An in vitro amplification approach for the expression of recombinant proteins in mammalian cells.
A method for the expression of recombinant DNA products in mammalian cells based on in vitro amplification of gene units is described. Gene cassettes containing either a selectable marker or the gene of interest are mixed at different molar ratios, and linear polymers are formed using forced head-to-tail ligation. After introduction of the polymers into mammalian cells, transformants with various amounts of the amplified gene unit are obtained. For a first characterization of the system, the gene coding for chloramphenicol acetyltransferase (CAT) has been used to produce polymers containing a single neomycin-resistance gene ligated to different numbers of CAT gene units and used for transfection into HeLa cells. All isolated G418 (gentamycin)-resistant cell transformants which received in vitro amplified DNA were found to express high levels of CAT activity in a stable manner. Southern-blot analysis of individual clones revealed multiple copies of the gene integrated head-to-tail in the genome. This system allowed us to express the gene coding for human prepro-endothelin-1 in A617 human vascular-smooth-muscle cells. The recombinant protein was shown to be correctly processed and biologically active endothelin-1.
Monaco
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L
Department of Biological and Technological Research, San Raffaele Scientific Institute, Milan, Italy.
Tagliabue
R
R
Soria
M R
MR
Uhlèn
M
M
D001483:Base Sequence; D001704:Biopolymers; D015139:Blotting, Southern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D002999:Clone Cells; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis* / Q000378:metabolism; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D001704:Biopolymers; D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase