Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells.
Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2 micrograms/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band of M(r) ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.
Sarcich
J L
JL
Biochemistry Unit, Upjohn Laboratories, Kalamazoo, Michigan 49001, USA.
Fischer
H D
HD
Babcock
M S
MS
Leone
J W
JW
Tomasselli
A G
AG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002455:Cell Division; D053480:Cholesterol Ester Transfer Proteins; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D003470:Culture Media; D015870:Gene Expression / Q000235:genetics; D006023:Glycoproteins; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis
D000596:Amino Acids; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D003470:Culture Media; D006023:Glycoproteins; D011994:Recombinant Proteins