한국생물공학회:학술대회논문집

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Comparison of various cell lysis methods for Pichia pastoris

  • Han, Kyung-Ah (Department of Material and Biochemical Engineering, BioProcess Technology lab., Research Center for BioPhotonics, Chonnam National University) ;
  • Kim, Sun-Yong (Faculty of Applied Chemical Engineering, BioProcess Technology lab., Research Center for BioPhotonics, Chonnam National University) ;
  • Rhee, Jong-Il (Faculty of Applied Chemical Engineering, BioProcess Technology lab., Research Center for BioPhotonics, Chonnam National University)
  • Published : 2005.10.27
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Abstract

Cell lysis method is very important to study intracellular metabolites in microorganisms. In this study, various cell lysis methods were compared to find a good method analysing intracellular metabolites in P. pastoris. P. pastoris was cultivated in YPD medium at 30 $^{\circ}C$, 200 rpm for 24 hours and its morphology as well as the change in strain's shape were observed by microscopy. The UV/vis spectrophotometer was used to measure intracellular protein concentrations.

10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose가 첨가된 YPD 배지를 이용하여 200 rpm, 30 $^{\circ}C$에서 24시간 배양한 Pichia pastoris x-33과 KM71H를 각각 대수증식기에 시료를 채취하여 4000 g, 4 $^{\circ}C$에서 5분간 원심분리하여 펠릿에 멸균된 증류수를 500 ${\mu}{\ell}$ 넣고 4000 g, 4 $^{\circ}C$에서 5분간 원심분리하여 2-3회 세척한 후 보관하여 각기 다른 4가지 방법으로 세포를 파쇄하여 얻은 시료를 현미경관찰을 통하여 형태의 변화를 관찰하였다. 12000 g, 4 $^{\circ}C$에서10분간 원심분리하여 얻은 상등액에 시약 500 ${\mu}{\ell}$와 시료 500 ${\mu}{\ell}$를 넣고 상온에서 10분간 반응하여 분광광도계를 이용하여 595 nm에서 세포 내 단백질 농도를 측정하였다. 이 결과를 바탕으로 효모를 가장 효과적으로 세포를 파쇄할 수 있는 방법을 조사하였다.

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