Production and purification of single chain human insulin precursors with various fusion peptides

Cho, Chung Woo; Park, Sun Ho; Nam, Doo Hyun

doi:10.1007/bf02931961

Cited by 7 publications

(3 citation statements)

References 16 publications

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“…In spite of its drawbacks, the inclusion body strategy for high expression and low proteolysis has become the default route for the production of target protein. Fusion partners such as ␤-galactosidase, protein A, glutathione-S-transferase, the immunoglobulin binding Z-domain of protein A, His-tagged sequences with a hexahistidine, H64A subtilisin or factor Xa have been also used to increase expression levels and the stability of target protein and to facilitate the purification process (Shen, 1984;Murby et al, 1991;Kang and Yoon, 1994;Cho et al, 2001). In addition, fusion proteins can increase the solubility of the target protein, and this higher solubility can also induce higher refolding yield.…”

Section: Introductionmentioning

confidence: 99%

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

Min

Son

Kim

et al. 2011

Journal of Biotechnology

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Section: Introductionmentioning

confidence: 99%

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

Min

Son

Kim

et al. 2011

Journal of Biotechnology

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“…Subsequently, the target insulin peptides are released from the fusion protein by either chemical or enzymatic cleavage at the corresponding site of carrier-peptide junction. Choosing an appropriate fusion protein partner is very important because according to previous studies, certain fusion protein partners could greatly improve the stability and the level of expression of the target proteins [ 12 – 14 ]. In the pancreatic tissue, a single chain proinsulin molecule is produced with the A-chain (21 amino acids) and B-chain (30 amino acids) linked together by the C-peptide (31 amino acids).…”

Section: Introductionmentioning

confidence: 99%

Human αB-crystallin as fusion protein and molecular chaperone increases the expression and folding efficiency of recombinant insulin

Akbarian

Yousefi

2018

PLoS ONE

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Low expression and instability are significant challenges in the recombinant production of therapeutic peptides. The current study introduces a novel expression and purification system for human insulin production using the molecular chaperone αB-crystallin (αB-Cry) as a fusion partner protein. Insulin is composed of A- and B-chain containing three disulfide bonds (one intarchain and two interchains). We have constructed two plasmids harboring the A- or B-chain of insulin joined with human αB-Cry. This system is suitable for cloning of the genes and for directing the synthesis of large amounts of the fusion proteins αB-Cry/A-chain (αB-AC) and αB-Cry/B-chain (αB-BC). The construction of vectors, their efficient expression in Escherichia coli and simple purification of the fusion proteins and two insulin chains are described. A large amount of the recombinant fusion proteins with high purity was obtained by applying a single step anion exchange chromatography or metal chelate affinity. The insulin A- and B-chain were released from the fusion proteins using cyanogen bromide cleavage. The insulin peptides were obtained with an appreciable yield and high purity using one-step gel filtration chromatography. To increase efficiency of chain combination to produce insulin, αB-Cry was used under oxidative conditions. The purification of natively folded insulin was performed by phenyl sepharose hydrophobic interaction chromatography. Finally, using an insulin tolerance test in mice and various biophysical methods, the structure and function of purified human recombinant insulin was compared with authentic insulin, to verify folding of insulin to its native state. Overall, the novel expression system using αB-Cry is highly demanding for producing human insulin and functional protein. The procedure for αB-Cry-mediated insulin folding could be also applicable for the large-scale production of this highly important therapeutic peptide hormone.

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“…One of the methods for increasing the human insulin expression yield and promoting efficient purification is using a fusion partner during human insulin expression [26][27][28]. 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].…”

Section: Hplc Profile Of Ircs From H27r Strainmentioning

confidence: 99%

Insulin related compounds and identification

Min

Lee

Kim

et al. 2012

Journal of Chromatography B

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Production and purification of single chain human insulin precursors with various fusion peptides

Cited by 7 publications

References 16 publications

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

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

Human αB-crystallin as fusion protein and molecular chaperone increases the expression and folding efficiency of recombinant insulin

Insulin related compounds and identification

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