• Biotechnology and Bioprocess Engineering:BBE
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• v.2 no.2
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• pp.86-89
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• 1997

Epidermal growth factor(EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6$\times$His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], an Escherichia coli host strain, in amount of 30-40% of total proteins pressent in E. coli extract by the addition of isopropylthio-$\beta$-galactopyranoside (IPTG). The recombinant EGF purified using a Ni2+-NTA affinity colume chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.559-568
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• 2020

Epidermal growth factor (EGF) is a hormone protein that affects cell growth and proliferation, and has various medical applications. In the present study, the gene of human EGF was codon-optimized with E. coli and the expression vector was constructed by cloning into pRSET. In order to obtain the recombinant human EGF in an active form rather than an inclusion body, chaperone co-expression was attempted along with codon optimization, for the first time. The expressed human EGF was isolated in the pure form by performing Ion Exchange Chromatography in two consecutive runs. ELISA analysis showed that the activity of purified EGF was greater than 99%, which is similar to commercially available EGF. Cell proliferation test confirmed that the recombinant human EGF has the ability to promote cell proliferation of human skin fibroblasts. The human EGF expression system of this study gives a significant amount of protein, and does not require the renaturation step and the additional chromatographic system to remove a fusion contaminant, thereby providing a very useful alternative to conventional expression systems for the preparation of recombinant human EGF.

• Archives of Pharmacal Research
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• v.24 no.4
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• pp.355-359
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• 2001

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.

• Archives of Pharmacal Research
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• v.26 no.8
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• pp.649-652
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• 2003

Polymeric nanoparticles composed of polystyrene (PS) as core and poly(methacrylic acid) (PMA) as corona were prepared by the dispersion copolymerization. The potential of the nanoparticles as carriers for recombinant human epidermal growth factor (EGF) was investigated. The nanoparticles showed monodispersity and good water-dispersibility. The loading content of EGF to the nanoparticles was very high due to electrostatic interaction between EGF and nanoparticles. EGF was released as a pseudo-zero order pattern after initial burst effect. The nanoparticles were sufficient for A431 cells proliferation.

• Archives of Pharmacal Research
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• v.20 no.1
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• pp.34-38
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• 1997

Recombinant human epidermal growth factor (rhEGF), a polypeptide of 53 amino acid residues, is subject to degradation by numerous enzymes, especially proteases, when it is applied on the skin for the treatment of open wound. Amastatin, aprotinin, bestatin, EDTA, EGTA, gabexate, gentamicin, leupeptin, and TPCK were investigated for the possible protease inhibitors, which may use to protect rhEGF from degradation by the enzymes in the skin. Skin homogenates containing protease inhibitors and rhEGF were incubated at $37^{\circ}C$ for 30 minutes. After the reaction was stopped with trifluoroacetic acid, the amount of rhEGF remaining in the sample was determined with an HPLC method. The percentages of rhEGF degraded, at the skin/PBS ratio of 0.25, in the mouse, rat, and human skin homogenate were 85%, 70%, and 46%, respectively. The degree of degradation of rhEGF in the cytosolic fraction was higher than that in the membrane fraction and these enzyme reactions were completed in 30 minutes. Bestatin, EGTA, and TPCK showed significant inhibitory effects on the degradation of rhEGF in the two fractions (p<0.05), while the other protease inhibitors had no significant inhibitory effects or, even resulted in deleterious effects. Therefore, the formulation containing one or several inhibitors among these effective inhibitors would be a promising topical preparation of rhEGF for the treatment of open wound.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.478-484
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• 1996

Recombinant human epidermal growth factor (rhEGF) was produced by E. coli BL21 harboring a plasmid pYHB101. The maximum production was 68.7 mg/l when the E. coli strain was cultured at 25$\circ$C for 48 hours in the modified MBL medium containing 10 g/l glucose with 1 mM IPTG induction at 2 hours after inoculation. The rhEGF was purified upto 267 folds by Amberlite XAD- 7 chromatography, ultrafiltration, and DEAE Sepharose fast flow ion exchange chromatography with an overall yield of 66.6%. The purified rhEGF was further separated into two fractions by HPLC. The N-terminal amino acid sequence of the second fraction was Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His. The effect of rhEGF on the DNA synthesis was examined using in vitro biological assay based on the incorporation of 5'-bromo-2'- deoxy-uridine (BrdU). The purified rhEGF shows no difference with natural human epidermal growth factor (nhEGF) in N-terminal amino acids residues and biological activity. From the results, we concluded that rhEGF produced from E. coli harboring the plasmid pYHB101 was apparently the same as nhEGF.

• Radiation Oncology Journal
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• v.24 no.3
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• pp.179-184
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• 2006

[ $\underline{Purpose}$ ]: To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. $\underline{Materials\;and\;Methods}$: Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. $\underline{Results}$: Number of fibroblast was significantly more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. $\underline{Conclusion}$: rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing.

• Korean Chemical Engineering Research
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• v.56 no.5
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• pp.711-717
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• 2018

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. Since the protein contains three intra-molecular disulfide bonds, the high expression of active hEGF in Escherichia coli has not been well researched, We fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in E. coli (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF with IPTG (Isopropyl-${\beta}$-D-Thiogalactopyranoside), was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.61-67
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• 1998

Recombinant human epidermal growth factor (rhEGF) was produced by E. coli BL2l (DE3) harboring a plasmid pYHB101. The production of rhEGF was 44.5 mg/L when the E. coli BL2l (pYHB101) was cultured at 27$^{\circ}C$ for 48 hr in the modified MBL medium containing 10 $\mu\textrm{g}$/L glucose with 10 $\mu\textrm{m}$ IPTG/lactose induction at 2 hr after inoculation. It was shown that lactose is able to induce the rhEGF expression of E. coli BL2l (pYHB101) with the same efficiency as IPTG. In the batch culture system, when induced with 10 $\mu\textrm{m}$ lactose, E. coli BL2l (pYHB101) produced maximum 45 mg/L of the rhEGF at 28 hr culture in the modified MBL medium containing 10 g/L glucose. In the semi-fed batch culture system, the volumetric yield was 160 mg/L when the culture was added with 0.5% (w/v) lactose and 0.25% (w/v) yeast extract in the late logarithmic phase and 94.3% of rhEGF was secreted as soluble form. However, when the culture was added with them in the early logarithmic phase, the volumetric yield was 120 mg/L and 20.9% of rhEGF was found in cytoplasmic insoluble aggregates. It was found that the addition time of lactose was important for production of soluble rhEGF from E. coli BL21 (pYHB101).

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.415.3-416
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• 2002

To improve the stability of recombinant human epidermal growth factor (rhEGF) as therapeutic agent. the N-terminal PEGylated rhEGF (N-PEG-rhEGF) was prepared by site-specific bioconjugation and the stability was investigated in rat skin wound homogenates. Two different N-PEG-rhGEFs (N-PEG5K- and N-PEG20K-rhEGF) were successfully prepared with the yields of above 70%. The PEGylation site was directly confirmed by determining the molecular mass of Lys-C digested samples using MALDI- TOF MS. (omitted)