Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells.
We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).
Koschinsky
M L
ML
Department of Cardiovascular Research, Genentech Inc., South San Francisco, California 94080.
Tomlinson
J E
JE
Zioncheck
T F
TF
Schwartz
K
K
Eaton
D L
DL
Lawn
R M
RM
D001053:Apolipoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D053241:Apoprotein(a); D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D017270:Lipoprotein(a); D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D001053:Apolipoproteins; D017270:Lipoprotein(a); D011994:Recombinant Proteins; D014415:Tunicamycin; D053241:Apoprotein(a)