Gene cloning, expression and purification of human mitochondrial transcription factor TFB1M = 인체 미토콘드리아 전사 인자의 클로닝과 정제

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Transcription from human mitochondrial DNA promoter has been recently reconstituted in vitro with three recombinant proteins: mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1, B2 (TFB1M, TFB2M) and the mitochondrial RNA polymerase (POLRMT). TFB1M is an essential component of the mitochondrial promoter dependent transcription process. Here we cloned the gene encoding TFB1M from human cDNA libraries and ligated it into the expression vector. The recombinant TFB1M was overexpressed in Escherichia coli and purified to near homogeneity, using metal chelate affinity chromatography on Ni-NTA affinity resin, followed by heparin-agarose chromatography. Expression of POLRMT alone in E.coli was not successful, because most of the protein was insoluble. We tried to stabilize POLRMT by coexpression POLRMT with TFB1M. The recombinant TFAM was purified by Ni-NTA affinity chromatography and phosphocellulose chromatography. Then the basic components of the mitochondrial transcription machinery in human now seem to be in our hands.
Advisors
Kang, Chang-Wonresearcher강창원researcher
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2004
Identifier
237817/325007  / 020023437
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생명과학과, 2004.2, [ iii, 32 p. ]

Keywords

CLONING; TRANSCRIPTION FACTOR TFB1M; MITOCHONDION; PURIFICATION; 정제; 클로닝; 전사 인자 TFB1M; 미토콘드리아

URI
http://hdl.handle.net/10203/28024
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=237817&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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