• 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).

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.493-501
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• 1998

The most significant direct role of estrogen in vivo is its ability to elicit receptor-mediated cellular proliferation in mammalian target tissues. However, the mechanism by which exogenously added estrogen causes the neoplastic transformation of renal cortical cells is yet to be uncovered. The present study was designed to evaluate interaction of $17{\beta}-estradiol\;(E_2)$ with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) on proliferation and $P_i$ uptake in primary cultured rabbit renal proximal tubular cells in phenol red-free, hormonally defined-medium. $[^3H]-thymidine$ incorporation increased markedly by about 133% and 141% more in the presence of $10^{-9}\;and\;10^{-6}\;M\;E_2$, respectively, than that of control. Cell count was 162% and 143% greater in the presence of $10^{-9}\;and\;10^{-6}\;M\;E_2$ , respectively, compared with control. Among all time points examined, there was an increase in $[^3H]-thymidine$ incorporation in the presence of $10^{-9}\;M\;E_2$ at day 9 or 13, respectively. However, $E_2$ ($10^{-9}\;M$) significantly drove up cell count to 160% of that of control at day 13, while it had a slight but statistically insignificant effect at day 9. $E_2-induced$ stimulation of $[^3H]-thymidine$ incorporation was completely reversed by $E_2$ antagonists (progesterone or tamoxifen). $E_2$ ($10^{-9}\;M$) or EGF ($10^{-8}\;M$) significantly stimulated $[^3H]-thymidine$ incorporation by 144% and 154% of control. $E_2$ plus EGF was synergistic on $[^3H]-thymidine$ incorporation (204% of control), while $E_2$ plus IGF-I showed a slight but no significant synergistic effect. Cell number also displayed similar pattern. $E_2$ ($10^{-9}\;M$) significantly stimulated $P_i$ uptake to 134% of control. $E_2$-induced stimulation of $P_i$ uptake was partially reversed by $E_2$ antagonists. EGF or IGF-I ($10^{-8}\;M$) significantly also increased $P_i$ uptake to 132% or 129% of control. $E_2$ plus EGF had synergistic effect on $P_i$ uptake, while $E_2$ plus IGF-I did not. In conclusion, $E_2$ may act not only directly interaction with its receptors but also indirectly as a modulator of EGF in proliferation and $P_i$ uptake of primary cultured rabbit renal proximal tubular cells.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.29-34
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• 2006

본 연구는 개 체외성숙 난자를 안정적으로 생산하기 위하여 채취시기, 난구세포 부착 여부 및 배양액에 EGF와 호르몬을 첨가 후 배양했을 때 체외성숙율에 미치는 영향을 조사하였다. 1. 미성숙 난포란을 TCM-199 배양액에서 24, 45시간 배양했을 때 체외성숙율은 각각 7.93%, 8.94%로서 48시간 배양했을 때 가장 높은 체외성숙율을 나타냈다. 2. 휴지기, 난포기, 황체기에 채취한 난소로부터 회수한 난자를 20 ng/ml의 EGF가 첨가된 TCM-199 배양액에서 배양했을 때 체외성숙율은 14.3%로서 0, 10 ng/ml의 EGF 첨가군(3.1%, 7.5%)에 비해 높은 체외성숙율을 나타냈다. 3. 난구세포 부착 및 미부착 난자를 48시간 배양했을 때 체외성숙율은 각각 18.8% 및 7.5%로서 난구세포 부착 난자가 미부착 난자보다 높은 체외성숙율을 나타냈다. 4. 난자의 체외성숙 배양 시 0.5 mg/ml FSH, 5 mg/ml LH, 1 mg/ml $E_2$와 FSH+LH, $FSH+LH+E_2$를 첨가한 TCM-199 배양액에서 배양했을 때 체외성숙율은 각각 1.2%, 10.0%, 2.0%와 10.0%, 31.2%로서 호르몬의 병용처리군이 높은 체외성숙율을 나타냈다. 5. 난자의 체외성숙 배양 시 EGF와 FSH, LH, $E_2$ 및 EGF와 FSH+LH, $FSH+LH+E_2$를 첨가한 TCM-199 배양액에서 배양했을 때 체외성숙율은 32.3%, 27.0%, 3.0%와 36.2%, 69.4%로서 EGF와 호르몬 병용 처리군이 높은 체외성숙율을 나타냈다.

• 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.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.33-39
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• 1996

This objective of this experiment was to test the effect of EGF on GVBD and MII of nuclear maturation of pig immature oocytes in vitro. Experiment 1 examined to the effect of EGF on nuclear maturation of pig immature oocytes according to different maturational times. The percentage of GVBD of EGF 10mg/ml treated groups were significantly higher than untreated groups after 24hr (p < 0.001). Experiment 2 examined to the effect of duration of exposure of oocytes to EGF supplement in maturation medium. Nuclear maturation rates (M II) of EGF treated groups (during 0-24: 72.8% and 0-42hr: 84.8%) were significantly higher than 53.5 and 26.1% of EGF treated group (during 24-42hr) and untreated group (p < 0.001). Also, experiment 3 examined to the effect of EGF on nuclear maturation of CEOs or CFOs. Nuclear maturation rate (M II) 84.6% of EGF treated group of CEOs was significantly higher than 53.0, 27.6, and 44.2% of EGF treated group of CFOs and untreated groups of CEOs and CFOs (p < O.001). These results conclude that EGF alone can stimulate GVBD and M II of nuclear maturation in pig immature oocytes.

• Journal of Life Science
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• v.11 no.1
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• pp.22-26
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• 2001

In estrogen-dependent MCF-7 human breast cancer cells, $E_2$ at 10 nM stimulated cell proliferation to over 200% compared to the untreated control. EGF and TGF${\alpha}$, which are known as the autocrine/paracrine growth factors induced by $E_2$, also directly stimulated the cell growth in almost as the same extent as $E_2$. DFMO which is the specific inhibitor of ODC could inhibit cell growth even at as low as 0.5 mM. In the treatment with 1 mM DFMO for 4 days, the cell growth was inhibited to 38% of the control. HO-TAM at 1 ${\mu}$M could inhibit the proliferation of MCF-7 cells to 19% of the control. Those inhibitory effects were also found in the cells stimulated with $E_2$, EGF, and TGF${\alpha}$. The inhibitory effects were found even in 2 days of treatment. However, $E_2$, EGF, and TGF${\alpha}$ did not give any effect in the protein synthesis. Neither DFMO or HO-TAM gave any effect on the total protein synthesis. But the pattern of protein secretion was noticeably influenced by the growth stimulants or inhibitors. Proteins of 160, 52, 42, 36, and 32 kDa belonged to the major secretory proteins. Especially, 42 and 36 kDa proteins were most significantly influenced by the treatment of $E_2$, EGF, or TGF$\alpha$. DFMO and HO-TAM inhibited the secretion of these major proteins.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.22 no.6
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• pp.542-549
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• 2021

The purpose of this study was to provide an economical and easy preparation method for recombinant human epidermal growth factor (rhEGF) without the need for an expensive enzyme to cleave the fusion part. However, the N-terminal fusion part is still useful for affinity chromatography. The hEGF is an important hormone in cell growth and proliferation in humans, and many studies on the expression and purification of this protein have been reported. In the present study, the hEGF gene was designed to be optimized with the E. coli codon usage preference and to contain Asn-Gly at the N-terminus of the protein. The gene was inserted into pRSET_A, an E. coli expression vector, and transformed into E. coli BL21 (DE3). The recombinant fusion protein was successfully co-expressed with pG-Tf2, a chaperone vector, in E. coli and purified by Ni-NTA column chromatography. The rhEGF was then released by hydroxylamine treatment and confirmed by SDS-PAGE. ELISA analysis showed that the activity of the free rhEGF was more than 92% similar to that of commercial EGF. The biological activity of the rhEGF was confirmed by a cell proliferation test with human skin fibroblasts.

• The korean journal of orthodontics
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• v.30 no.3 s.80
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• pp.317-333
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• 2000

The purpose of this study was to determine the proliferative activity of the osteoblasts and fibroblasts in the midpalatal area and to investigate the adjacent periodontal tissues of individual tooth following rapid expansion of the palate. Ten young adult dogs, aged approximately ten months, were used in the experiment. The experimental design was consisted of 1 week expansion group(Group E1, 3 dogs), 2 week expansion group(Group E2, 3 dogs), 2 week expansion and 2 week retention group(Group E3, 3 dogs), and control group(Group C, 1 dog). For each group, expansion screw was activated one time per day(1/4 turn;$90^{\circ}$) following Hyrax-screw application. The experimental animals in each group were sacrificed at 1, 2 and 4 weeks following palatal expansion. Maxillary tissue blocks were obtained and prepared ior the histomorphologic and immunohistochemical studies. Light mcroscope, polarizing microscope, and soft X-ray apparatus were used in this study, and following results were obtained. 1. In polarizing microscopic study, the expansion groups(E1 & E2) showed blue color representing bone resorption and new bone formation in midpalatal suture area. E3 groups skewed less blue color compared to the E1 and E2 group. But yellow color increased by calcification in the E3 groups. 2. Immunohistochemical study revealed that positive responses of the osteoblasts to PCNA and undifferentiated fibroblasts to EGF in E1 group were somewhat increased. Positive response to PCNA and EGF were increased in fibroblasts and the osteoblasts forming new bone in E2 group. In E3 group, the positive response cell concentrated the periphery of edge of palatal process in both PCNA and EGF. 3. Throughout the expansion period(E1 & E2), light microscopic study showed the edges of the extensive resorption and new palatal processes, indicating bone remodeling within the suture. E3 group exhibited less remodeling of midpalatal suture area. E2 group and E3 group showed cementum formation and resorption at the apex of 3rd premolar and 1st molar E3 group exhibited extensive hyalinized zone on the cervical portion of buccal side of 1st molar. 4. Soft X-ray analysis of E1 group showed hypomineralized defect and microfractures in various parts of the suture areas when compared with control animals. There was no significant difference in the degree of mineralization in the midpalatal suture region between the C and E3 groups. Tooth axis showed tipping of 3rd premolar and 1st molar in the E2 group and E3 group. Based upon these experimental results, it is concluded that the undifferentiated mesenchymal cells always presented in midpalatal suture area following RPE. Differentiated osteoblasts and fibroblasts possess proliferating cellular activity until the 2 week retention period. The posterior teeth are tend to tip buccally as RPE force applied. Retention group exhibited irreversible response with severe hyalinized zone on the buccal surface of the first molar.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.13-20
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• 1997

The objective of this study was to determine the effect of EGF on the preimplantation development of mouse IVF embryos and their ICM and TE cell number. And also, we examined the expression of EGF-R protein on embryonic development by indirect immunofluorescence. The results obtained in these experiments were summarized as follows; Group culture (5 embryos/ 25 ${\mu}l$) showed more improved development rate to blastocyst than singly culture. This inferior development of singly cultured 2-cell embryos improved by the addition of EGF. Especially, 2-cell embryos cultured singly in 10 ng/ml of EGF (62.4%) indicated significant difference in development to blastocyst compared with control group (47.9%). Also, cell number of ICM and TE by differential labelling showed the increased pattern in the EGF treatment group. The stimulating effect of EGF with the development level was significantly increased after 4-cell stage (p<0.05). ICM proportion also showed the increased pattern with the developmental level in the EGF treatment group. In addition, expression of EGF-R by indirect immunofluorescence detected after 4-cell stage. Therefore, EGF could stimulate preimplantation mouse embryo development by binding with expressed EGE-R after 4-cell stage and produce the more increased ICM and TE cell number of blastocyst.