title: Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance.
abstract: Expression vectors were designed and constructed to achieve optimum production of two different isozymes of rat glutathione S-transferase (GST) (EC 2.5.1.18) in COS cells, for studies of drug resistance. Promoter-enhancer elements from the simian virus 40 (SV40) early-region or the mouse alpha 2(I)-collagen gene, GST cDNAs encoding the rat Ya or Yb1 isozymes, and an SV40 replicative origin (ori) were positioned in the vector to express two GSTs at high levels in the same cell. The optimized construct yielded levels of both GST proteins (1% of postmitochondrial protein fraction) that were up to 1.3-fold greater than the sum of those produced individually by two single-unit expression constructs. The best production of the tandem recombinant gene products was observed when the genes were placed in a head to head orientation in close proximity (1 kilobase). With the recombinant genes configured in this way, the plasmid DNA was also amplified in COS cells to higher levels (30% increase over single-unit expression constructs), as ori elements were placed on both DNA strands. Cells expressing the recombinant GSTs were viably sorted by flow cytometry on the basis of a GST-catalyzed conjugation of glutathione to monochlorobimane. Sorted COS cells that expressed both GST Ya and Yb1 from recombinant genes in a tandem, head to head configuration were 25 or 70% more resistant to the alkylating agent chlorambucil than cells that expressed GST Ya or Yb1 alone.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
authors.forename: T H
authors.initials: TH
authors.lastname: Manoharan
authors.forename: P J
authors.initials: PJ
authors.lastname: Welch
authors.forename: A M
authors.initials: AM
authors.lastname: Gulick
authors.forename: R B
authors.initials: RB
authors.lastname: Puchalski
authors.forename: A L
authors.initials: AL
authors.lastname: Lathrop
authors.forename: W E
authors.initials: WE
authors.lastname: Fahl
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002469:Cell Separation / Q000379:methods; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000502:physiology; D005982:Glutathione Transferase / Q000235:genetics*; D000882:Haplorhini; D007527:Isoenzymes / Q000235:genetics*; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics
chemical_list: D000477:Alkylating Agents; D004274:DNA, Recombinant; D007527:Isoenzymes; D004247:DNA; D005982:Glutathione Transferase