Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.
The CKS1At gene product, p10CKS1At from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At, a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21(DE3) cotransformed with the CKS1At gene and the E. coli argU gene that encoded the arginine acceptor tRNAUCU produced a sufficient amount of p10CKS1At to start the structural study by NMR. Replacement of four rare codons in the CKS1At gene sequence, including a tandem arginine, by highly used codons in E. coli, restored also a high expression of the recombinant protein. Double-isotopic enrichment by 13C and 15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1At was purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1At protein.
Landrieu
I
I
Unité de Microbiologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, B-5030, Belgium.
Casteels
P
P
Odaert
B
B
De Veylder
L
L
Portetelle
D
D
Lippens
G
G
Van Montagu
M
M
Inzé
D
D
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002247:Carbon Isotopes; D003062:Codon / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D002247:Carbon Isotopes; D003062:Codon; D017931:DNA Primers; D018744:DNA, Plant; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D011994:Recombinant Proteins; D018844:Cyclin-Dependent Kinases