The construction of a synthetic<i>Escherichia coli trp</i>promoter and its use in the expression of a synthetic interferon gene

Windass, John D.; Newton, Clive R.; Maeyer‐Guignard, Jaqueline De; Moore, Valerie; Markham, Alexander F.; Edge, Michael D.

doi:10.1093/nar/10.21.6639

Cited by 29 publications

(7 citation statements)

References 43 publications

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“…The pICI vectors used in this study are a series of expression vectors derived from pSTPl [38], which incorporate one of a number of alternative ribosome-binding site sequences downstream of the trp promoter. pICI 0020 incorporates a trp leader (AAAAAGGGTATCGACAm) ribosome-binding site sequence [39]; pICI 0027 incorporates a bicistronic ribosome-binding site sequence [40] ; pICI 0029 incorporates a ribosome-binding sequence (ACACAGGAACAGATCTA, -TG) used to increase the expression of IFN-y in E. coli [41].…”

Section: General Dna Techniquesmentioning

confidence: 99%

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Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Sancho

Tonge

Hockney

et al. 1994

European Journal of Biochemistry

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Plasminogen-activator inhibitor type 1 (PAI-l), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-Mitre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37°C with a half-life of approximately 48 h. At 20°C, PAI-1 activity was stable for a week and at 4°C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of highlevel expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.Activation of plasminogen to form the serine protease plasmin is central to the processes of fibrinolysis [l], cell migration, angiogenesis and tumour metastasis [2]. Activation of plasminogen is accomplished by either tissue-type or urokinase-type plasminogen activator. Regulation of the system occurs at several levels, including assembly of the components and stimulation of activation on fibrin [l] or at cell surfaces [3, 41. The inhibition of the active enzymes is an important element in the control of the plasminogedplasmin system. Plasminogen-activator inhibitor type 1 (PAI-1) is the main physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator, with association rate constants of the order of 0.1 pM s- ' [5]. PAI-1 is present in a wide variety of tissues and cultured cells, where regulation of its expression by several stimuli has been observed [6]. It is present in the circulation both in cell-free plasma and in platelets, these two pools being clearly distinct [7]. The plasma concentration of PAI-1 is normally low, approximately 20 ng/ml, but higher levels are observed in a range of diseases, suggesting an association with thrombotic disorders [S]. The platelet pool of PAI-1 is released during aggregation, so that it protects the newly formed thrombus against premature dissolution. Platelet PAI-1 contributes to the resistance PAI-1 was identified as a member of the superfamily of serine protease inhibitors known as serpins, on the basis of its sequence [16-181. The serpins are the major class of inhibitors regulating the fibrinolytic, coagulation and complement cascades. They act as pseudosubstrates and form an SDS-stable covalent 1 : 1 complex with their target...

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Section: General Dna Techniquesmentioning

confidence: 99%

“…Purification of PAI-Samples detailed inTable 2were analysed by SDSPAGE (10% polyacrylamide gel) followed by staining with Coomassie-brilliant blue (lanes a-c) or by immunoblotting (lane d) with rabbit anti-PAI-1[38]. Lane a, molecular mass markers ( m a ) ; lane b, 10 pl high-speed supernatant; lane c, 7.5 pg purified PAL1 ; lane d, 150 ng purified PAI-1.…”

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confidence: 99%

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Sancho

Tonge

Hockney

et al. 1994

European Journal of Biochemistry

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“…The trp promoter is 2-3 times stronger than the lac UV5 promoter (Windass et al, 1982). The tac promoter combines the -10 region of the lac UV5 promoter and the -35 region of the trp promoter.…”

Section: 2 Transcriptionmentioning

confidence: 99%

Microbial Enzymes and Biotechnology

Fogarty¹,

Kelly²

1990

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“…The oligonucleotides shown in Fig. 1 (0.4nmol), with the exceptions of 1,10,11,20,21,30,31,40,41,50,51,60,61 and 68 were phosphorylated to a specific activity of 8.4 Ci/miiol with [y-32P] ATP (11). Reaction mixtures were either combined to give the appropriate small sub-fragments and ethanol precipitated or ethanol precipitated then fractionated on 20% polyacrylamide/7M urea gels.…”

Section: Ligation Of Oligonucleotides To Construct the Synthetic Ifn-a2 Genementioning

confidence: 99%

“…E.coli strains PA340 and C600 were grown at 37°C to a stationary phase in L broth (28). JA221 cultures were grown at 37°C in Spizizen salts (29) as previously described (30). Lysates were purified by precipitation with trichloroacetic acid and size exclusion chromatography on Ultrogel AcA 54 (31) to a final specific activity of 5.4 x 105 U of interferon/mg of protein before determination of growth inhibition and natural killer cell activity.…”

Section: Ligation Of Oligonucleotides To Construct the Synthetic Ifn-a2 Genementioning

confidence: 99%

Chemical synthesis of a human interferon-α², gene and its expression inEscherichia coli

Edge¹,

Greene²,

Heathcliffe³

et al. 1983

Nucl Acids Res

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A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.

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The construction of a syntheticEscherichia coli trppromoter and its use in the expression of a synthetic interferon gene

Cited by 29 publications

References 43 publications

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Microbial Enzymes and Biotechnology

Chemical synthesis of a human interferon-α², gene and its expression inEscherichia coli

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The construction of a syntheticEscherichia coli trppromoter and its use in the expression of a synthetic interferon gene

Cited by 29 publications

References 43 publications

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Microbial Enzymes and Biotechnology

Chemical synthesis of a human interferon-α2, gene and its expression inEscherichia coli

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About

Chemical synthesis of a human interferon-α², gene and its expression inEscherichia coli