Transcription efficiency along the tissue-plasminogen-activator cDNA gene in Escherichia coli

Rothstein, David M.; Bertonis, Jeanne M.

doi:10.1016/0378-1119(87)90363-5

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…The plasminogen activator activity of t-PA is greatly enhanced in the presence of fibrin because both t-PA and plasminogen specifically bind to fibrin to form a ternary complex in which plasminogen activation takes place (5). This catalytic enhancement by fibrin, so-called fibrin acceleration, is a characteristic feature of t-PA not mammalian cells (e.g., CHO cells) is required for the large-scale production of t-PA, because the expression of nt-PA cDNA in Escherichia coli was very low, and the inactive t-PA produced was barely renatured to an active form (3, 6,7). Therefore, our aim was to create new t-PA derivatives that are stable in plasma, effective by bolus administration, and easily produced in E .…”

Section: Introductionmentioning

confidence: 99%

Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

Saito

Ishii

Sasaki

et al. 1994

Biotechnology Progress

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We have created a novel thrombolytic agent by the combination of mutation with partial deletion of tissue-type plasminogen activator (t-PA). We constructed Escherichia coli expression vectors for (i) native t-PA (nt-PA) and its derivatives; (ii) K1K2P, consisting of kringle 1 (K1), kringle 2 (K2), and protease (P) domains; (iii) K2P, consisting of K2 and P domains; (iv) D-nt-PA; (v) D-K1K2P; and (vi) D-K2P. The latter three are point mutants of nt-PA, K1K2P, and K2P, respectively, in which Arg275 (number corresponds to that of nt-PA) has been mutated to Asp. The production of nt-PA and its derivatives was remarkably improved by (i) removal of the 3' noncoding region of nt-PA cDNA from expression vectors and (ii) expression in mutant E. coli derived from E. coli HB101, which is insensitive to heat-shock inductions. The proteins produced were precipitated as insoluble aggregates in the cells and were renatured to active forms by extraction with 8 M urea followed by dialysis against a redox buffer containing GSH and GSSG. The renaturation yield depended on the pH of the buffer and the number of disulfide bonds of the proteins (nt-PA << K1K2P < K2P). The mutation of Arg275 (the plasmin cleavage site) caused an increase in the catalytic enhancement by fibrin and a decrease of the interaction with plasminogen activator inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Introductionmentioning

confidence: 99%

Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

Saito

Ishii

Sasaki

et al. 1994

Biotechnology Progress

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“…Herein, we report the measurement of gene expression efficiency in terms of transcription and translation efficiencies by using the met hod developed by Brosius and Holy,' which was employed to measure the transcription efficiency of the tpa gene. 37 To apply this method to our recombinant fermentation system, we constructed a new plasmid. The newly constructed plasmid allowed us to simultaneously investigate the separate and independent effects of plasmid content, transcription efficiency and translation efficiency on the productivity of a cloned gene product.…”

Section: Introductionmentioning

confidence: 99%

The effects of plasmid content, transcription efficiency, and translation efficiency on the productivity of a cloned gene protein in Escherichia coli

Kim

Ryu

1991

Biotech & Bioengineering

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In order to investigate how plasmid content, transcription efficiency, and translation efficiency affect the productivity of a cloned gene protein, a new vector (pPLc-RP4.5) was constructed. The vector has PL promoter and lacZ as a structure gene 4.5S RNA gene between PL promoter and lacZ gene. We took advantage of the characteristic that the 4.5S RNA is accumulated inside E. coli cells and can be quantitatively measured. A two-stage continuous culture system in combination with a temperature-sensitive gene switching system was used to study the performance of the recombinant fermentation. It was found that the plasmid content as varied by the dilution rate in the production stage showed a different pattern from that in the growth stage. The result showed that promoter strength had a greater influence on the overall gene expression efficiency of a cloned gene than the plasmid content, and the overall gene expression efficiency was largely dependent upon translation efficiency when a multicopy plasmid (pBR322 derivative and rop-) and a strong promoter (PL) were used to express a heterologous protein in E. coli.

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Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y₇₃₀ and Y₇₃₁ in Radical Propagation

et al. 2007

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E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.

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Transcription efficiency along the tissue-plasminogen-activator cDNA gene in Escherichia coli

Cited by 3 publications

References 21 publications

Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

The effects of plasmid content, transcription efficiency, and translation efficiency on the productivity of a cloned gene protein in Escherichia coli

Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y₇₃₀ and Y₇₃₁ in Radical Propagation

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Transcription efficiency along the tissue-plasminogen-activator cDNA gene in Escherichia coli

Cited by 3 publications

References 21 publications

Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli

The effects of plasmid content, transcription efficiency, and translation efficiency on the productivity of a cloned gene protein in Escherichia coli

Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y730 and Y731 in Radical Propagation

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Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y₇₃₀ and Y₇₃₁ in Radical Propagation