Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations

Abstract Background Plasmid propagation in recombination strains such as Escherichia coli DH5α is regarded as a beneficial instrument for stable amplification of the DNA materials. Here, we show trans-conjugation of pGEM-T cloning vector (modified Promega PCR product cloning vector with tra genes, transposable element (Tn5)) and M13 sequence via α-complementation mechanism in order to activate β-d-galactosidase gene in DH5α strain (non-lactose-fermenting host). Results Trans-conjugation with pGEM-T allows correction of LacZ gene deletion through Tn5, and successful trans-conjugants in DH5α host cells can be able to produce active enzyme, thus described as lactose fermenting strain. The intracellular β-galactosidase was subjected to precipitation by ammonium sulfate and subsequently gel filtration, and the purified enzyme showed a molecular weight of approximately 72-kDa sodium dodecyl sulfate-polyacrylamid gel electrophoresis. The purified enzyme activity showed an optimal pH and temperature of 7.5 and 40 °C, respectively; it had a high stability within pH 6–8.5 and moderate thermal stability up to 50 °C. Conclusion Trans-conjugant of E. coli DH5α- lacZ∆M15 was successfully implemented. UV mutagenesis of the potent trans-conjugant isolate provides an improvement of the enzyme productivity. The enzymatic competitive inhibition by d-galactose and hydrolysis of lactose at ambient temperatures could make this enzyme a promising candidate for use in the dairy industry.

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Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

KnE Life Sciences ◽

10.18502/kls.v3i5.986 ◽

2017 ◽

Vol 3 (5) ◽

pp. 139

Author(s):

Mariana Wahjudi ◽

Catherina . ◽

Nita Marcelia Wangunhardjo ◽

Ernest Suryadjaja ◽

Xavier Daniel

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Sodium Dodecyl ◽

Xylanase Activity ◽

Cellular Protein ◽

Host Cells ◽

Chromosomal Dna ◽

Clear Zone ◽

Sds Page

The xynB gene of Bacillus subtilis subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the xynB gene from Bacillus subtilis subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the xynB gene in Escherichia coli Origami as host cells. The xynB gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The xynB gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for xynB gene and for the vector, then continued by restriction analyses. The result showed that transformants clone 9 and 10 bear the recombinant pMMB-xynB plasmid. The xylanase activity of xynB gene in Escherichia coli Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-xynB recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the xynB gene of Bacillus subtilis subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the Escherichia coli Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.

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Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

Canadian Journal of Biochemistry ◽

10.1139/o79-101 ◽

1979 ◽

Vol 57 (6) ◽

pp. 813-821 ◽

Cited By ~ 49

Author(s):

Peter Dickie ◽

Joel H. Weiner

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Sodium Cholate ◽

Defined Medium ◽

Exchange Chromatography ◽

Fumarate Reductase ◽

Molar Ratios ◽

Membrane Bound

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

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The purification from Escherichia coli of a protein relaxing superhelical DNA

Canadian Journal of Biochemistry ◽

10.1139/o76-045 ◽

1976 ◽

Vol 54 (4) ◽

pp. 301-306 ◽

Cited By ~ 12

Author(s):

M. G. Burrington ◽

A. R. Morgan

Keyword(s):

Escherichia Coli ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Rapid Screening ◽

Intact Protein ◽

Winding Number ◽

Circular Dna ◽

E Coli ◽

The Individual ◽

Negative Supercoils

The Escherichia coli omega protein was first described by Wang (Wang, J.C.: J. Mol. Biol. 55, 523–533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, α. We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity. It appears to be an αβ-ype subunit protein with a molecular weight of the intact protein of about 80 000 (determined by gel filtration) and of the individual subunits of 56 000 and 31 000 (sodium dodecyl sulfate polyacrylamide gels). We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on positive supercoils. Its characteristics with respect to pH, salts, temperature and chromatography are described. A method for rapid screening of E. coli for omega mutants is described.

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Lectin-Like Glycoprotein PsNLEC-1 Is Not Correctly Glycosylated and Targeted in Boron-Deficient Pea Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.5.663 ◽

2001 ◽

Vol 14 (5) ◽

pp. 663-670 ◽

Cited By ~ 31

Author(s):

Luis Bolaños ◽

Arancha Cebrián ◽

Miguel Redondo-Nieto ◽

Rafael Rivilla ◽

Ildefonso Bonilla

Keyword(s):

Escherichia Coli ◽

Gel Electrophoresis ◽

Root Nodules ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Host Cells ◽

Immunogold Localization ◽

Electrophoretic Mobilities ◽

Cytoplasmic Vesicles ◽

Infected Cells

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

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Arginase from human full-term placenta

Biochemical Journal ◽

10.1042/bj1590579 ◽

1976 ◽

Vol 159 (3) ◽

pp. 579-583 ◽

Cited By ~ 11

Author(s):

R Porta ◽

C Esposito ◽

A Martin ◽

G D Pietra

Keyword(s):

Gel Electrophoresis ◽

Competitive Inhibition ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Disc Electrophoresis ◽

Full Term ◽

Term Placenta ◽

A Value ◽

Optimum Ph

Arginase was purified about 1800-fold from extracts of human full-term placenta; the enzyme appeared to be homogenous by disc electrophoresis and molecular-sieve chromatography. The mol. wt. determination by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis yielded a value of 70000 for the most pure and the partially purified enzyme. The human placenta arginase is a metalloenzyme with an optimum pH of 9.1. The Km for L-arginine is 27 mM. L-Ornithine and L-lysine show competitive inhibition with Ki values of 6.3 and 14 mM respectively.

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Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Biochemical Journal ◽

10.1042/bj2130187 ◽

1983 ◽

Vol 213 (1) ◽

pp. 187-191 ◽

Cited By ~ 37

Author(s):

A Lewendon ◽

J R Coggins

Keyword(s):

Escherichia Coli ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Purification Step ◽

Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

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In Vitro Labeling Strategies for Identifying Primary Neural Tissue and a Neuronal Cell Line after Transplantation in the Cns

Cell Transplantation ◽

10.1177/096368979300200207 ◽

1993 ◽

Vol 2 (2) ◽

pp. 131-149 ◽

Cited By ~ 51

Author(s):

Stephen M. Onifer ◽

Linda A. White ◽

Scott R. Whittemore ◽

Vicky R. Holets

Keyword(s):

Escherichia Coli ◽

Cell Line ◽

Neuronal Cell ◽

Fluorescein Isothiocyanate ◽

Initial Step ◽

Host Cells ◽

Lacz Gene ◽

Raphe Neurons

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS transplantation studies were tested by first characterizing the labels in vitro. The labels that were tested included 4,6-diamidino-2-phenylindole hydrochloride, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate-conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3′-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or tetanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in vivo after CNS transplantation. In vitro, 1,1-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neurons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Most labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retained in cellular debris. In vivo, DiI was visualized in transplanted, DiI-labeled raphe neurons, but not in astrocytes up to 1 mo posttransplant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. β-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No β-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels for use in CNS transplantation studies should be optimized for the specific population of donor cells under study, with the initial step being characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be assessed.

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Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase

Biochemical Journal ◽

10.1042/bj1910209 ◽

1980 ◽

Vol 191 (1) ◽

pp. 209-219 ◽

Cited By ~ 121

Author(s):

J Hughes ◽

G Mellows

Keyword(s):

Escherichia Coli ◽

Exchange Reaction ◽

Rat Liver ◽

Equilibrium Dialysis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Trna Synthetase ◽

Stable Complex ◽

E Coli ◽

Escherichia Coli B

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9′-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9′-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.

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Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

Biochemical Journal ◽

10.1042/bj1650417c ◽

1977 ◽

Vol 165 (3) ◽

pp. 417-423 ◽

Cited By ~ 2

Author(s):

Dobrivoje V. Marinkovic ◽

Jelka N. Marinkovic

Keyword(s):

Escherichia Coli ◽

Sodium Dodecyl Sulphate ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Galactosidase Activity ◽

Terminal Amino Acid ◽

E Coli ◽

Molecular Weights ◽

Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

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Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

Biochemical Journal ◽

10.1042/bj1750555 ◽

1978 ◽

Vol 175 (2) ◽

pp. 555-563 ◽

Cited By ~ 14

Author(s):

Carol M. Blackwell ◽

John M. Turner

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Coenzyme B12 ◽

Apparent Homogeneity ◽

Purification And Properties ◽

Protein Components ◽

Inhibited Enzyme

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, Km values for ethanolamine and coenzyme B12 were 44μm and 0.42μm respectively. The Km for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (Ki 0.86μm), dl-1-aminopropan-2-ol (Ki 2.2μm) and dl-1,3-diaminopropan-2-ol (Ki 88.0μm) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (Ki 1.4nm), hydroxocobalamin (Ki 2.1nm) and cyanocobalamin (Ki 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K+ ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s20,w of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.

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