Expression and purification of recombinant human insulin from E. coli 20 strain

Zieliński, Marcin; Romanik-Chruścielewska, Agnieszka; Mikiewicz, Diana; Łukasiewicz, Natalia; Sokołowska, Iwona; Antosik, Jarosław; Sobolewska-Ruta, Agnieszka; Bierczyńska-Krzysik, Anna; Zaleski, Piotr; Płucienniczak, Andrzej

doi:10.1016/j.pep.2019.02.002

Cited by 30 publications

(27 citation statements)

References 6 publications

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“…In this study, the protein was expressed mainly in the soluble fraction; therefore, the inclusion body recovery and renaturation of the protein steps were eliminated, which decreased the expenses associated with these steps. Therefore, the method of human insulin biosynthesis employed in this study is a more efficient process than the current methods (Zieliński et al 2019).…”

Section: Discussionmentioning

confidence: 99%

“…The extraction of recombinant biosynthesised proteins from inclusion bodies usually results in low yields of the bioactive protein, and this process is laborious (Singh et al 2015). Inclusion bodies require downstream processing such as isolation from the cell, solubilisation, purification, and refolding of the protein (Redwan et al 2007;Singh et al 2015;Zieliński et al 2019). In this study, the protein was expressed mainly in the soluble fraction; therefore, the inclusion body recovery and renaturation of the protein steps were eliminated, which decreased the expenses associated with these steps.…”

Section: Discussionmentioning

confidence: 99%

“…The proinsulin biosynthesis strategy of human insulin is the preferred method. However, this procedure results in the formation of inclusion bodies and requires the use of enzymes such as carboxypeptidase B to remove the C-peptide (Redwan et al 2007;Zieliński et al 2019) and the use of affinity tags (Redwan et al 2007). The inclusion body formation requires additional steps to be taken, such as the renaturation of the protein (Redwan et al 2007;Zieliński et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…However, this procedure results in the formation of inclusion bodies and requires the use of enzymes such as carboxypeptidase B to remove the C-peptide (Redwan et al 2007;Zieliński et al 2019) and the use of affinity tags (Redwan et al 2007). The inclusion body formation requires additional steps to be taken, such as the renaturation of the protein (Redwan et al 2007;Zieliński et al 2019). This study aimed to create a novel and more efficient method for the biosynthesis of human insulin employing a polymerase chain reaction (PCR)-based strategy using the pET21b expression vector in E. coli for the optimisation of human insulin expression.…”

Section: Introductionmentioning

confidence: 99%

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A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

et al. 2020

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Insulin has captured researchers' attention worldwide. There is a rapid global rise in the number of diabetic patients, which increases the demand for insulin. Current methods of insulin production are expensive and time-consuming. A PCR-based strategy was employed for the cloning and verification of human insulin. The human insulin protein was then overexpressed in E. coli on a laboratory scale. Thereafter, optimisation of human insulin expression was conducted. The yield of human insulin produced was approximately 520.92 (mg/L), located in the intracellular fraction. Human insulin was detected using the MALDI-TOF-MS and LC-MS methods. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of human insulin was tested in vitro using a MTT assay, which revealed that the crude biosynthesised human insulin displayed a similar degree of efficacy to the standard human insulin. This study eliminated the use of affinity tags since an untagged pET21b expression vector was employed. Tedious protein renaturation, inclusion body recovery steps, and the expensive enzymatic cleavage of the C-peptide of insulin were eliminated, thereby making this method of biosynthesising human insulin a novel and more efficient method.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

et al. 2020

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“…Moreover, every protein needs a specific purification method appropriate to its structure. We have developed a unique and effective method for protein purification that allows to obtain a desired protein with a high purity [26]. Based on our experience, we are able to adjust the individual method of protein purification for various heterologous proteins.…”

Section: Discussionmentioning

confidence: 99%

Novel Expression Vectors Based on the pIGDM1 Plasmid

et al. 2019

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Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIG-DM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIG-DMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals. Keywords Expression vectors • Recombinant protein production • Bacterial expression system • E. coli Abbreviations bla gene AMPICILLIN RESISTANCE GENE cat gene Chloramphenicol resistance gene kan gene Kanamycin resistance gene MCS Multiple cloning site SD Shine Dalgarno sequence IPTG Isopropyl ß-D-1-thiogalactopyranoside CPH The yeast cyclophilin gene IFNA13 The interferon alpha 13 gene Ubi HGH: ubiquitin-human growth hormone fusion gene

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Inclusion Bodies: Status Quo and Perspectives

Kopp

Spadiut

2023

Methods in Molecular Biology

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Expression and purification of recombinant human insulin from E. coli 20 strain

Cited by 30 publications

References 6 publications

A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

Novel Expression Vectors Based on the pIGDM1 Plasmid

Inclusion Bodies: Status Quo and Perspectives

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