• Journal of Embryo Transfer
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• v.24 no.3
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• pp.145-150
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• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.236-241
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• 2005

A decolorization process using ion exchange chromatography was developed to refine rhEGF as a cosmetic ingredient. A macroreticular resin (D314) was selected, with respect to its high decolorization rate and recovery yield of rhEGF, and the operational conditions of the decolorization process optimized. The optimum conditions were as follows: the rhEGF effluent was ion exchanged at a flow rate of 60.0mL/h, with an effluent pH 5.0, using a chromatographic column (i.d. 16mm) packed with D314, with a 7.5cm in bed height. The decolorization process was carried out under the optimum conditions, and halted when the effluent volume reached 350mL, giving a decolorization rate and recovery yield of rhEGF higher than 67 and $80\%$, respectively. When the decolorization rate exceeded $67\%$, the final product turned out to be white or light yellowish, which was to the satisfaction of the cosmetic standard.

• Journal of Pharmaceutical Investigation
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• v.28 no.2
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• pp.99-107
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• 1998

The objective of this study was to develop effective oral formulations of rhEGF for gastric ulcer healing using polycarbophil. hydroxypropylcellulose(HPC) and sucralfate as its bioadhesive bases. Cytoprotective effects of rhEGF, cell proliferation and differentiation. on the ulcers induced by ethanol or acetic acid in rats were studied. rhEGF release from HPC formulation was much faster than that from polycarbophil formulation. HPC formulation combined with small amount of sucralfate showed much slower release of rhEGF than only HPC base only. rhEGF preparations with bioadhesive polymers showed better effects on the healing of gastric ulcers than EGF solution when administered orally. When rhEGF preparations were administered at once and the animals were under starvation, polycarbophil formulation showed better effect on gastric ulcers than HPC formulation. Otherwise, when rhEGF preparations were given more than three times and the rats were fed normally, HPC formulation showed good healing efficacy of ulcers compared to polycarbophil formulation. rhEGF showed dose-dependent effect on the healing of both chronic and acute ulcers.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.1-5
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• 2002

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

• KSBB Journal
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• v.17 no.2
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• pp.153-157
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• 2002

A fusion protein, consisting of a human epidermal growth factor as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as an inclusion body in recombinant E. coli, yet when the conventional solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably because of the opposite surface charge resulting from the vastly different pl values of each domain. Accordingly the solid-phase refolding process, which exploits the ionic interactions between a solid matrix and the protein, was tried, however the ionic binding yield was also very low regardless of the resins and pH conditions used. Therefore, to provide a higher affinity toward the solid matrix, six Iysine residues were tagged to the N-terminus of the hEGF domain. When cation exchange resins, such as heparin- or CM-Sepharose, were used as the matrix, the adsorption capacity increased 2.5~3-fold and the subsequent refolding yield increased nearly 15-fold compared to the conventional process. A similat result was also obtained when an Ni-NTA metal affinity resin was used.

• Korean Chemical Engineering Research
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• v.43 no.1
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• pp.103-109
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• 2005

Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. $1.92{\pm}0.59{\times}10^6$ viable cells were isolated by trypsin enzymatic digestion method from $0.25cm^2$ biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 0.15 mM $Ca^{2+}$ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn't have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 2.0 mM $Ca^{2+}$, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.

• KSBB Journal
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• v.23 no.3
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• pp.205-212
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• 2008

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and peri plasm region. Three fusion partners for cytoplasmic expression such as acidic tail of synuclein (ATS), thioredoxin (Trx) and lipase, and three fusion partners for periplasmic expression such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein (MBP) were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, yielding about 18.1 mg/L and three-fold higher level compared to that of insoluble non-fusion hEGF in cytoplasm. Thus, heterologous proteins containing complex disulfide bond and many hydrophobic amino acids can effectively be produced as an active form in E. coli by introducing a suitable peptide or protein.