Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.
Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.
Hussain
A
A
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Lewis
D
D
Sumbilla
C
C
Lai
L C
LC
Melera
P W
PW
Inesi
G
G
D000818:Animals; D001693:Biological Transport, Active; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005493:Folic Acid Antagonists; D015870:Gene Expression; D005822:Genetic Vectors; D000882:Haplorhini; D008727:Methotrexate / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D016296:Mutagenesis; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection
D005493:Folic Acid Antagonists; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D000252:Calcium-Transporting ATPases; D002118:Calcium; D008727:Methotrexate