A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site

Liu, Yang; Ren, Liping; Ge, Lingmiao; Cui, Qingxin; Cao, Xiaofang; Hou, Yuanyuan; Bai, Fang; Bai, Gang

doi:10.1007/s10529-014-1526-1

Cited by 10 publications

(4 citation statements)

References 14 publications

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“…As noted above, hESCs have a particularly long S phase, however, the half‐life of the injected S 54 ‐Cre in hESCs is 7.2 hr, much shorter than the required 30 hr. One of our future goals is to extend the half‐life of the translocated S 54 ‐Cre, using such approaches as optimization of the N‐terminal amino acids sequence as they are known to have the greatest impact on the half‐lives of proteins (Gutfreund, ; Hirel, Schmitter, Dessen, Fayat, & Blanquet, ) or fusing with various stabilizing protein domains (Liu et al, ).…”

Section: Discussionmentioning

confidence: 99%

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High‐efficiency protein delivery into transfection‐recalcitrant cell types

Cai

et al. 2019

Biotech & Bioengineering

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Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS.This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.

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Section: Discussionmentioning

confidence: 99%

“…Cre, using such approaches as optimization of the N-terminal amino acids sequence as they are known to have the greatest impact on the half-lives of proteins (Gutfreund, 1993;Hirel, Schmitter, Dessen, Fayat, & Blanquet, 1989) or fusing with various stabilizing protein domains (Liu et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

High‐efficiency protein delivery into transfection‐recalcitrant cell types

Cai

et al. 2019

Biotech & Bioengineering

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“…A previous report showed that a recombinant glucagon-like peptide-1 analogue (KGLP-1), also used on diabetes therapy, was expressed in E. coli cells as a fusion protein with GST (Liu et al 2014). In this study, digestion reactions were performed to remove GST from glucagon and confirmed by Tris-Tricine gel (Figure 1C).…”

Section: Discussionmentioning

confidence: 99%

Recombinant glucagon: a differential biological activity

et al. 2015

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In Brazil, there is a growing demand for specialised pharmaceuticals, and the high cost of their importation results in increasing costs, reaching US$ 1.34 billion in 2012 and US$ 1.61 billion in 2013. Worldwide expenses related to drugs could reach US$ 1.3 trillion in 2018, especially due to new treatments for hepatitis C and cancer. Specialised or high-cost pharmaceutical drugs used for the treatment of viral hepatitis, multiple sclerosis, HIV and diabetes are distributed free of charge by the Brazilian government. The glucagon peptide was included in this group of high-cost biopharmaceuticals in 2008. Although its main application is the treatment of hypoglycaemia in diabetic patients, it can also be used with patients in an alcoholic coma, for those patients with biliary tract pain, and as a bronchodilator. Therefore, in order to reduce biopharmaceutical production costs, the Brazilian government passed laws focusing on the development and increase of a National Pharmaceutical Industrial Centre, including the demand for the national production of glucagon. For that reason and given the importance and high cost of recombinant glucagon, the purpose of this study was to develop methods to improve production, purification and performance of the biological activity of recombinant glucagon. Glucagon was recombined into a plasmid vector containing a Glutathione S-transferase tag, and the peptide was expressed in a heterologous Escherichia coli system. After purification procedures and molecular analyses, the biological activity of this recombinant glucagon was examined using in vivo assays and showed a highly significant (p < 0.00001) and prolonged effect on glucose levels when compared with the standard glucagon. The experimental procedure described here facilitates the high level production of recombinant glucagon with an extended biological activity.

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“…Additionally, small peptides are difficult to obtain by conventional bioexpression and purification systems due to their high susceptibility to fast degradation. Generally, conjugating peptides with an appropriate partner protein significantly improves the level of expression and their pharmacokinetic profiles [ 13 , 29 – 32 ]. Therefore, we developed a novel strategy of liraglutide precursor peptide (LPP) production via forming a fusion construct using human αB-crystallin (αB-Cry), as the partner protein (αB-lir).…”

Section: Introductionmentioning

confidence: 99%

A novel strategy for production of liraglutide precursor peptide and development of a new long-acting incretin mimic

et al. 2022

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Nowadays, a small number of incretin mimics are used to treat type 2 diabetes mellitus (T2DM) due to their longer half-life. The present study aimed to introduce a novel method for producing the liraglutide precursor peptide (LPP) and developing a potentially new incretin mimic. Here, human αB-crystallin (αB-Cry) was ligated to the LPP at the gene level, and the gene construct was expressed in Escherichia coli with a relatively good efficiency. The hybrid protein (αB-lir) was then purified by a precipitation method followed by anion exchange chromatography. After that, the peptide was released from the carrier protein by a chemical cleavage method yielding about 70%. The LPP was then purified by gel filtration chromatography, and HPLC estimated its purity to be about 98%. Also, the molecular mass of the purified peptide was finally confirmed by mass spectroscopy analysis. Assessment of the secondary structures suggested a dominant α-helical structure for the LPP and a β-sheet rich structure for the hybrid protein. The subcutaneous injection of the LPP and the αB-lir hybrid protein significantly reduced the blood sugar levels in healthy and diabetic mice and stimulated insulin secretion. Also, the hybrid protein exerts its bioactivities more effectively than the LPP over a relatively longer period of time. The results of this study suggested a novel method for the easy and cost-effective production of the LPP and introduced a new long-acting incretin mimic that can be potentially used for the treatment of T2DM patients.

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A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site

Cited by 10 publications

References 14 publications

High‐efficiency protein delivery into transfection‐recalcitrant cell types

High‐efficiency protein delivery into transfection‐recalcitrant cell types

Recombinant glucagon: a differential biological activity

A novel strategy for production of liraglutide precursor peptide and development of a new long-acting incretin mimic

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