Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Min, Cheol-Ki; Son, Young–Jin; Kim, Chang-Kyu; Park, Sang-Joong; Lee, Jinwon

doi:10.1016/j.jbiotec.2010.12.023

Cited by 23 publications

(39 citation statements)

References 33 publications

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“…The expression plasmid pPT, in which the g-csf ( isoform B ) gene was inserted into the Nde I and Hind III sites of the vector, was used for protein production as previously described [13,23]. …”

Section: Methodsmentioning

confidence: 99%

“…The expression of rhG-CSF in E. coli frequently results in the formation of insoluble aggregates, which are called inclusion bodies (IBs). These aggregates are solubilized in a buffer containing chaotropic agents such as urea [13,14] or guanidine [15]. Thus, it is important to renature the denatured forms to be biologically active forms with apposite 3D structure [16,17].…”

Section: Introductionmentioning

confidence: 99%

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Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Kim

Lee

et al. 2013

PLoS ONE

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Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Kim

Lee

et al. 2013

PLoS ONE

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“…The vector maps of the pPT-B5Kpi and pPT-H27Rpi plasmids and the cloning methods for their construction were described in our previous patent and paper [21,22].…”

Section: Cell Construction and Related Compounds Purificationmentioning

confidence: 99%

“…We carried out experiments in which the length and type of fusion partner were modified to increase the preproinsulin expression level and purification yield and then constructed the H27R strain whose expression rate was 30% higher than that of the B5K strain [21]. The H27R strain expressed preproinsulin with a 28-amino acid fusion partner.…”

Section: Hplc Profile Of Ircs From H27r Strainmentioning

confidence: 99%

“…Preproinsulin from the H27R strain was more hydrophilic than that from the B5K strain, so preproinsulin from H27R had a tendency for increased contact with ␤-mercaptoethanol, and then its hydrophilic character led to the cleavage of the disulfide bond of preproinsulin. Actually, the preproinsulin from the H27R strain was very sensitive to the amount of ␤-mercaptoethanol, and the yield for refolding was decreased with high concentrations of ␤-mercaptoethanol [21]. Aside from IRC4, the major IRCs of the H27R strain were IRC 5 and IRC 6.…”

Section: Tablementioning

confidence: 99%

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Insulin related compounds and identification

Min

Lee

Kim

et al. 2012

Journal of Chromatography B

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Se‐Glargine: Chemical Synthesis of a Basal Insulin Analogue Stabilized by an Internal Diselenide Bridge

Weil‐Ktorza,

Dhayalan,

Chen

et al. 2024

ChemBioChem

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Insulin provides a model for studies of protein folding and stability, enabling enhanced treatment of diabetes mellitus via analogue design. We describe the chemical synthesis of a basal insulin analogue stabilized by substitution of an internal cystine (A6–A11) by a diselenide bridge. The studies focused on insulin glargine (formulated as Lantus® and Toujeo®; Sanofi). Prepared at pH 4 in the presence of zinc ions, glargine exhibits a shifted isoelectric point due to a basic B chain extension (ArgB31‐ArgB32). Subcutaneous injection leads to pH‐dependent precipitation of a long‐lived depot. Pairwise substitution of CysA6 and CysA11 by selenocysteine was effected by solid‐phase peptide synthesis; the modified A chain also contained substitution of AsnA21 by Gly, circumventing acid‐catalyzed deamidation. Although chain combination of native glargine yielded negligible product, in accordance with previous synthetic studies, the pairwise selenocysteine substitution partially rescued this reaction: substantial product was obtained through repeated combination, yielding a stabilized insulin analogue. This strategy thus exploited both (a) the unique redox properties of selenocysteine in protein folding and (b) favorable packing of an internal diselenide bridge in the native state, once achieved. Such rational optimization of protein folding and stability may be generalizable to diverse disulfide‐stabilized proteins of therapeutic interest.

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Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Cited by 23 publications

References 33 publications

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Insulin related compounds and identification

Se‐Glargine: Chemical Synthesis of a Basal Insulin Analogue Stabilized by an Internal Diselenide Bridge

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