < 0.00001). The variability of env constant domains appeared to be more relevant in the non-B virus population. Conclusions In conclusion, gp120 substitutions were detected in different subtypes and in both R5 and non-R5 variants. Despite the great variability of gp120, the frequency of mutations was low overall and the predominant substitution was S375T, the role of which in reducing fostemsavir efficacy is less substantial.

scholarly journals Rational molecular engineering of l-amino acid deaminase for production of α-ketoisovaleric acid from l-valine by Escherichia coli

RSC Advances ◽  
2017 ◽  
Vol 7 (11) ◽  
pp. 6615-6621 ◽  
Author(s):  
Ruoxi Li ◽  
Hossain Gazi Sakir ◽  
Jianghua Li ◽  
Hyun-dong Shin ◽  
Guocheng Du ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Engineering

The targeted modification of enzymatic efficiency can drive an increased production of desired metabolites.

scholarly journals Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid

2020 ◽  
Vol 21 (5) ◽  
pp. 1681 ◽  
Author(s):  
Beatriz Rodríguez-Alonso ◽  
Veronica Maillo ◽  
Omar Salvador Acuña ◽  
Rebeca López-Úbeda ◽  
Alejandro Torrecillas ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Amino Acid Content ◽  
Phosphoglycerate Kinase ◽  
Cell System ◽  
Early Embryo ◽  
Carbohydrate Composition ◽  
Protein Amino Acid ◽  
Oviduct Fluid ◽  
Post Hoc

Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (І) oviduct anatomy and (ІІ) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 µl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (І) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (ІІ) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ protein concentrations. In conclusion, data indicate that the composition of bovine OF is anatomically dynamic and influenced by the presence of an early embryo.

Sign in / Sign up

Export Citation Format

Share Document