Expression of biologically active, mature human granulocyte-macrophage colony stimulating factor with anE. coli secretory expression system

Greenberg, Robert S.; Lundell, Daniel; Alroy, Yair; Bonitz, S G; Condon, Russell G. G.; Fossetta, James; Frommer, Beth R.; Gewain, Keith M.; Katz, Mitchell H.; Leibowitz, Paul J.; Narula, Satwant K.; Kastelein, Robert A.; Kimmenade, Anita van

doi:10.1007/bf01570872

Cited by 20 publications

(15 citation statements)

References 38 publications

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“…The rhGM-CSF (E. coli-derived; Schering-Plough Research Institute/Sandoz Research Institute) was expressed periplasmically in E. coli with the secretory vector pINIIIOmpA2 (Greenberg et al, 1988) and purified by ion-exchange and molecular size-exclusion chromatography as described previously (Trotta et al, 1987). Ammonium bicarbonate, 4-vinylpyridine, and tri-n-butylphosphine were purchased from Aldrich Chemical Co. (Milwaukee, Wisconsin).…”

Section: Methodsmentioning

confidence: 99%

“…We also apply MS-based mapping methods in combination with chromatographic separation in order to check the cDNA-predicted protein sequence for any posttranslational modifications, and to establish whether the OmpA 21-amino acid leader sequence of rhGM-CSF (Greenberg et al, 1988) was indeed cleaved at the predicted site. This work also illustrates the power of the ES MS method for accurate measurement of the molecular weight of the protein and determination of the number of disulfide bonds.…”

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confidence: 99%

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Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry

Tsarbopoulos¹,

Pramanik²,

Labdon³

et al. 1993

Protein Science

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A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs' liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with P-mercaptoethanol.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry

Tsarbopoulos¹,

Pramanik²,

Labdon³

et al. 1993

Protein Science

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“…The mGM-CSF polypeptide (124 residues; Mr, 14,346) is 54% identical to the hGM-CSF polypeptide (127 residues; Mr, 14,650). Despite the high degree of homology, the two polypeptides are species specific (5).…”

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confidence: 97%

“…Proteolytic digestion of both proteins identified two disulfide bonds, linking the first and third, and second and fourth cysteine residues (6). Using chemically synthesized hGM-CSF protein fragments and analogs, Clark-Lewis et al (7) identified residues 1-13 and residues 122-127 as being not critical and residues [14][15][16][17][18][19][20][21][22][23][24][25] as being critical for biological activity. Mutagenesis of mGM-CSF by Gough et al (8) revealed that residues 11-15, 24-37, 47-49, and 81-89 are obligatory for function.…”

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confidence: 99%

Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

Shanafelt

Kastelein

1989

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

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Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals offive amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating fac gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional threeamino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The dislide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed.

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“…GM-CSF produced recombinantly in E. coli ends up in inclusion bodies (IBs) and has certain drawbacks, including complex processing, low specific activity, and poor in vitro renaturation (2). The GM-CSF product obtained as an IB has an added methionine at the N terminus that leads to stimulation of antibody production in the human body, hence influencing the therapeutic value (8,33). Interferon alpha 2b (IFN-␣2b) belongs to the IFN family of cytokines, which can induce antiproliferative, immunomodulatory, and potent antiviral activities against a wide range of mammalian viruses (7).…”

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confidence: 99%

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

Sletta

Tøndervik

Hakvåg

et al. 2007

Appl Environ Microbiol

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Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-␣2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-␣2b expression remained poor even when fused to a signal sequence, and an alternative IFN-␣2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

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Expression of biologically active, mature human granulocyte-macrophage colony stimulating factor with anE. coli secretory expression system

Cited by 20 publications

References 38 publications

Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry

Isolation and characterization of a resistant core peptide of recombinant human granulocyte‐macrophage colony‐stimulating factor (gm‐csf); confirmation of the gm‐csf amino acid sequence by mass spectrometry

Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

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