Expression and site-directed mutagenesis of hepatic glucokinase.
Ontology highlight
ABSTRACT:
SUBMITTER: Lange AJ
PROVIDER: S-EPMC1151205 | biostudies-other | 1991 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.
| S-EPMC2175162 | biostudies-literature
Project description:Quercetin-4'-O-glucoside is one of the major quercetin derivatives in the mature red onion bulb. It has an adjuvant effect on allergies, asthma, arthritis, and cancer. The present study aimed to use uridine diphosphate glycosyltransferase 88A1 (UGT88A1) from Arabidopsis thaliana to achieve the enzymatic synthesis of quercetin-4'-O-glucoside from quercetin. The results showed that UGT88A1 was most active at pH 9.0. The optimum temperature of UGT88A1 for synthesizing quercetin-4'-O-glucoside was 45°C, which was a little lower than that for synthesizing quercetin-3-O-glucoside (50°C). One mutant, V18R, of UGT88A1 was obtained by site-directed mutation and showed a greater affinity (Km 0.20 mM) and twice the enzyme activity (552.3 mU/mg) towards quercetin compared with the wild-type enzyme (0.36 mM and 227.6 mU/mg, respectively). The possible reason could be attributed to the distance change between the 18th amino-acid residue of UGT88A1 and the substrate quercetin, as deduced by molecular simulation.
| S-EPMC6527070 | biostudies-literature
Project description:Abortive infection mechanisms of Lactococcus lactis form a heterogeneous group of phage resistance systems that act after early phage gene expression. One of these systems, AbiK, aborts infection of the three most prevalent lactococcal phage groups of the dairy industry. In this study, it is demonstrated that the antiphage activity depends on the level of expression of the abiK gene and on the presence of a reverse transcriptase (RT) motif in AbiK. The abiK gene was shown to be part of an operon that includes two additional open reading frames, with one of these encoding a phage-related transcriptional repressor named Orf4. Expression of AbiK is driven by two promoters, PabiK and Porf3, the latter being repressed by Orf4 in vivo. Binding of the purified Orf4 to the Porf3 promoter was demonstrated in vitro by gel retardation assays. The N-terminal half of the deduced AbiK protein possesses an RT motif that was modified by site-directed mutagenesis. Conservative mutations in key positions resulted in the complete loss of the resistance phenotype. These data suggest that an RT activity might be involved in the phage resistance activity of AbiK. A model for the mode of action of AbiK is proposed.
| S-EPMC1112063 | biostudies-literature
Project description:SITE-DIRECTED MUTAGENESIS OF MYCOBACTERIUM TUBERCULOSIS AND FUNCTIONAL VALIDATION TO INVESTIGATE POTENTIAL BEDAQUILINE RESISTANCE CAUSING MUTATIONS
| PRJEB52049 | ENA
Project description:Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.
| S-EPMC4707547 | biostudies-literature
Project description:Primer design is essential to conduct whole plasmid site-directed mutagenesis for protein study. Traditionally, primers of mutagenesis are designed manually that is time-consuming and fallible. Here, we present a Python script for searching primers by presetting parameters of nucleotide composition and percentage of guanine-cytosine (GC content). The running results showed that the script is able to search primers with mutations of target residue automatically. This script may facilitate primer design for whole plasmid site-directed mutagenesis and aid protein mutant construction.
| S-EPMC7096704 | biostudies-literature
Project description:Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA.A CotA double mutant, K316N/D500G, was constructed by combining random and site-directed mutagenesis. It can be functionally expressed at an 11.4-fold higher level than the wild-type enzyme. In addition, it is able to convert ferulic acid much faster than the wild-type enzyme (21% vs. 14%) and is far more efficient in decolorizing a range of industrial dyes. The investigation of the effects of the mutations K316N and D500G showed that amino acid at position 316 had a major influence on enzyme activity and position 500 had a major influence on the expression of the laccase.The constructed double mutant K316N/D500G of the Bacillus licheniformis CotA laccase is an appropriate candidate for biotechnological applications due to its high expression level and high activity in dimerization of phenolic acids and decolorization of industrial dyes.
| S-EPMC2653023 | biostudies-literature
Project description:C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.
| S-EPMC3271008 | biostudies-literature
Project description:The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
| S-EPMC5138438 | biostudies-literature
Project description:The protein cross-reactive material 197 (CRM197) is known to catalyze the hydrolytic cleavage of DNA (DNase activity). A suspected metal-binding site (S109, T111, and E112) and suspected DNA-binding motif (T89, K90, and V91) were predicted within the CRM197 protein X-ray crystal structure (4AE0) using METSITE and DNABindProt, respectively. Between these two predicted sites is a groove (K103, E116, T120, E122, F123, and R126) that may assist in DNase activity. Alanine scanning was performed at these sites to determine which amino acids might be important for DNase activity. These mutations individually or in combination either maintained or increased the overall DNase activity compared to the unmodified CRM197. Mutation at the suspected metal-binding site showed similar fluctuations to the overall DNase activity whether the DNase assays were run with Mg2+ and Ca2+ or Mn2+. However, many of the mutations within the suspected DNA-binding motif saw significant differences depending on which metal was used. Only some of the improvements in DNase activity could be attributed to improved folding of the mutants compared to the unmodified CRM197. This study should provide a basis for further mutagenesis studies to remove the DNase activity of CRM197.
| S-EPMC6682014 | biostudies-literature