• 한국산학기술학회논문지
• /
• 제21권9호
• /
• pp.559-568
• /
• 2020

세포 분열 및 성장 촉진에 영향을 주는 상피세포 성장인자(Epidermal Growth Factor, EGF)는 다양한 의학적 용도를 갖고 있는 호르몬 단백질이다. 본 연구에서는 human EGF 유전자를 대장균 코돈에 최적화 하고 pRSET 벡터에 클로닝하여 발현벡터를 구축하였다. Human EGF를 봉입체가 아닌 활성이 있는 형태로의 과량 발현을 위해 코돈의 최적화와 더불어 최초로 샤페론 공발현이 시도되었다. 발현된 Native protein 형태의 재조합 human EGF는 고순도로 정제하기 위해 Ion Exchange Chromatography를 2번 연속적으로 수행하여 순수 분리 정제되었고, ELISA 분석결과 99% 이상으로 재조합 EGF의 활성도가 상업용 EGF와 유사하게 나타났으며, 세포증식시험 결과 인간 재조합 EGF는 인체 피부 섬유아세포의 세포증식을 촉진하는 것으로 확인 되었다. 본 연구의 인간 EGF 발현 시스템은 양적인 측면 뿐 아니라 성공적인 활성형태의 발현으로 추가적인 재접힘 과정 및 N 말단의 융합부분을 제거하기 위한 크로마토그래피 작업이 필요가 없다는 점에서 기존의 방법들에 대체 될 수 있는 효과적인 인간 EGF 발현 시스템을 제공하고 있다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권8호
• /
• pp.4859-4864
• /
• 2013

Background: The purpose of this study was to assess the effectiveness of a recombinant human epidermal growth factor (EGF)-based cream for the prevention of acute radiation dermatitis in breast cancer patients receiving radiotherapy (RT). Materials and Methods: Between December 2012 and April 2013, 40 breast cancer patients who received postoperative RT were prospectively enrolled in this study and randomly assigned to receive human recombinant EGF-based cream (intervention group) or general supportive skin care (control group). The grade of radiation dermatitis and pain score were examined at weekly intervals during RT and 6 weeks after RT completion. Results: All patients completed the planned RT and complied well with instructions for applying the study cream and general supportive skin care. In the intervention group, radiation dermatitis of maximum grade 3, 2, and 1 developed in 3 (15%), 11 (55%), and 6 patients (30%), respectively. In comparison, in the control group, radiation dermatitis of maximum grade 3, 2, and 1 developed in 8 (40%), 10 (50%), and 2 patients (10%), respectively. The intervention group showed lower incidence of grade 3 radiation dermatitis than the control group (p=0.068 in univariate analysis and p=0.035 in multivariate analysis). There was no statistically significant difference in the maximal pain score between the two groups (p=0.934). Conclusions: This single-blind randomized preliminary study showed that recombinant human EGF-based cream can have a beneficial role in preventing or minimizing radiation dermatitis in breast cancer patients. To confirm the results of our study, additional studies with a large sample size are required.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
• /
• pp.415.3-416
• /
• 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)

• 약학회지
• /
• 제39권5호
• /
• pp.471-479
• /
• 1995

The recombinant human epidermal growth factor(DWP 401) was investigated on the pharrnacological actions. DWP 401 had no effects on the hexobarbital-induced sleeping time, locomotor activity, rotarod test, body temperature, analgesic action and anticonvulsant action in mice. It also had no influences on the isolated tracheal muscle and ileum of guinea-pig, isolated uterus and fundus strip of rats. Slight hypotensive action with effect on respiration was revealed at a dose of 8 g/kg i.v. of DWP 401 in rabbits. DWP 401 exhibited a weak inhibitory action of glucose tolerance in normal rats, significantly lowered the blood glucose contents in adrenalectomized rats at .a concentration of 160 g,/kg, and produced a significant inhibitory effect on leucocyte migration in CMC-pouch of rats at a concentration of 32 g/rat. Furthermore, DWP 401 showed a significant decrease on gastric juice volume and acidity. However. DWP 401 had no intestinal propulsion rate and influence on urine excretion.

• 한국미생물·생명공학회지
• /
• 제24권4호
• /
• pp.472-477
• /
• 1996

Natural human epidermal growth factor (nhEGF) was purified from pregnant human urine by benzoic acid adsorption, DEAE-Sepharose ion exchange, and immunoaffinity chromatography. The purified nhEGF was further separated into four fractions using Bondapak C$_{18}$ HPLC system. Following characterization by Western blot analysis and double immu- nodiffusion, we found that each fraction corresponds to four derivatives of the nhEGF. For biological analysis of nhEGF, we optimized the labeling time and serum concentration for the incorporatioin of 5-bromo-2'-deoxy uridine (BrdU), a non-radioactive alternative for [$^{3}$H]-thymidine uptake, into NIH 3T3 cells. The DNA synthesis of NIH 3T3 cells was gradually increased at the nhEGF concentrations between 0.1 - 10 ng/ml in the Dulbecco's Modified Eagles Medium (DMEM) containing 0.2% Fetal calf serum (FCS). When we assayed the biological activity of four fractions, the activity of the second fraction was superior to that of the others. Based on the results from the HPLC analysis spiked with recombinant human epidermal growth factor (rhEGF) and amino acid sequencing, we concluded that the second fraction was nhEGF and the other three fractions were the derivatives of nhEGF. In addition, the proportion of nhEGF was approximately 46% is compared with that of the other three derivatives.

• Journal of Microbiology and Biotechnology
• /
• 제25권1호
• /
• pp.119-126
• /
• 2015

Among the various human growth factors, epidermal growth factor (hEGF, consisting of 53 amino acids) has various effects on cell regeneration, stimulation of proliferation, migration of keratinocytes, formation of granulation tissues, and stimulation of fibroblast motility, which are important for wound healing. Owing to their multiple activities, EGFs are used as pharmaceutical and cosmetic agents. However, their low productivity, limited target specificity, and short half-life inhibit their application as therapeutic agents. To overcome these obstacles, we fused the collagen-binding domain (CBD) of Vibrio mimicus metalloprotease to EGF protein. About 18 or 12 amino acids (aa) (of the 33 total amino acids), which were essential for collagen-binding activity, were combined with the N- and C-termini of EGF. We constructed, expressed, and purified EGF (53 aa)-CBD (18 aa), EGF (53 aa)-CBD (12 aa), CBD (18 aa)-EGF (53 aa), and CBD (12 aa)-EGF (53 aa). These purified recombinant proteins increased the numbers of cells in treated specimens compared with non-treated specimens and control hEGF samples. The collagen-binding activities were also evaluated. Furthermore, CBD-hybridized hEGF induced phosphorylation of the EGF receptor. These results suggested that these fusion proteins could be applicable as small therapeutic agents in wound tissue healing.

• 약학회지
• /
• 제41권3호
• /
• pp.381-388
• /
• 1997

Metabolism of DWP401, recombinant juman epidermal growth factor, was examined in vivo and in vitro in rats. When $^{125}I$-labeled DWP401 was administered at a dose of 50 ${\mu}g$/kg by i.v. injection. $^{125}I$-labeled DWP401 was rapidly degraded within 30 minytes above 93%. Thin layer chromatography analysis of urine collected for 24 hr after i.v. administration of $^{125}I$-labeled DWP401 showed ohly one spot on a X-ray film which was considered as diiodo-tyrosine. This result suggests tha $^{125}I$-labeled DWP401 was completely digested into free amino acids without any specific intermediate polypeptides. About 42.1% of the administered iodine was recovered in 24 hr. For in vitro degradation study, $^{125}I$-labeled DWP401 was added to plama and tissue homogenates of rats and incubated at $37^{\circ}C$. Almost 98% of the added radioactivity recovered from the protein fraction of the liver, kidey, small intestine, stomach and spleen decreased rapidly. For examplem the recovery rates of $^{125}I$-labeled DWP401 were 58.6, 63.2, 39.9, 52.9 and 66.8% after 4hrs of incubation in respective organ homogenates.

• Archives of Pharmacal Research
• /
• 제20권1호
• /
• pp.34-38
• /
• 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.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2001년도 Proceedings of the Pharmaceutical Society of Korea
• /
• pp.223.1-223.1
• /
• 2001

• Archives of Plastic Surgery
• /
• 제41권5호
• /
• pp.466-471
• /
• 2014

Background Bleeding can be a problem in wound debridement. In search for an effective hemostatic agent, we experimented with a chitosan film combined with the recombinant human epidermal growth factor (rh-EGF), hypothesizing that it would achieve effective hemostasis and simultaneously enhance arterial healing. Methods Forty-eight Sprague-Dawley rats were used, and 96 puncture wounds were made. The wounds were divided into the following four groups: treated with sterile gauze, treated with gelatin sponge, treated with chitosan, and treated with chitosan combined with rh-EGF. Immediate hemostasis was evaluated, and arterial healing was observed histologically. Results Groups B, C, and D showed a significant rate of immediate hemostasis as compared to group A (P<0.05), but there were no significant differences among groups B, C, and D. Histologically, only group D showed good continuity of the vessel wall after 1 week. It was the only group to show smooth muscle cell nuclei of the vessel wall. Conclusions We observed that chitosan has an effective hemostatic potential and the mix of rh-EGF and chitosan does not interfere with chitosan's hemostatic capabilities. We also identified enhanced healing of vessel walls when rh-EGF was added to chitosan. Further research based on these positive findings is needed to evaluate the potential use of this combination on difficult wounds like chronic diabetic ulcerations.