Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone

Goeddel, David V.; Heyneker, Herbert L.; Hozumi, Toyohara; Arentzen, René; Itakura, Keiichi; Yansura, Daniel G.; Ross, Michael J.; Miozzari, Giuseppe; Crea, Roberto; Seeburg, Peter H.

doi:10.1038/281544a0

Cited by 502 publications

(144 citation statements)

References 25 publications

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“…41,42 Consistent with this view, recombinant hirudin that we expressed using the hGH signal peptide (recHV-1.2) was cleaved as predicted between Ala −1 and Val +1 . The hGH precursor protein does not contain a propeptide sequence; 43 therefore, aberrant cleavage may be less likely when the hGH signal sequence is used to direct heterologous protein secretion.…”

Section: Discussionmentioning

confidence: 99%

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

et al. 1999

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We constructed a hirudin cDNA cassette, HV-1.1, that and mass spectroscopic analysis revealed the presence of encodes mature hirudin variant-1 fused to the signal pepan extra N-terminal serine residue, indicating aberrant tide of human tissue-type plasminogen activator (t-PA).cleavage of the t-PA signal peptide and likely accounting The cassette was subcloned into retroviral vectors and for the diminished activity. We therefore constructed a used to transduce human vascular endothelial cells in vitro.second cDNA cassette, HV-1.2, in which hirudin secretion Hirudin antigen and activity were measured by ELISA and was directed by the signal peptide of human growth horthrombin inhibition assays, respectively. Transduced cells mone. Hirudin expressed from the HV-1.2 cassette had a secreted up to 35 ± 2 ng/10 6 cells/24 h of biologically active specific activity of 13.5 ± 0.2 ATU/ g. Protein sequencing hirudin; expression was stable for at least 7 weeks.and mass spectroscopic analysis demonstrated proper Recombinant hirudin, expressed from the HV-1.1 cassette, cleavage of the growth hormone signal peptide. Thus, we had a specific activity of 7.1 ± 0.2 antithrombin units per achieved high level retrovirus-mediated secretion of biomicrogram (ATU/ g), compared with specific activities of logically active hirudin from endothelial cells in vitro. Use approximately 12 ATU/ g for both native leech hirudin and of these vectors may permit sustained local antagonism of recombinant hirudin produced in yeast. Protein sequencing thrombin activity in vivo.

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Section: Discussionmentioning

confidence: 99%

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

et al. 1999

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“…Both the E.coli lac and trp promoters have been used to ensure good expression of cloned genes in prokaryotes (26,37). We chose the trp promoter region for local mutagenesis for two main reasons.…”

Section: Vector Constructionmentioning

confidence: 99%

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Warburton¹,

Boseley²,

Porter³

1983

Nucl Acids Res

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A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E.coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased a-galactosidase synthesis, were selected for DNA sequencing. One clone has a G--A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C--T and T--C) between the Shine-Dalgarno sequence and initiation codon which raise expression of a-galactosidase two-fold.A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present.These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

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“…50ml cultures were grown at 370 to a stationary phase in Spizizen salts (32), supplemented with 0.2% glucose; 0.05% acid casein hydrolysate (Oxoid); 204g/ml L-leucine; 1004g/ml L-tryptophan; 4±g/ml thiamine and 33jig/ml ampi- Cultures were harvested by centrifugation at 8000rpm for 6 minutes at 4. Bacterial extracts were prepared for IFN assays by a modification of the method described by Goeddel et al (7). Thus The former assays were performed as recently described (13), in the latter the relative extent of cell damage was assessed by the dye uptake method (33).…”

Section: -13 (Fig 4amentioning

confidence: 99%

“…This signal can be provided either by joining the eukaryotic gene to the beginning of a bacterial structural gene, resulting in a fused polypeptide product (1)(2)(3)(4)(5), or by constructing a hybrid ribosomebinding site with a bacterial "Shine Dalgarno" (SD)(6) sequence followed by a translation initiation codon at the beginning of the eukaryotic gene (7)(8)(9).…”

Section: Introductionmentioning

confidence: 99%

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

Windass

Newton²,

Maeyer‐Guignard

et al. 1982

Nucl Acids Res

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Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone

Cited by 502 publications

References 25 publications

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

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

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