tailieunhanh - Báo cáo Y học: Balanced expression of single subunits in a multisubunit protein, achieved by cell fusion of individual transfectants

To establish stable cell lines that produce recombinant multisubunit proteins, it is usually necessary to cotransfect cells with several independent gene constructs. Here, we show that a stepwise fusion of individually transfected cells, results in a fused cell-line that secretes a complete multisubunit protein. Functional expression of recombinant multisubunit proteins may require a defined expression ratio between each protein subunit. The cell-fusion technology described allows a predefined expression level of each subunit. Using SIgA as a model protein we demonstrate that the majority of the fused cells inherit the molar expression ratio of the parental transfected cells. These results indicate that. | Eur. J. Biochem. 269 3205-3210 2002 FEBS 2002 doi Balanced expression of single subunits in a multisubunit protein achieved by cell fusion of individual transfectants Lars Norderhaug1 Finn-Eirik Johansen2 and Inger Sandlie3 1 Antibody Design AS Nesoddtangen Norway department of Pathology Rikshospitalet Norway 3Department of Biology University of Oslo Norway To establish stable cell lines that produce recombinant multisubunit proteins it is usually necessary to cotransfect cells with several independent gene constructs. Here we show that a stepwise fusion of individually transfected cells results in a fused cell-line that secretes a complete multisubunit protein. Functional expression of recombinant multisubunit proteins may require a defined expression ratio between each protein subunit. The cell-fusion technology described allows a predefined expression level of each subunit. Using SIgA as a model protein we demonstrate that the majority of the fused cells inherit the molar expression ratio of the parental transfected cells. These results indicate that simplified screening of clones expressing the expected subunit ratios may be possible using the cell-fusion technology. This technology may therefore be an alternative to generic transfection methods for the establishment of cells that produce multiprotein complexes such as antibodies receptors ion channels and other multisubunit proteins. Keywords antibody expression fusion SIgA transfection. To establish stable cell lines that produce recombinant multisubunit proteins it is generally necessary to introduce vectors that direct the expression of each subunit gene. For multisubunit proteins such as secretory antibodies 1 2 receptors 3 4 and ion channels 5-8 a defined expression of each unit may be essential for the specific function of the mature protein product. This is usually achieved by cotransfecting cells with several independent constructs or by introducing a single vector .