site directed mutagenesis using oligonucleotide based recombineering

... unless otherwise specified, were purchased from Sigma or Merck Site- directed mutagenesis Site- directed mutagenesis was performed using a plasmid pT7-7 [13] containing an inserted DNA fragment ... Letters inside the boxes indicate residues introduced through site- directed mutagenesis at positions 39 and 451 The diagram was based on the structure of the Sinapis alba myrosinase [24] noncovalent ... Experiments were carried out according to the instructions included with the ÔQuikChange site- directed mutagenesis kit (Stratagene) The sequence of the mutagenic primers contained a common segment...

Ngày tải lên: 16/03/2014, 18:20

... in the vicinity of the active site and could form a binding site for a glucosyl unit in subsite )1 (Fig 4B) Together with the observations from the site- directed mutagenesis, Trp402 of IPU appears ... identification of active site residues by site- directed mutagenesis J Biol Chem 274, 30474–30480 20 Shimizu, T., Nakatsu, T., Miyairi, K., Okuno, T & Kato, H (2002) Active -site architecture of endopolygalacturonase ... participating in subsite )1 The residues that form potential subsites )1 and )2 are reportedly more conserved in GH family 49 than the residues in subsites +1 and +2 [17] Comparison of the active sites of...

Ngày tải lên: 19/02/2014, 16:20

... This study provides the first investigation of the importance of key active -site residues of ACE2 through site- directed mutagenesis, with the aim of providing practical evidence for their role in ... small However, site- directed mutagenesis studies of the equivalent residue R1098Q in somatic ACE (sACE; equivalent to Arg522 in tACE) have a dramatic effect [18], with the mutation causing increased ... one chloride-binding site (CL1) whereas ACE has two sites (one in each subdomain) In addition to this, the CL1 site is found in subdomain II, some distance from the active site, and, with a likely...

Ngày tải lên: 20/02/2014, 01:20

... HCl pH 7.5 Site- directed mutagenesis The His120Asn, His145Asn and Asn149Asp mutant forms of human arginase II were obtained by a two-step PCR [40], using the QuickChange site- directed mutagenesis ... knowledge, this is the first report in which the substrate specificity of arginase was altered by using sitedirected mutagenesis Experimental procedures Materials All reagents were of the highest quality ... with substrate and metal ions was examined by sitedirected mutagenesis and kinetic studies Selection of target residues (Asp149, His120 and His145) was based on the roles assigned to the equivalent...

Ngày tải lên: 20/02/2014, 02:21

... Conserved active site motif derived from the crystal structure of P putida E1b (D) Conserved active site motif derived from the crystal structure of human E1b Ó FEBS 2003 Active site of E1 component ... phosphorylation sites and in the Ela sequence alignment (Fig 2) Non-equivalence of active sites has been observed in the crystal structure of the thiamin-dependent yeast pyruvate decarboxylase; one active site ... dimer with two active sites of equal catalytic efficiency [10,11] and two ThDP per E1, albeit bound with different affinities [11–13] There is some evidence that the two active sites of pigeon breast...

Ngày tải lên: 20/02/2014, 23:20

... computer-fitted to the appropriate equations Site- directed mutagenesis The D153N mutant form of E coli agmatinase was obtained by a two-step PCR [20], using the plasmid pKA5 containing the speB ... added to agmatinase using the same superposition matrix calculated for manganese ions, in order to conserve the ligand-manganese distances RESULTS AND DISCUSSION By site- directed mutagenesis, a D153N ... 248–254 Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K & Pease, L.R (1989) Site- directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77, 51–59 Vriend, G (1990) WHAT...

Ngày tải lên: 21/02/2014, 01:21

... function [45] Site- directed mutagenesis This step was performed with the QuickChangeTM SiteDirected Mutagenesis method from Stratagene Briefly, this was based on a PCR with two complementary oligonucleotide ... expressed aromatase following site- directed mutagenesis Biochim Biophys Acta 1163, 195–200 52 Zhou, D.J., Korzekwa, K.R., Poulos, T & Chen, S.A (1992) A site- directed mutagenesis study of human ... P-450 using molecular modeling and site- directed mutagenesis J Biol Chem 266, 11939– 11946 61 Ganguly, M., Cheo, K.L & Brodie, H.J (1976) Estrogen biosynthesis and 1beta-hydroxylation using C19...

Ngày tải lên: 21/02/2014, 15:20

... molecular docking on the above A Site- directed mutagenesis of aryl-alcohol oxidase model, site- directed mutagenesis and kinetic studies were used to identify the enzyme active site and evaluate the role ... resulting vector (pALAAO) was used for site- directed mutagenesis, and transformation of E nidulans biA1, metG1, argB2 (IJFM A729), as described below [17] Site- directed mutagenesis AAO variants were obtained ... Sweden) Site- directed mutagenesis variants and wild-type AAO were purified from the induction medium after 48 h Purification included Sephacryl S-200 Table Oligonucleotides used as primers for PCR site- directed...

Ngày tải lên: 07/03/2014, 11:20

... binding site for HbpR and to examine the necessity of the sensing A-domain of HbpR for DNA binding The role of individual nucleotides and nucleotide motifs was characterized by site- directed mutagenesis ... binding site, which did not per se FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS D Tropel and J R van der Meer A HbpR-binding site Combining mutations in the distal and the proximal site very ... Sequence numbers refer to the locations of the transcriptional start site of hbpC (B) Alignments of the sequences of three HbpR-binding sites within the hbp gene region [19] The lane ‘cons-1’ indicates...

Ngày tải lên: 07/03/2014, 17:20

... expressed in lmặmg)1ặmin)1 Site- directed mutagenesis, protein expression and purication Presteady-state kinetic parameters for the acetylation of NAT Site- directed mutagenesis of the hamster ... to Ile (Y190I) and to Ala (Y190A), was carried out using the pPH70D vector and QuickChange site- directed mutagenesis kit (Stratagene)[52] The oligonucleotide primers used for Y190F, Y190I and ... (AcCoA) in the absence of amine [21] and the active site Cys68 or Cys69 has been further identied through thiol-specic modication and site- directed mutagenesis [2224] The rst crystal structure of...

Ngày tải lên: 16/03/2014, 00:20

... serve as Ca2+-binding sites [19] The aim of the present study was to identify the exact Ca2+-binding sites of human TG2, by using sitedirected mutagenesis and targeting sites homologous to FXIIIa ... enzymological properties of TG2 [36], were left on the protein Site- directed mutants were constructed using the QuikChange Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) Mutant constructs ... Ca2+-binding sites of TG2 A Bound Ca2+ (mol/mol) ´ly R Kira et al C277S WT 0 [Ca2+] free (mM) B Strong binding site of wild typeTG2 0.0 Weak binding sites of wild type TG2 Weak binding sites of S1...

Ngày tải lên: 16/03/2014, 03:20

... and CL2 sites of ACE and ACE2 were changed by site- directed mutagenesis, and the effects on chloride sensitivity, substrate selectivity and inhibitor potency were observed Results PCR mutagenesis ... region Table tACE and ACE2 residues subjected to site- directed mutagenesis For all residues subjected to PCR mutagenesis, the chloride-binding site at which they are located and their role are ... of critical active -site residues in angiotensin-converting enzyme-2 (ACE2) by site- directed mutagenesis FEBS J 272, 3512–3520 27 Reinalter SC, Jeck N, Brichausen C, Watzer B, Nusing ¨ RM, Seyberth...

Ngày tải lên: 16/03/2014, 04:20

... NaCl) Table Oligonucleotides for site- directed mutagenesis The replaced nucleotides are bold-face and the introduced restriction sites are underlined Mutation Oligonucleotides Restriction site S21L ... QuikChangeTM site- directed mutagenesis kit (Stratagene) to obtain the mutations N34D, K31A/R33S, L35A, L35S, and F46Y For the mutations K31A/R33S/F46Y, L35A/F46Y, and L35S/F46Y, site- directed mutagenesis ... carry the mutations for K31A/R33S, L35A, or L35S using the oligonucleotides for the F46Y mutation The oligonucleotides and the introduced restriction sites to facilitate the selection of positive...

Ngày tải lên: 16/03/2014, 18:20

... improved PCR -based method for site directed mutagenesis using megaprimers Mol Cell Probes 6, 345–348 Tyagi R, Lai R & Duggleby RG (2004) A new approach to ‘megaprimer’ polymerase chain reaction mutagenesis ... (1995) Evidence for two catalytically different magnesiumbinding sites in acetohydroxy acid isomeroreductase by site- directed mutagenesis Biochemistry 34, 6026–6036 11 Ahn HJ, Eom SJ, Yoon H-J, ... al Mutagenesis of the active site of E coli KARI established independent assays for each activity In addition, for inactive mutants, we developed methods to measure NADPH and Mg2+ binding using...

Ngày tải lên: 16/03/2014, 18:20

... important amino acids of glycosyltransferases, which is based on sequence alignment studies, is not satisfactory due to the results of the site- directed mutagenesis experiments presented here The question ... catalytic amino acids and proposed reaction mechanisms of glycosyltransferases [8] Because site- directed mutagenesis studies have not so far been performed for eucaryotic glycosyltransferases in ... 270) 537 the nucleophile We were able to prove for the first time the suggested model by site- directed mutagenesis of arbutin synthase First the E368D mutein was generated Determination of the...

Ngày tải lên: 17/03/2014, 09:20

... Restriction enzymes and T4 DNA ligase were from Promega Life Sciences Site- directed mutagenesis reactions were made using the Altered SitesTM II Kit (Promega Life Sciences) D-amino acids, xanthine, ... characterized two single-point mutations at site Y238 of RgDAAO generated using a dual primer method to simultaneously introduce ampicillin resistance and a site- directed mutation (Y238F: 5Â-GGCGGGACGTTCGGCGTGGGAG-3Â, ... investigated by site- directed mutagenesis experiments, but only by replacing a phenylalanine (a nondisruptive mutation) In the case of RgDAAO we also changed the spatial arrangement in the active site by...

Ngày tải lên: 17/03/2014, 17:20

... obtained from Perkin-Elmer (Boston, MA, USA) Site- directed mutagenesis Site- directed mutagenesis was carried out using the QuikChange Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) ... (rhGCPII; amino acids 44–750) using site- directed mutagenesis Individual amino acid changes were created by modifying the rhGCPII sequence using two complementary oligonucleotide primers harboring ... Conclusions In conclusion, we report a detailed analysis of the active site of glutamate carboxypeptidase II using sitedirected mutagenesis as a tool Amino acid residues important for substrate ⁄...

Ngày tải lên: 23/03/2014, 07:20

... contains two HAKN1 binding sites in opposite orientations After HAKN1 binding to the 94-bp oligonucleotide, labeled specifically at one of its 3¢ ends by filling-in the HindIII site, the complex was ... polyacrylamide gel electrophoresis (Fig 2A) Because the oligonucleotide contains two sites in opposite orientation, both strands of the binding site can be observed in a single footprinting assay ... HAKN1 binding to DNA An oligonucleotide containing two HAKN1 binding sites (BS1) in opposite orientations was labeled in the 3¢ end of either strand (HindIII or XbaI sites) and subjected to hydroxyl...

Ngày tải lên: 23/03/2014, 13:20

... Initial mutagenesis studies suggested that 314 R A Rothery et al there may be two Q-sites present – a polar QB site (equivalent to the QP site) , and an apolar QA site (equivalent to the QD site) ... redox-active Q -site that is formally located on the cytoplasmic side of the membrane Menaquinol (MQH2) oxidation by FrdABCD has been studied using a combination of site- directed mutagenesis, enzymology, ... the cytoplasmic side of the membrane The other site, the ˚ QD site (the distal Q -site) is located approximately 25 A from the QP site on the opposite (periplasmic) side of the membrane [3,10]...

Ngày tải lên: 23/03/2014, 13:20

... from AppliChem (Darmstadt, Germany) Site- directed mutagenesis Site- directed mutagenesis of (His)6PNAE was achieved using the QuikChangeTM in vitro Site- Directed Mutagenesis Kit, according to the ... kinetics The enzyme activity tests using PNA as substrate were performed with the wild-type enzyme and its individual muteins after purification The site- directed mutagenesis results verified that ... hydrolytic activity of PNAE In order to gain evidence for these suggestions, inhibitor studies, site- directed mutagenesis experiments and modelling of PNAE were performed Inhibitor studies Several known...

Ngày tải lên: 24/03/2014, 04:21