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Titolo:
Expression of cloned cDNA for the human mitochondrial RNA polymerase in Escherichia coli and purification
Autore:
Nam, SC; Kang, C;
Indirizzi:
Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea Korea Adv Inst Sci & Technol Taejon South Korea 305701 5701, South Korea
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 3, volume: 21, anno: 2001,
pagine: 485 - 491
SICI:
1046-5928(200104)21:3<485:EOCCFT>2.0.ZU;2-4
Fonte:
ISI
Lingua:
ENG
Soggetto:
TRANSCRIPTION FACTOR-A; HMG-BOX PROTEIN; SPECIFICITY FACTOR; SACCHAROMYCES-CEREVISIAE; CELL MITOCHONDRIA; XENOPUS-LAEVIS; SIGMA-FACTORS; HEAVY-STRAND; FACTOR-I; PROMOTER;
Keywords:
human mitochondrial RNA polymerase; in vitro transcription; mitochondrial transcription factor A; histidine-tagged protein; soluble expression of cloned cDNA;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
30
Recensione:
Indirizzi per estratti:
Indirizzo: Kang, C Korea Adv Inst Sci & Technol, Dept Biol Sci, Taejon 305701, South Korea Korea Adv Inst Sci & Technol Taejon South Korea 305701 uth Korea
Citazione:
S.C. Nam e C. Kang, "Expression of cloned cDNA for the human mitochondrial RNA polymerase in Escherichia coli and purification", PROT EX PUR, 21(3), 2001, pp. 485-491

Abstract

A full-length cDNA for the mature, mitochondrial form of human mitochondrial RNA polymerase was cloned and expressed under the control of T5 or tac promoter in Escherichia coli, The cDNA was efficiently expressed at 37 degreesC, but virtually all the polymerase produced was insoluble, and renaturation of the inclusion bodies was unsuccessful. When the cells were grown at 25 degreesC, however, a portion of approximately 10% was soluble and active. The protein was purified 100-fold from the soluble lysates to homogeneityby two-step chromatography using Ni-nitrilotriacetic acid-Sepharose and heparin-agarose columns, as an N-terminal histidine tag attached and as the tag cleaved away. The purified polymerases with and without the histidine tag were both active in RNA polymerization in vitro as measured with poly(dA-dT) template, and specific activity was 140,000 units/mg. The purified enzyme has the same biochemical properties as the polymerase fraction partiallypurified from the human mitochondria, except for the promoter-specific activity that was not observed with the purified polymerase in the presence ofmitochondrial transcription factor A. Additional factor(s) and/or mammalian-specific or regulatory modification(s) of the polymerase should be necessary for promoter-specific transcription. (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 27/09/20 alle ore 06:51:55