STABLE, RECOMBINANT EXPRESSION OF HUMAN INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-1 (HIGFBP-1) IN CHINESE-HAMSTER OVARY (CHO) CELLS

Stable expression of human insulin-like growth factor binding protein- 1 (hTGFBP-1) at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of exp ression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reduc tase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX ). Expression of hIGFBP-1 in mixed clones was found to increase with i ncreasing copy number and an apparent correlation between intra- and e xtracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIG FBP-1 25 times. The productivity did not increase further after five m ore months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplif ication in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production .