Baculovirus-mediated expression of rat liver CTP:phosphocholine cytidylyltransferase.
We describe herein the expression and purification of milligram quantities of rat liver CTP:phosphocholine cytidylyltransferase in recombinant baculovirus-infected insect cells. The enzyme was purified by incorporating modifications to a previously published procedure (P. A. Weinhold and D. A. Feldman, 1992, in "Methods in Enzymology" (E. A. Dennis and D. E. Vance, Eds.), Vol. 209, pp. 248-258, Academic Press, San Diego, CA). Like cytidylyltransferase purified from rat liver, the purified recombinant cytidylyltransferase has the same molecular weight (42 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and reacts to antibodies generated against the N and C termini of rat liver cytidylyltransferase. In addition, like the purified rat liver enzyme, the recombinant cytidylyltransferase is activated by lipids. We also expressed rat liver cytidylyltransferase as a fusion protein with glutathione-S-transferase and with a thrombin cleavage site between the two enzymes for rapid isolation of cytidylyltransferase. Thrombin cleavage was, however, incomplete and prolonged exposure to thrombin resulted in the degradation of cytidylyltransferase itself.
MacDonald
J I
JI
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Kent
C
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D019916:Choline-Phosphate Cytidylyltransferase; D003001:Cloning, Molecular; D004247:DNA; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009036:Moths; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats
D004247:DNA; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase