Expression, purification, and circular dichroism analysis of human CDK9.
The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.
Faculdade de Medicina de São José do Rio Preto, SP, Brazil.
Andreia Machado
AM
Leopoldino
Fernanda
F
Canduri
Hamilton
H
Cabral
Magno
M
Junqueira
Alessandra Bernadete Trovó
AB
de Marqui
Luciano H
LH
Apponi
Isabel Osório
IO
da Fonseca
Gilberto Barbosa
GB
Domont
Diógenes S
DS
Santos
Sandro
S
Valentini
Gustavo Orlando
GO
Bonilla-Rodriguez
Marcelo Andrés
MA
Fossey
Walter Filgueira
WF
de Azevedo
Eloiza Helena
EH
Tajara
D002942:Circular Dichroism; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9