The influence of mRNA primary and secondary structure on human IFN-γ gene expression in<i>E. coli</i>

Tessier, Luc-Henri; Sondermeyer, P.; Faure, Thérèse; Dreyer, Dominique; Benavente, Annie; Villeval, Dominique; Courtney, Michael; Lecocq, Jean‐Pierre

doi:10.1093/nar/12.20.7663

Cited by 94 publications

(22 citation statements)

References 35 publications

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“…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]. A full-length PAI-1 cDNA clone was isolated from the human placental cDNA library by screening with synthetic oligonucleotide probes with the sequences CAATCGCAAGG-CACCTCTGAGAACTTCCAGG and TCTCCAGTTTGTC-CCAGATGAAGGCGTG, which hybridize to the 5' and 3' ends, respectively, of the published sequence coding for P.41-1.…”

Section: General Dna Techniquesmentioning

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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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 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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“…More or less stable structures of the mRNA translation initiation region have been investigated (10,14,20). These studies, which focused on the ribosome binding site and its tendency to hybridize with nearby sequences, reported that such structures, if their free energy was weaker than ±6 kcal/mol, did not affect the ribosome.…”

Section: Introductionmentioning

confidence: 99%

RNA stem-loop enhanced expression of previously non-expressible genes

Paulus¹

2004

Nucleic Acids Research

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The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem±loop (stem length, 7 bp; DG 0 = ±9.9 kcal/mol)

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“…The complete coding sequence of mPAI-2 was placed under the control of the A PL promoter in the vector pLK58 (27) with a synthetic oligonucleotide inserted upstream of the ATG to provide a bacterial ribosome binding site at an appropriate distance from the start codon (28), giving the plasmid pBTA447. Cell extracts of induced E. coli N4830 containing pBTA447 showed biologically active mPAI-2, as evidenced by the shift in electrophoretic mobility in the presence of urokinase, characteristic of the formation of a urokinase-mPAI-2 complex (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Antalis¹,

Clark²,

Barnes³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the A PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

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The influence of mRNA primary and secondary structure on human IFN-γ gene expression inE. coli

Cited by 94 publications

References 35 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

RNA stem-loop enhanced expression of previously non-expressible genes

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

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