미생물학회지 (Korean Journal of Microbiology)

  • 제31권4호
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  • Pages.274-278
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  • 1993
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  • 0440-2413(pISSN)
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  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

대장균에서의 T7 발현체계에 의하여 과잉생산된 클로람페니콜 아세틸전이효소와 베타-락타메이즈의 수용성과 활성

Solubilities and Activities of Chloramphenicol Acetyltransferase and $\beta$-Lactamase Overproduced by the T7 Expression System in Escherichia coli

  • 발행 : 1993.08.01
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초록

단백질이 어떻게 비수용성이 되는지를 알기위해, 클로람페니콜 아세틸전이효소와 베타-락타메이즈를 과잉생산하여 그들의 수용성과 활성을 측정하였다. 클로람페니콜 아세틸전이효소는 총단백질의 9에서 45%를 차지하였으며, inclusion body 형성없이 완전히 수용성이었으며, 효소활성은 만들어진 양과 비례하였다. 또한 30℃에서 T7 발현체계에 의해 생성된 베타-락타메이즈는 수용성의 숙성체였으나, 37℃에서는 비수용성이 되었다. 세포질에 있는 대부분의 베타-락타메이즈는 비수용성이었고. 페리플라즘 공간에서는 대부분이 수용성이었다. 단백질의 올바른 폴딩을 도와주는 chaperone의 일종인 GroEL 단백질은 본 실험조선에서는 베타-락타베이즈의 수용성을 별로 높이지는 못했다. 세포 내에서 inclusion body의 형성은 단백질의 높은 종도보다는 각각 단밸질 자체의 특성과 관련된 듯하다.

Overproduced proteins in many cases result in forming insoluble inclusion bodies, and their formation might be due to high concentration of protein. To investigate how proteins become insoluble, chloramphenicol acetyltransferase (CAT) and .betha.-lactamase were overproduced, and their solubilities and activities were determined. CAT was accumulated from 9 to 45% of total cellular protein in a fully soluble form without inclusion body formation. CAT specific activity was shown to be proportional to the amount of the protein produced. Moderately produced .betha.-lactamase by the phase T7 expression system at 30.deg.C comprised only mature forms in a soluble form. However, overproduced .betha.-lactamase at 37.deg.C became insoluble. Most precursor forms of .betha.-lactamase in the cytoplasm were insoluble, whereas majority of the mature forms in the periplasm space were soluble. Also, chaperone GroE proteins which assist proper protein folding and translocation did not increase .betha.-lactamase solubility significantly under the experimental condition. It seems that the formation of inclusion bodies in the cell is related to the nature of protein itself rather than just to high concentration of protein.

키워드

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