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.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J I
JI
MacDonald
C
C
Kent
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