High-level expression of human fibroblast growth factor-9 N33 in Escherichia coli

Kuriyama, Masato; Sakamoto, Jyun-Ichi; Nakatu, Masanori; Kurokawa, Tsutomu; Sawada, Hideki

doi:10.1016/0922-338x(95)94199-2

Cited by 6 publications

(5 citation statements)

References 9 publications

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“…The full-length coding region for human FGF9 (Miyamoto et al, 1993) cDNA was isolated as a BamHI/blunt fragment from pET vector (Kuriyama et al, 1995) and was subcloned into the vector pBacPAK9 digested with BglII and SmaI. The FGF9 cDNA was used without changes in the coding region.…”

Section: Methodsmentioning

confidence: 99%

Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces

Hecht¹,

Adar

Hofmann³

et al. 2001

Acta Crystallogr D Biol Cryst

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Fibroblast growth factors (FGFs) constitute a family of at least 20 structurally related heparin-binding polypeptides active in regulating cell growth, survival, differentiation and migration. FGF9, originally discovered as a glia-activating factor, shares 30% sequence identity with other FGFs and has a unique spectrum of target-cell speci®city. FGF9 crystallized in the tetragonal space group I4 1 , with unit-cell parameters a = b = 151.9, c = 117.2 A Ê . The structure of the glycosylated protein has been re®ned to an R value of 21.0% with R free = 24.8%) at 2.6 A Ê resolution. The four molecules in the asymmetric unit are arranged in two non-crystallographic dimers, with the dimer interface composed partly of residues from N-and C-terminal extensions from the FGF core structure. Most of the receptor-binding residues identi®ed in FGF1± and FGF2±receptor complexes are buried in the dimer interface, with the 8± 9 loop stabilized in a particular conformation by an intramolecular hydrogen-bonding network. The potential heparin-binding sites are in a pattern distinct from FGF1 and FGF2. The carbohydrate moiety attached at Asn79 has no structural in¯uence.

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Section: Methodsmentioning

confidence: 99%

Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces

Hecht¹,

Adar

Hofmann³

et al. 2001

Acta Crystallogr D Biol Cryst

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“…The production of hFGF‐9 N33 in a 50 litre fermentor culture was more than 500 mg/l. All the accumulated protein was in a soluble form [6]. The purification and characterization scheme is summarized in Scheme 1 and Table 1.…”

Section: Resultsmentioning

confidence: 99%

“…E.coli MM294(DE3)/pTG931 harbouring the hFGF‐9 N33 gene was cultured as previously described [6]. The cells were collected by centrifugation and stored at −80°C until use.…”

Section: Methodsmentioning

confidence: 99%

“…These species also had similar growth‐stimulating effects on glial cells in vitro [1] to native hFGF‐9. Kuriyama et al previously established a recombinant Escherichia coli for the efficient production of 25 kDa hFGF‐9 (hFGF‐9 N33) [6] and Naruo et al purified hFGF‐9 from the culture supernatant of a human glioma cell line by a combination of heparin‐affinity chromatography, gel filtration chromatography and reversed‐phase HPLC [1]. We wished to prepare recombinant hFGF‐9, expressed in E. coli , for investigating its biological role and clinical application, and in the present study we describe an improved large‐scale purification of hFGF‐9 N33 from E. coli .…”

Section: Introductionmentioning

confidence: 99%

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Improved preparation and crystallization of 25 kDa human fibroblast growth factor‐9

Koyama

Ohmae

Tsuji

et al. 2001

Biotech and App Biochem

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We prepared 25 kDa human fibroblast growth factor-9 (hFGF-9 N33) on a large scale after overproduction in Escherichia coli MM294 (DE3)/pTG931. The purification was performed by a combination of hydrophobic chromatography and HPLC with an ion exchange column, a heparin affinity column and a gel filtration column. This improved procedure was rapid and simple, and the purified hFGF-9 N33 was found to be homogeneous as judged by various criteria, such as amino acid analysis, N-terminal amino acid sequence, C-terminal amino acid analysis and biological activity. Furthermore, as determined by low endotoxin and DNA content, the protein was of high purity. In addition, the hFGF-9 N33 prepared in the present study was easily crystallized by the vapour diffusion method.

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“…Expression vectors that are available for E. coli have different characteristics that differentiate one vector from another and are important for efficient and regulated expression of heterologous protein. Successful expression of a gene in E. coli depends on different parameters such as plasmid copy number [6], antibiotic selection [7], promoters [8], codon usage [9], growth conditions [10] and fusion protein [11]. However, no system has been analysed to be perfectly suitable for all recombinant protein over-expression owing to the relative diversity of model proteins to be studied.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Expression ofα-Amylase Gene inEscherichia coli: Effect on Specific Oxygen Uptake Rate and Host Cell Morphology during Batch Fermentation

Mathur

Chand

2011

Indian Chemical Engineer

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Cloning and expression of any heterologous gene product always impart metabolic load on the recombinant host for utilization of its metabolites that results in variations in cellular physiology. An understanding of such stress is important for robust process design and scaleup. In the present communication, an attempt has been made to explain the metabolic load imposed on the recombinant strain of Escherichia coli BL21(DE3) owing to over-expression of amyE gene, using different expression vectors of varying size. Current studies show that metabolic quotient or specific oxygen uptake rate (SOUR) of different recombinant hosts increases with an increase in vector size, showing correlation between vector size and cellular metabolic load. Further increase in SOUR on protein expression shows even higher metabolic load for protein expression than plasmid maintenance. Increase in bacterial size and decrease in specific growth rate on transformation and recombinant protein expression are cellular responses to additional metabolic load.

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High-level expression of human fibroblast growth factor-9 N33 in Escherichia coli

Cited by 6 publications

References 9 publications

Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces

Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces

Improved preparation and crystallization of 25 kDa human fibroblast growth factor‐9

Cloning and Expression ofα-Amylase Gene inEscherichia coli: Effect on Specific Oxygen Uptake Rate and Host Cell Morphology during Batch Fermentation

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