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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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The Secretion Optimization of Oligopeptide with His-Pro Repeats in Bacillus subtilis and Its Anti-Diabetic Effects

고초균에서 His-Pro 반복서열을 갖는 Oligopeptide의 분비 최적화 및 항당뇨 효과

  • 정선화 (대구대학교 생명환경대학 바이오산업학과) ;
  • 최장원 (대구대학교 생명환경대학 바이오산업학과)
  • Received : 2017.03.13
  • Accepted : 2017.03.27
  • Published : 2017.03.31
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Abstract

To verify anti-diabetic effect of oligopeptide with His-Pro repeats (mHP peptide), the oligopeptide was first secreted and optimized using the secretion vector, pRBAS with alkaline protease gene promoter and the signal sequence in Bacillus subtilis and directly the anti-diabetic effect of the mHP peptide was investigated in insulinoma cell, RINm5F cell line. The oligopeptide gene was obtained by annealing oligonucleotides with repeated His-Pro sequence and finally was constructed as 18 dipeptides (108 bp and 4.0 kDa) coding gene, named oligopeptide with His-Pro repeats (mHP peptide) to make cyclo(His-Pro) known to be anti-diabetic effects. The region encoding the oligopeptide gene was subcloned into the pRBAS secretion vector (E.coli-Bacillus shuttle vector) after PCR amplification using the designed primers including initiation and termination codons and His tag, named pRBAS-mHP (6.56 kb). To optimize secretion of the oligopeptide, various culture conditions were investigated in Bacillus subtilis LKS. As a result, the secreted oligopeptide was maximally measured (approximately $59.6{\mu}g/mL$) in 3 L batch culture and the highest secretion was achieved at $30^{\circ}C$, PY medium, and carbon sources (particularly barley and glycerol). In the RINm5F cells treated with 2 mM STZ, the oligopeptide treatment (0.1 mg/mL) restored the cell viability (10%) and reduced the nitric oxide (NO) generation (35%) and DNA fragmentation (90%). And also, insulin secretion level was increased to 17% higher than in STZ-treated RINm5F cells. These results suggest that the oligopeptide with His-Pro repeats could be a candidate material for anti-diabetic agent against STZ-induced diabetes.

Keywords

References

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