Expression in Escherichia coli: purification and characterization of cyclin H, a subunit of the human general transcription/DNA repair factor TFIIH.
The human cyclin H, a protein normally associated with the cyclin-dependent kinase cdk7, was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified recombinant cyclin H is similar to the endogenous one according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to interact with cdk7, and regulatory kinase activity. The scale-up of cyclin H purification is described.
Poterszman
A
A
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch, France.
Andersen
G
G
Busso
D
D
Rossignol
M
M
Egly
J M
JM
Thierry
J C
JC
D000345:Affinity Labels; D000595:Amino Acid Sequence; D015153:Blotting, Western; D056748:Cyclin H; D016213:Cyclins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019473:Transcription Factors, TFII / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D000345:Affinity Labels; C534751:CCNH protein, human; D056748:Cyclin H; D016213:Cyclins; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII