• 한국미생물·생명공학회지
• /
• 제26권1호
• /
• pp.61-67
• /
• 1998

재조합 인간상피세포 성장인자(rhEGF)가 E. coli BL21(pYHB101) 균주를 사용하여 발현되었다. 10g/L glucose를 첨가한 변형된 MBL 배지를 사용하여 10 $\mu\textrm{m}$ IPTG/lactose로 2시간 동안 유도배양한 후 27$^{\circ}C$에서 48시간 동안 배양하였을 때 44.5 mg/L의 rhEGF가 발현되었다. 상기의 결과는 E. coli BL2l(pYHB101)를 사용하여 rhEGF를 발현시 lactose를 IPTG와 동일한 유도 물질로 사용 가능하다는 것을 시사하는 것이다. 회분식 배양에서 glucose를 10 g/L 첨가한 변형된 MBL 배지에 유도물질로 10 $\mu\textrm{m}$ lactose를 사용하였으며 28시간 동안 배양하였을 때 최대 45 mg/L의 rhEGF가 발현되었다. 유가식 배양에서 정지기에 0.5%(w/v) lactose와 0.25%(w/v) yeast extract를 첨가하였을 때 160mg/L의 rhEGF가 발현되었으며 94.3%가 분비되었다. 이에 비하여 유도기에 lactose를 첨가한 경우는 120 mg/L의 rhEGF가 발현되었으며 cytoplasm으로 발현된 불용성 봉입체의 비율은 20.9%에 달하였다. 이것은 lactose의 첨가시기가 E. coli BL2l(pYHB101)로부터 soluble rhEGF의 생성에 중요하다는 것을 확인한 결과이다.

• 한국미생물·생명공학회지
• /
• 제24권5호
• /
• pp.553-559
• /
• 1996

We have designed nucleotide sequences of hEGF structural gene to eliminate the N-terminal methionine residue incorporated during the translation initiation step, and constructed recombinant human epidermal growth factor (rhEGF) secretion plasmids pYHB101, and pYHB2 in which pelB signal sequence-hEGF gene was expressed under the control of the T7, and tac promoter, respectively. We also constructed pYHB1 vector which contains rhEGF gene controlled by T7 promoter. The transformant with pYHB101 showed relatively slow growth pattern compared to the transformant with pYHB1. However, we observed that the transformant with pYHB101 secreted rhEGF of 13 mg/l significantly after 5 hr induction with 1 mM IPTG and that the T7 promoter was more effective than tac promoter when connected to pelB signal sequence. The amount of rhEGF was 14 mg/l under the sub-optimized condition.

• 한국미생물·생명공학회지
• /
• 제24권4호
• /
• pp.478-484
• /
• 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.