Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli

Pagès, Jean‐Marie; Belaı̈ch, Anne; Anba, Jamila; Lazdunski, Claude

doi:10.1111/j.1432-1033.1987.tb13411.x

Cited by 5 publications

(1 citation statement)

References 28 publications

(20 reference statements)

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“…The Pi-starved cells will now produce the desired protein. If the protein to be made is not toxic to the cells, continuous culture can also be an attractive approach to make large quantities of protein of interest by the pho-regulated expression system (Pages et al, 1987).…”

Section: (D) Relative Strength Of the P~mentioning

confidence: 99%

A novel phosphate-regulated expression vector in Escherichia coli

Su¹,

Schweizer²,

Oxender³

1990

Gene

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The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.

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Section: (D) Relative Strength Of the P~mentioning

confidence: 99%

A novel phosphate-regulated expression vector in Escherichia coli

Su¹,

Schweizer²,

Oxender³

1990

Gene

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Purification of an L‐asparaginase—atrial natriuretic peptide fusion protein expressed in Escherichia coli

Wang

Roe

et al. 1995

Biotech & Bioengineering

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A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.

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