Optimization of secreted recombinant human epidermal growth factor production using pectate lyase B from Escherichia coli BL21(DE3) by central composite design and its production in high cell density culture

Sriwidodo, Sriwidodo; Subroto, Toto; Maksum, Iman Permana; Wathoni, Nasrul; Rostinawati, Tina; Ulya, Himmatul; Putri, Indah U

doi:10.4103/jpbs.jpbs_207_19

Cited by 14 publications

(9 citation statements)

References 5 publications

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“…The material used in this study is two bags of blood samples (Palang Merah Indonesia [PMI]), sucrose (Sigma Aldrich, USA), physiological NaCl (Sigma Aldrich, USA), aquadest (IKA), recombinant hEGF from the previous study,[ 8 14 15 16 ] sodium pyrosulfite (Sigma Aldrich, USA), Vitamin E (Arrow Pharm), tween 80 (Sigma Aldrich, USA), fluid thioglycolate medium (FTM) (Sigma Aldrich, USA), tryptone soya broth (TSB) (Sigma Aldrich, USA), limulus amebocyte lysate (LAL) (Pyrotell, Cape Cod Inc., East Falmouth, USA), NIH3T3 fibroblast cell (ATCC ® CRL-1658™, ATCC Inc., Manassas, Virginia), and WST-8 Reagent (Dojindo Molecular Technologies Inc., USA).…”

Section: Methodsmentioning

confidence: 99%

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Stabilization of eye drops containing autologous serum and recombinant human epidermal growth factor for dry eye syndrome

Sriwidodo

Syah

Maksum

et al. 2020

J Adv Pharm Technol Res

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Section: Methodsmentioning

confidence: 99%

“…The hEGF used is a recombinant product using Escherichia coli (BL21) obtained from the previous studies. [ 8 14 15 16 ]…”

Section: Introductionmentioning

confidence: 99%

Stabilization of eye drops containing autologous serum and recombinant human epidermal growth factor for dry eye syndrome

Sriwidodo

Syah

Maksum

et al. 2020

J Adv Pharm Technol Res

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“…Human epidermal growth factor (hEGF) is a protein with 53 amino acid residues with 6.2 kDa of molecular weight. This polypeptide has three intramolecular disulfide bonds and heat resistance properties (Eissazadeh et al, 2017;Sriwidodo et al, 2019;Tang et al, 2016). hEGF plays a role in stimulating cell proliferation, differentiation, and migration in the wound-healing process.…”

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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“…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. 31,[35][36][37][38] The total levels of hEGF produced reached 416 μg/mL. However, the activity and effectiveness of the obtained hEGF on diabetic wound healing was not yet known.…”

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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Optimization of secreted recombinant human epidermal growth factor production using pectate lyase B from Escherichia coli BL21(DE3) by central composite design and its production in high cell density culture

Cited by 14 publications

References 5 publications

Stabilization of eye drops containing autologous serum and recombinant human epidermal growth factor for dry eye syndrome

Stabilization of eye drops containing autologous serum and recombinant human epidermal growth factor for dry eye syndrome

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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