OVER-EXPRESSION AND CHARACTERIZATION OF ACTIVE RECOMBINANT RAT-LIVER CARNITINE PALMITOYLTRANSFERASE-II USING BACULOVIRUS

The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect c ell system. Unlike Escherichia call, the baculovirus-infected insect c ells expressed mostly soluble active recombinant CPT-II (rCPT-II), CPT activity from crude lysates of recombinant baculovirus-infected insec t cells was maximal between 50 and 72 h post-infection, with a peak sp ecific activity of 100-200 times that found in the mock- or wild-type- infected control lysates. Milligram quantities (up to 1.8 mg/l of cult ure) of active rCPT-II were chromatographically purified from large-sc ale cultures of insect cells infected with the recombinant baculovirus . The rCPT-II was found to be: (1) similar in size to the native rat l iver enzyme( similar to 70 kDa) as judged by SDS/PAGE; (2) immunoreact ive with a polyclonal serum raised against rat liver CPT-II; and (3) n ot glycosylated. Kinetic analysis of soluble rCPT-II revealed K-m valu es for carnitine and palmitoyl-CoA of 950 +/- 27 pM and 34 +/- 5.6 mu M respectively.