Expression and purification of human epidermal growth factor (hEGF) fused with GB1

Zheng, Xueming; Wu, Xin; Fu, Xingli; Dai, David Yun; Wang, Feihu

doi:10.1080/13102818.2016.1166984

Cited by 21 publications

(22 citation statements)

References 21 publications

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“…Fusi dengan protein lain yang dapat meningkatkan kelarutannya dan menambah bobot molekul diperlukan untuk meningkatkan keberhasilan ekspresi rh-EGF. rh-EGF pernah diekspresikan dengan bantuan fusi protein SUMO (Ma et al 2016), GB1 (Zheng et al 2016), dan TrpE (Allen et al 1985 sehingga kelarutannya meningkat dalam sitoplasma E. coli. rh-EGF_SUMO dan rh-EGF_GB1 diekspresikan dengan induksi IPTG, masingmasing 0,6 mM dan 0,2 mM, sedangkan rh-EGF _TrpE diekspresikan dengan induksi asam akrilik indol.…”

Section: Pendahuluanunclassified

Optimasi Ekspresi Human Epidermal Growth Factor (h-EGF) Rekombinan dalam Escherichia coli BL21(DE3) dengan Variasi Media dan Konsentrasi Penginduksi

Pratiwi¹

2019

Chim. Nat. Acta

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Epidermal growth factor (EGF) merupakan protein yang sangat penting dalam proliferasi, diferensiasi, dan pematangan sel. EGF sintetik atau recombinant human EGF (rh-EGF) telah digunakan secara klinis untuk mengatasi kondisi kekurangan EGF alami seperti mempercepat pemulihan luka pada penderita diabetes dan teravaksin untuk pengobatan kanker paru. Ekspresi rh-EGF dilaporkan tidak mudah karena protein ini sensitif terhadap proteolisis intraselular. Kondisi yang optimal sangat diperlukan untuk ekpsresi rh-EGF agar diperoleh protein dalam jumlah yang tinggi. Hingga saat ini, sejumlah studi ekspresi rh-EGF telah dilaporkan, namun harus difusi dengan protein peningkat solubilitas. Pada penelitian ini, rh-EGF berhasil diekspresikan, meskipun tanpa protein peningkat solubilitas. Ekspresi rh-EGF dikontrol oleh promotor T7 yang tergantung pada isopropil β-D-1-tiogalaktopiranosid (IPTG). Konsentrasi IPTG yang optimal menentukan keberhasilkan ekspresi protein yang diinduksinya. Ekspresi rh-EGF dilakukan dalam dua media, yaitu Luria Bertani (LB) dan Terrific Broth (TB) dengan kondisi non induksi dan induksi IPTG konsentrasi 0,1 mM; 0,5 mM; dan 1 mM. Lama induksi juga divariasikan, yakni 3 dan 6 jam. Konsentrasi rh-EGF tertinggi (0,21 mg/ml) diperoleh dengan ekspresi menggunakan TB yang induksi IPTG 0,5 mM selama 6 jam. Kata kunci: recombinant human epidermal growth factor, induksi IPTG, pET21b(+), Escherichia coli BL21(DE3)

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Section: Pendahuluanunclassified

Optimasi Ekspresi Human Epidermal Growth Factor (h-EGF) Rekombinan dalam Escherichia coli BL21(DE3) dengan Variasi Media dan Konsentrasi Penginduksi

Pratiwi¹

2019

Chim. Nat. Acta

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“…hEGF plays a role in stimulating cell proliferation, differentiation, and migration in the wound-healing process. This has led to the high demand for hEGF in the clinical field, thus encouraging efforts to increase hEGF production through recombinant DNA technology (Ma et al, 2016;Zheng et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Expression of human epidermal growth factor using Ssp DnaB mini-intein as fusion partner in Escherichia coli BL21(DE3)

Yosua¹,

Rima²,

Sriwidodo³

et al. 2021

J Appl Pharm Sci

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The human epidermal growth factor (hEGF) is widely used clinically as a wound healer, as it has a vital role in stimulating cell proliferation, differentiation, and migration. Consequently, the large-scale production of recombinant hEGF in E. coli has been developed to meet the high demand for hEGF clinically. However, intracellular proteins in E. coli, especially small proteins like hEGF, are degraded by proteases. To overcome this issue, hEGF was fused with CBD-Ssp DnaB to construct a fusion protein CBD-Ssp DnaB-hEGF. This study was conducted to obtain refolded hEGF from the inclusion bodies of CBD-Ssp DnaB-hEGF. The experiment was carried out using E. coli BL21(DE3) containing plasmid pD861-CBD-Ssp DnaB-hEGF. The CBD-Ssp DnaB-hEGF gene was constructed by fusing CBD-Ssp DnaB and hEGF gene and then was optimized. The method was started with E. coli transformation, CBD-Ssp DnaB-hEGF expression, inclusion bodies solubilization, refolding, and simultaneous cleavage to release hEGF. The CBD-Ssp DnaB-hEGF was expressed as inclusion bodies, which can then be purified by washing with Triton X-100 and 1 M urea. The inclusion bodies were solubilized in 8 M urea, the solubilized CBD-Ssp DnaB-hEGF was reformed by dialysis, and then hEGF was spliced by shifting the pH from 8.5 to 6.0 to yield a concentration of 0.163 mg/ml. Therefore, we concluded that hEGF was obtained from the solubilized CBD-Ssp DnaB-hEGF from inclusion bodies produced by E. coli BL21(DE3).

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“…[23][24][25][26][27][28] One way to produce recombinant hEGF with three disulfide bonds that perfectly fold is by using Escherichia coli (E. coli) with the PelB signal peptide. [29][30][31] The use of E. coli is also beneficial because recombinant hEGF is secreted directly into the periplasmic space and does not mix with cytoplasmic components, making it easy to purify and streamline production costs. 29,[32][33][34] In this study, the hEGF used was obtained based on the results of optimization in previous studies using E. coli BL21 (DE3) as a recombinant vector.…”

Section: Introductionmentioning

confidence: 99%

<p>Activity and Effectiveness of Recombinant hEGF Excreted by <em>Escherichia coli</em> BL21 on Wound Healing in Induced Diabetic Mice</p>

Sriwidodo¹,

Maksum²,

Subroto³

et al. 2020

JEP

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Context: Human epidermal growth factor (hEGF) has biological activities and can be used in medicines and cosmetics. A high level of effectiveness of hEGF can be obtained when three disulfide bonds fold perfectly. Extracellular secretion from E. coli BL21 using the PelB signal peptide is a new way to obtain hEGF with a structure that folds appropriately. Object: This study aimed to determine the activity and effectiveness of recombinant hEGF excreted by E. coli BL21 on wound healing in induced diabetic mice. Methods: Cell proliferation and migration tests were performed on NIH3T3 cells, followed by wound healing tests in induced diabetic mice, along with histological and endotoxin test at various hEGF concentrations (25, 50, and 75 µg/mL). Results: Based on the results, hEGF at a level of 50 μg/mL showed optimal proliferation and migration activities. Wound healing in induced diabetic mice showed faster-wound closure within 12 days at hEGF 50 and 75 µg/mL with a percentage wound closure of 95% and 98.5%, respectively, which was significant versus control. In the histology test, the number of fibroblasts showed an increase and was significant at hEGF 75 µg/mL compared to the control group. The single test vial (STV) showed that hEGF solution was free of endotoxin. Conclusion: Recombinant hEGF produced by extracellular secretion using E. coli BL21 has optimal diabetic wound healing activity through increased fibroblast proliferation.

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Expression and purification of human epidermal growth factor (hEGF) fused with GB1

Cited by 21 publications

References 21 publications

Optimasi Ekspresi Human Epidermal Growth Factor (h-EGF) Rekombinan dalam Escherichia coli BL21(DE3) dengan Variasi Media dan Konsentrasi Penginduksi

Optimasi Ekspresi Human Epidermal Growth Factor (h-EGF) Rekombinan dalam Escherichia coli BL21(DE3) dengan Variasi Media dan Konsentrasi Penginduksi

Expression of human epidermal growth factor using Ssp DnaB mini-intein as fusion partner in Escherichia coli BL21(DE3)

<p>Activity and Effectiveness of Recombinant hEGF Excreted by <em>Escherichia coli</em> BL21 on Wound Healing in Induced Diabetic Mice</p>

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