Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter

The mouse mammary tumor virus (MMTV) promoter is induced by the addition of a glucocorticoid hormone or analog such as dexamethasone. The hormone bind s to its specific transcription factor, glucocorticoid receptor (GR), and t he activated complex then binds to the glucocorticoid response elements (GR Es) in the enhancer region of the MMTV promoter to induce the overexpressio n of downstream genes. We have constructed an expression vector for a repor ter protein, secreted alkaline phosphatase (SEAP),controlled by the MMTV pr omoter and co-transfected this vector along with a GR expression cassette i nto Chinese hamster ovary (CHO) cells. High producers were cloned and grown in suspension cultures. A very high titer, over 0.4 mg/mL, of SEAP was obt ained from this inducible overexpression system, about ten times that achie vable with the same reporter protein using the strong constitutive SV40 pro moter in CHO cells. A peak production rate of 187 pg SEAP per cell per day was observed within 3 days after induction, compared to the peak rate of 23 pg SEAP per cell per day expressed using the constitutive SV40 promoter. W ith the reduced or zero growth rate during the protein production phase, th is novel MMTV overexpression system is highly suited for optimizing glycopr otein synthesis rates in high cell density fed-batch or perfusion bioreacto rs. (C) 2000 John Wiley & Sons, Inc.