Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells.
We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002999:Clone Cells / Q000378:metabolism; D006224:Cricetinae; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D010053:Ovary; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014162:Transfection
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D008668:Metallothionein