GROWTH AND INDUCTION KINETICS OF INDUCIBLE AND AUTOINDUCIBLE EXPRESSION OF HETEROLOGOUS PROTEIN IN SUSPENSION-CULTURES OF RECOMBINANT MOUSEL CELL-LINES

Citation
Mb. Gu et al., GROWTH AND INDUCTION KINETICS OF INDUCIBLE AND AUTOINDUCIBLE EXPRESSION OF HETEROLOGOUS PROTEIN IN SUSPENSION-CULTURES OF RECOMBINANT MOUSEL CELL-LINES, Biotechnology progress, 12(2), 1996, pp. 226-233
Citations number
24
Categorie Soggetti
Biothechnology & Applied Migrobiology","Food Science & Tenology
Journal title
ISSN journal
87567938
Volume
12
Issue
2
Year of publication
1996
Pages
226 - 233
Database
ISI
SICI code
8756-7938(1996)12:2<226:GAIKOI>2.0.ZU;2-L
Abstract
Mouse Lth(-) cells were transfected with four different plasmids for a utoinducible and highly-inducible expression of the bacterial lacZ gen e and cultivated in suspension. Two selection genes, thymidine kinase (th) 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 LacZ gene expression in the M4 cell line (''autoinducible''), while the constitu tive rous sarcoma virus (RSV) promoter controls gr gene expression and the MMTV promoter controls LacZ gene expression in the R2 cell line ( ''highly-inducible''). Both cell lines were stable with respect to rep roducibility of growth rate in spinner flasks and inducibility of P-ga lactosidase expression. The exponential growth rate of R2 cells was sl ower than that of M4 cells before induction because the R2 cell line c ontinuously expressed gr genes under the constitutive RSV promoter, an d the percent reduction of exponential growth rate mainly caused by gr gene expression was about 20%. The inducibility of the M4 cell line w as greater than that of the R2 cell line because in the M4 cell line M MTV promoter controlled gr and LacZ gene expression autoinducibly. Max imum induction of the M4 cell line occurred after induction with the h ormone dexamethasone (Dex) at 10(-7) M, and the final beta-galactosida se content increased 400-fold after induction. The optimum conditions for inducer concentration and induction time were determined, and the highest production of beta-galactosidase occurred when Dex was added a fter the cell concentration had reached its maximum in batch culture. Dex(10(-9) M) is a critical inducer concentration in view of inducibil ity 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 i nducibility of M4 was higher than that of the R2 cell line at Dex conc entrations of more than 10(-9) M.