Purification of functional recombinant YY1 expressed in Sf9 insect cells.
We expressed a gene for YY1, the multifunctional mammalian transcription factor, with a recombinant baculovirus in insect cells and obtained high levels of protein in cell lysates. A simple and efficient purification method was developed. In the final step, we used DNA affinity chromatography with concatermerized sequence derived from the upstream region of the B19 parvovirus P6 promoter, which has strong affinity for YY1. Approximately 1.8 mg of highly purified YY1 was obtained from 10(9) Sf9 cells. Highly purified YY1 behaved as authentic YY1 on electrophoretic mobility shift assays and in enhancing transcription from the P6 promoter. YY1 produced in a baculovirus system should prove useful for in vitro transcription assays of YY1 and related regulatory proteins.
Momoeda
M
M
Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kawase
M
M
Young
N S
NS
Kajigaya
S
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D050981:Erythroid-Specific DNA-Binding Factors; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D016732:Parvovirus B19, Human / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D018411:Spodoptera; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D050986:YY1 Transcription Factor
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050986:YY1 Transcription Factor; C494698:YY1 protein, human