고초균에서 His-Pro 반복서열을 갖는 Oligopeptide의 분비 최적화 및 항당뇨 효과 (The Secretion Optimization of Oligopeptide with His-Pro Repeats in Bacillus subtilis and Its Anti-Diabetic Effects)

서지정보

· 발행기관 : 한국생물공학회
· 수록지 정보 : KSBB Journal / 32권 / 1호 / 71 ~ 82페이지
· 저자명 : 정선화, 최장원

초록

To verify anti-diabetic effect of oligopeptide withHis-Pro repeats (mHP peptide), the oligopeptide was first secretedand optimized using the secretion vector, pRBAS withalkaline protease gene promoter and the signal sequence inBacillus subtilis and directly the anti-diabetic effect of the mHPpeptide was investigated in insulinoma cell, RINm5F cell line.
The oligopeptide gene was obtained by annealing oligonucleotideswith repeated His-Pro sequence and finally was constructedas 18 dipeptides (108 bp and 4.0 kDa) coding gene,named oligopeptide with His-Pro repeats (mHP peptide) tomake cyclo(His-Pro) known to be anti-diabetic effects. Theregion encoding the oligopeptide gene was subcloned into thepRBAS secretion vector (E.coli-Bacillus shuttle vector) afterPCR amplification using the designed primers including initiationand termination codons and His tag, named pRBAS-mHP(6.56 kb). To optimize secretion of the oligopeptide, variousculture conditions were investigated in Bacillus subtilis LKS87. As a result, the secreted oligopeptide was maximally measured(approximately 59.6 μg/mL) in 3 L batch culture and thehighest secretion was achieved at 30°C, PY medium, and carbonsources (particularly barley and glycerol). In the RINm5Fcells treated with 2 mM STZ, the oligopeptide treatment (0.1mg/mL) restored the cell viability (10%) and reduced the nitricoxide (NO) generation (35%) and DNA fragmentation (90%).
And also, insulin secretion level was increased to 17% higherthan in STZ-treated RINm5F cells. These results suggest thatthe oligopeptide with His-Pro repeats could be a candidate materialfor anti-diabetic agent against STZ-induced diabetes.

영어초록

To verify anti-diabetic effect of oligopeptide withHis-Pro repeats (mHP peptide), the oligopeptide was first secretedand optimized using the secretion vector, pRBAS withalkaline protease gene promoter and the signal sequence inBacillus subtilis and directly the anti-diabetic effect of the mHPpeptide was investigated in insulinoma cell, RINm5F cell line.
The oligopeptide gene was obtained by annealing oligonucleotideswith repeated His-Pro sequence and finally was constructedas 18 dipeptides (108 bp and 4.0 kDa) coding gene,named oligopeptide with His-Pro repeats (mHP peptide) tomake cyclo(His-Pro) known to be anti-diabetic effects. Theregion encoding the oligopeptide gene was subcloned into thepRBAS secretion vector (E.coli-Bacillus shuttle vector) afterPCR amplification using the designed primers including initiationand termination codons and His tag, named pRBAS-mHP(6.56 kb). To optimize secretion of the oligopeptide, variousculture conditions were investigated in Bacillus subtilis LKS87. As a result, the secreted oligopeptide was maximally measured(approximately 59.6 μg/mL) in 3 L batch culture and thehighest secretion was achieved at 30°C, PY medium, and carbonsources (particularly barley and glycerol). In the RINm5Fcells treated with 2 mM STZ, the oligopeptide treatment (0.1mg/mL) restored the cell viability (10%) and reduced the nitricoxide (NO) generation (35%) and DNA fragmentation (90%).
And also, insulin secretion level was increased to 17% higherthan in STZ-treated RINm5F cells. These results suggest thatthe oligopeptide with His-Pro repeats could be a candidate materialfor anti-diabetic agent against STZ-induced diabetes.

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