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Growth and Induction Kinetics of Inducible and Autoinducible Expression of Heterologous Protein in Suspension Cultures of Recombinant Mouse L Cell Lines
Author(s) -
Gu Man Bock,
Todd Paul,
Kompala Dhinakar S.
Publication year - 1996
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp950074s
Subject(s) - microbiology and biotechnology , cell culture , biology , gene expression , rous sarcoma virus , expression vector , gene , mouse mammary tumor virus , heterologous , transfection , cell growth , recombinant dna , genetics
Abstract Mouse L tk − cells were transfected with four different plasmids for autoinducible and highly‐inducible expression of the bacterial lac Z gene and cultivated in suspension. Two selection genes, thymidine kinase ( tk ) and neomycin resistance ( neo r ), were used to select the clones in both cell lines. The resulting two cell lines, designated M4 and R2, differ in that the inducible MMTV promoter from mouse mammary tumor virus (MMTV) controls glucocorticoid receptor ( gr ) gene and lac Z gene expression in the M4 cell line (“autoinducible”), while the constitutive rous sarcoma virus (RSV) promoter controls gr gene expression and the MMTV promoter controls lac Z gene expression in the R2 cell line (“highly‐inducible”). Both cell lines were stable with respect to reproducibility of growth rate in spinner flasks and inducibility of β‐galactosidase expression. The exponential growth rate of R2 cells was slower than that of M4 cells before induction because the R2 cell line continuously expressed gr genes under the constitutive RSV promoter, and the percent reduction of exponential growth rate mainly caused by gr gene expression was about 20%. The inducibility of the M4 cell line was greater than that of the R2 cell line because in the M4 cell line MMTV promoter controlled gr and lac Z gene expression autoinducibly. Maximum induction of the M4 cell line occurred after induction with the hormone dexamethasone (Dex) at 10 −7 M, and the final β‐galactosidase content increased 400‐fold after induction. The optimum conditions for inducer concentration and induction time were determined, and the highest production of β‐galactosidase occurred when Dex was added after the cell concentration had reached its maximum in batch culture. Dex (10 −9 M) is a critical inducer concentration in view of inducibility between M4 and R2 cell lines. The inducibility of R2 cell line is higher than that of the M4 cell line from 0 to 10 −9 M Dex, but the inducibility of M4 was higher than that of the R2 cell line at Dex concentrations of more than 10 −9 M.