Recombinant production of the p10(CKS1At) protein from Arabidopsis thaliana and C-13 and N-15 double-isotopic enrichment for NMR studies

The CKS1At gene product, p10(CKS1At) from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CHS) family of small proteins. The se proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and pla y an essential, but still not precisely known role in cell cycle progressio n. To solve the structure of p10(CKS1At), a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) s pectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21 (DE3) cotra nsformed with the CKS1At gene and the E. coli argU gene that encoded the ar ginine acceptor tRNA(UCU) produced a sufficient amount of p10(CKS1At) to st art the structural study by NMR. Replacement of four rare codons in the CKS 1At gene sequence, including a tandem arginine, by highly used codons in E, coli, restored also a high expression of the recombinant protein. Double-i sotopic enrichment by C-13 and N-15 is reported that will facilitate the NM R study. Isotopically labeled p10(CKS1At) 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 structura l analysis using triple-resonance spectra is feasible for the labeled p10(C KS1At) protein. (C) 1999 Academic Press.