An in vitro amplification approach for the expression of recombinant proteins in mammalian cells

• Published in: Biotechnology and applied biochemistry Vol. 20; no. 2; p. 157
• Main Authors: Monaco, L, Tagliabue, R, Soria, M R, Uhlèn, M
• Format: Journal Article
• Language: English
• Published: United States 01.10.1994
• Subjects: Base Sequence; Biopolymers; Blotting, Southern; Cell Line; Chloramphenicol O-Acetyltransferase - biosynthesis; Chloramphenicol O-Acetyltransferase - genetics; Clone Cells; Endothelin-1; Endothelins - biosynthesis; Endothelins - metabolism; Genome, Human; HeLa Cells; Humans; Molecular Sequence Data; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - metabolism; Nucleic Acid Amplification Techniques; Protein Precursors - metabolism; Recombinant Proteins - biosynthesis; Transfection
• ISSN: 0885-4513
• DOI: 10.1111/j.1470-8744.1994.tb00311.x

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.