selective isotope labeling of recombinant proteins in escherichia coli

... to explain the large variability observed in the levels of recombinant protein yield Recent studies on the transcriptomic profiling of recombinant cultures has improved our understanding on the ... (1996) Probing the structural role of an alpha beta loop of maltose-binding protein by mutagenesis: heat-shock induction by loop variants of the maltose-binding protein that form periplasmic inclusion ... Betton JM, Mazodier P, Benaroudj N (2009) Production of recombinant proteins in the lon-deficient BL21(DE3) strain of Escherichia coli in the absence of the DnaK chaperone Appl Environ Microbiol 75(11):3803–3807...

Ngày tải lên: 20/06/2014, 23:20

... permeability of the IM of E coli, · 106 cells were mixed with lm SYTOX Green in NaCl ⁄ Pi for in the dark After addition of peptide, the increase in fluorescence, owing to the binding of the dye to intracellular ... of Esc(1–18) into E coli LPS films could be derived by extrapolating the Dp)p0 slope to Dp = 0, yielding a value of  47 mNÆm)1 (Fig 3B) The kinetics of the insertion of the peptide into the LPS ... 10) The exposure of · 107 cellsÆmL)1 at the corresponding MIC of Esc(1–18) resulted in an irregular rod form with a deep wrinkling of the cell surface (within min) However, all of these changes...

Ngày tải lên: 16/03/2014, 00:20

... for recombinant proteins Targeting signals retain recombinant proteins within distinct compartments of 28 the cells, preserve integrity and protect them from proteolytic degradation leading to increased ... of proteins in seeds, plant suspension cells, phyllosecretion and by rhizosecretion from engineered plant roots For production of recombinant proteins in seeds, such as expression of avidin in ... aided by targeting signals Retaining recombinant proteins within the distinct compartments of the cell preserves integrity, protects proteins from proteolytic degradation and thereby increases accumulation...

Ngày tải lên: 12/09/2015, 11:08

... cloning of the foreign gene in this shuttle vector, we created a unique enzyme site There were two HindIII sites in pASVNPT A single HindIII site was generated in pASVNPT by destroying one of ... constructs were selected in E coli using ampicillin (50 mg/ml) and in plant cells using kanamycin (50 mg/ml) Plasmid pASVGUS, a derivative of pASV82 (8.2 kb) containing the GUS expression cassette, ... region The recombinant plasmids described in the Results are indicated in boxes The unique HindIII in pASV82 can be used to clone expression cassettes with the gene of interest Optimization of electroporation...

Ngày tải lên: 16/09/2015, 17:13

... metal binding Specificity of Zn(II) binding by the purified Zn(II)-binding proteins The specificity of Zn(II) binding was also examined for the purified Zn(II)-binding proteins The binding of radioactive ... systematic search for E coli proteins Ó FEBS 2002 Escherichia coli zinc-binding proteins (Eur J Biochem 269) 2411 Table Zinc-binding proteins in Escherichia coli Protein Function Reference Ada ... E coli proteins, the total number of zinc-binding proteins in E coli could be about 200–400 (or 5–10% of a total of 4000 open reading frames on the E coli genome) Most of the Zn(II)binding proteins...

Ngày tải lên: 22/02/2014, 04:20

... Protein expression from the pSS101 plasmid as measured by 3H ⁄ 14C double labeling Proteins in the parental MG1655 strain were labeled with [14C]lysine Proteins in CVR40D and CVR69L or in MG1655 in ... kasugamycin-binding site on the 30S subunit or on the 70S ribosome in the region of the mRNA-binding tunnel in the E-site and P-site [32,33] As no effect of kasugamycin on mRNA binding to the ... 277, 225–230 Stringer EA, Sarkar P & Maitra U (1977) Function of initiation factor in the binding and release of initiation factor from ribosomal initiation complexes in Escherichia coli J Biol Chem...

Ngày tải lên: 29/03/2014, 09:20

... taking the ratio of σ in each time point and dividing by σ in the reference, and scaling that value such that a sigma of corresponds to a model ratio of 2.5 Nor, norfloxacin; Novo, novobiocin; ... expression profile representing the supercoiling of the reporter plasmid in each experiment (Table 1), with black indicating no change in plasmid supercoiling and bright green indicating complete ... effects of translocating proteins, such as RNA polymerase, that create (+) supercoils in front and (-) supercoils behind the moving protein [12,13] The balanced activities of these enzymes result in...

Ngày tải lên: 14/08/2014, 14:21

... chains are correctly targeted to the SecY protein in the translocon via the SRP pathway SRP dependency of (G-10L)prePhoE in vivo Fig Cross-linking of soluble E coli proteins to PhoE nascent chains ... exposing the signal-anchor domain just outside the ribosome To compensate for the loss of methionines from the deleted domains of the proteins, both constructs contain a C-terminal tetra-methionine ... these nascent chains are targeted to SecY via SRP by performing cross-linking experiments in vitro in the presence of IMVs To obtain a high cross-linking efficiency, reconstituted E coli SRP was added...

Ngày tải lên: 08/03/2014, 09:20

... sequence, confirming the presence of five of the six additional residues at the N-terminus of recombinant chimeric GO (the starting methionine is not present) The recombinant GO produced in E coli was ... glycine, glycine-ester and D-pipecolic acid was only observed using GO Interestingly, D-proline is the only amino acid in which the a-amino group is involved in a covalent bond with the side chain ... native PAGE in the presence of: (1) sarcosine; (2) glycine; (3) N-ethylglycine; (4) N,N-dimethylglycine; (5) D-alanine; (6) L-alanine; (7) D-proline; (8) L-proline; (9) D-valine; (10) L-valine, as...

Ngày tải lên: 08/03/2014, 16:20

... produce 55kD fusion of the cleavable intein tag to N-terminus of a synthetic inhibitor As known, Mr of MCoTI-II is about 3.4kD, hence Mr of the obtained recombinant fusion protein should be about ... following IMPACT-CN system manufactures instruction by using chitin beads column chromatography The obtained purified recombinant MCoTI-II showing inhibitory activity against trypsin,and proteinases ... pattern of PI-17 proteins Mc Standard molecular markers 2, Uninduced 1, 3, 5, Induced (containing 58kD fusion protein band) Cells were broken by sonication in 10mM Tris-HCl buffer pH8 containing...

Ngày tải lên: 12/02/2014, 10:20

... composed of EIIA, B and C domains, with the EIIA and B domains being part of the phosphorylation chain and the EIIC domain representing the membrane domain As all components of the PTS, depending ... phosphorylation of glucose and a number of other carbohydrates (Fig 1) It consists of two common cytoplasmic proteins, enzyme I (EI) and histidine-containing protein (HPr), as well as an array of carbohydrate-specific ... phosphoryl group of phosphoenolpyruvate is transferred to the incoming glucose The degrees of phosphorylation of the various PTS proteins represent starting points for a number of signal transduction...

Ngày tải lên: 18/02/2014, 13:20

... out of 20 uridine residues in 23S rRNA and one out of eight uridine residues in 16S rRNA to pseudouridine [9] We have analyzed the substrate specificity of the pseudouridine synthase RluD in vivo ... presence of pseudouridines in 70S but not in free 50S subunits can be explained by reduced rates of pseudouridine formation due to the transition of A1916 to G The effects of the mutations in 23S ... base flipping of three bases including the substrate uridine 32 These structural elements are absent in normal tRNA RluA appears to recognize its substrate by indirect readout of a protein induced...

Ngày tải lên: 18/02/2014, 16:20

... by hematoxylin and eosin staining We examined the expression of these proteins in a cancer-free layer of squamous epithelial cells, carcinoma in situ (CIS) and invasive cancer, all of which were ... cellular proteins but also in chromatin-bound proteins The Mcm proteins bound to chromatin probably function in DNA replication, but the role of the proteins not tightly bound to chromatin remains to ... comparing the amounts of Mcm in total cellular proteins and chromatin-bound proteins, approximately one-third of total Mcm protein was found to be recovered in the chromatin-bound fraction in HeLa...

Ngày tải lên: 20/02/2014, 23:20

... rates in the presence of kirromycin The effect of kirromycin on the growth of GRd.tsf was investigated by measuring growth rates of cultures in LB containing various concentrations of kirromycin ... translation indirectly by inhibition of mRNA synthesis during amino acid starvation [20–23] Kirromycin is one in a family of antibiotics that inhibits protein synthesis by binding EF-Tu–GTP and preventing ... hydrolysis of the GTP at the ribosome [25] Kirromycin has been shown to stimulate the nucleotide exchange in EF-Tu by increasing the dissociation of GDP and binding of GTP [26] In addition, the binding...

Ngày tải lên: 21/02/2014, 00:20

... effects of site-specific mutations of the UhpC protein on both the transport and sensing functions of this molecule We mutated arginine residues as these are known to be involved in binding of anions ... mutations of a membrane protein can in uence the efficiency of integration into the native membrane and thus in uence the apparent transport activity The efficiency of protein incorporation into the E coli ... proteins and UhpT proteins substantially similar Arginine 204 is present in all UhpC proteins and in the Glc6P transporter HPTcp, whereas R437 is present only in the UhpC proteins from E coli and S...

Ngày tải lên: 08/03/2014, 08:20

... active This may induce the cascade reaction of GluV8 activation, because recombinant proteins remain inside E coli cells, instead of being secreted from S aureus The conversion of amino acids adjacent ... mature form of GluV8 could be renatured even after exposure to heat in the presence of SDS Role of N-terminal Val69 in processing of the GluV8 proform Finally, the role of N-terminal Val69 of mature ... cultured in LB broth containing 50 lgÆmL)1 of ampicillin and 25 lgÆmL)1 of kanamycin at 37 °C overnight Protein expression was induced by dilution of the culture with two volumes of LB broth containing...

Ngày tải lên: 16/03/2014, 06:20

... muscle (Roche) and a sample of purified AcnB in a final volume of mL Gel electrophoresis and blotting of proteins The protein fractions obtained from the purification of 2-methylisocitrate dehydratase ... 5.68 35.7 Galactose-binding protein MalE 5.22 40.7 Maltose-binding protein Proteins induced at a lower level as compared with growth on acetate: AdhP 5.94 35.4 Propanol-preferring alcohol dehydrogenase ... specifically induced by iron and redox stress [29] Interestingly, two enzymes are involved in the conversion of methylcitrate into 2-methylisocitrate PrpD is involved in the dehydration of (2S,3S)-methylcitrate...

Ngày tải lên: 17/03/2014, 10:20

... previously [17] Supercoiling assay DNA supercoiling was assessed in terms of the linking number of intracellular plasmid pBR322 [18] DNA supercoiling was expressed as the active linking number, aLk, ... increase upon inhibition of gyrase with coumermycin [27] Our results are in good agreement with this: Extrapolation of our ®ts in Fig to an aLk of (corresponding to coumermycin inhibition) indicates ... (2000) Roles of topoisomerases in maintaining steady state DNA supercoiling in Escherichia coli J Biol Chem 275, 8103±8113 Menzel, R & Gellert, M (1983) Regulation of the genes for E coli DNA gyrase:...

Ngày tải lên: 18/03/2014, 01:20

... may be caused by binding to zinc binding proteins, e.g proteins that contain zinc finger protein structures [30] Zinc and cadmium are in the same group in the Periodic Table, contain the same common ... aqua-glyceroporins (glycerol transport proteins) in bacteria, yeast and mammals [26], and its toxicity lies in its ability to bind sulfhydryl groups of cysteine residues in proteins, thereby inactivating ... attachment of GMP to the phosphate group of Moco, forming the MPT guanine dinucleotide cofactor (MGD) [14,15] In total, more than 10 genes are involved in the biosynthesis of Moco in E coli, and...

Ngày tải lên: 23/03/2014, 06:20

... function of the refolding time Refolding reaction was carried out at 10 °C in the absence (dotted line), or presence of 21 nM of C-domain+ (thick solid line), 210 nM of C-domain+ (thin solid line) ... a4 helix, and a C-terminal region of a3 helix The N-domain is unique to the MIP-like FKBP subfamily proteins This domain is involved in dimerization of the protein and the interface between two ... rather C Fig Estimation of the amount of the proteins in soluble and insoluble forms by SDS ⁄ PAGE N-domain+ (A), C-domain– (B), and C-domain+ (C) were overproduced in E coli as described for SIB1...

Ngày tải lên: 23/03/2014, 13:20