Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach

Kim, Dae-Young; Shin, Nam-Kyu; Chang, Seung-Gu; Shin, Hang-Cheol

doi:10.1007/bf00168477

Cited by 8 publications

(4 citation statements)

References 4 publications

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“…coli cells. A general methodology to circumvent such an issue is to attach the peptide to a protein partner to generate a fusion protein with an affinity tag for the recovery and separation of the desired protein. − The desired PKC or TAT fragments were prepared using such fusion proteins followed by specific cleavage by either chemical or enzymatic processes. The basic model of the fusion protein is shown in Figure .…”

Section: Resultsmentioning

confidence: 99%

Biotechnology Based Process for Production of a Disulfide-Bridged Peptide

Goswami¹,

Goldberg²,

Hanson³

et al. 2016

Bioconjugate Chem.

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A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.

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Section: Resultsmentioning

confidence: 99%

Biotechnology Based Process for Production of a Disulfide-Bridged Peptide

Goswami¹,

Goldberg²,

Hanson³

et al. 2016

Bioconjugate Chem.

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“…Therefore, the difference in the expression levels of insoluble and soluble forms shown in the above cases cannot be simply explained. Human glucagon was produced in E. coli as a fusion protein in which the glucagon was fused to the 57-residue N-terminal portion of the human tumor necrosis factor-␣ (19). As a result, about 80% of the expressed fusion glucagon was present in the soluble fraction.…”

Section: Figmentioning

confidence: 99%

Use of Carboxypeptidase Y Propeptide as a Fusion Partner for Expression of Small Polypeptides in Escherichia coli

Hahm

Chung

1999

Protein Expression and Purification

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“…In our previous study [12], the 57 N-terminal residues of human TNF-α were used as a fusion partner to produce glucagon with high-level expression in E. coli. The property of high-level expression along with the characteristic β-sheet structure of TNF-α has been utilized in the production of peptide hormones as fusion proteins [13].…”

Section: Cloning and Expression Of Mini-proinsulinmentioning

confidence: 99%

Human insulin production from a novel mini-proinsulin which has high receptor-binding activity

Chang¹,

Kim²,

Choi³

et al. 1998

Biochemical Journal

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To increase the folding efficiency of the insulin precursor and the production yield of insulin, we have designed a mini-proinsulin (M2PI) having the central C-peptide region replaced with a sequence forming a reverse turn. The mini-proinsulin was fused at the N-terminus to a 21-residue fusion partner containing a His10 tag for affinity purification. The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 25% of the total cell protein. The protein was sulphonated, cleaved by CNBr and the M2PI mini-proinsulin was purified using ion-exchange chromatography. The refolding yield of M2PI was 20-40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was successfully generated by subsequent enzymic conversion of mini-proinsulin. The mini-proinsulin exhibited high receptor-binding activity, about 50% as potent as insulin, suggesting that this single-chained mini-proinsulin may provide a foundation in understanding the receptor-bound structure of insulin as well as the role of C-peptide in the folding and activity of proinsulin.

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Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach

Cited by 8 publications

References 4 publications

Biotechnology Based Process for Production of a Disulfide-Bridged Peptide

Biotechnology Based Process for Production of a Disulfide-Bridged Peptide

Use of Carboxypeptidase Y Propeptide as a Fusion Partner for Expression of Small Polypeptides in Escherichia coli

Human insulin production from a novel mini-proinsulin which has high receptor-binding activity

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