title: Tandem gene amplification in vitro for rapid and efficient expression in animal cells.
abstract: Plasmid vectors pHSG293 and pHSG747, suitable for in vitro gene amplification for subsequent animal-cell expression, were developed. A cosmid vector pHSG293 confers Km resistance to Escherichia coli host cells and G418 resistance to animal cells and contains a single BstXI recognition/cleavage site, CCACGGGG/CTGG, near the cos site (the recognition site is underlined). The cassette vector plasmid pHSG747 contains a multiple cloning site (MCS) between the simian virus 40 early promoter and the poly(A) signal sequence flanked by the same BstXI sites and confers Cm resistance to E. coli host cells. After inserting a coding fragment for human protein C or its derivative in the appropriate orientation in the MCS of pHSG747, the BstXI expression unit fragment was purified, mixed with BstXI-digested pHSG293 DNA at a molecular ratio of 20 to 40:1 and ligated. This allowed for tandem gene amplification due to asymmetric cohesive ends. Ligation products were packaged in lambda phage particles, amplified in E. coli cells as large cosmid molecules, and then introduced into CHO cells. G418R transformants were found to produce and secrete recombinant protein molecules at a high level. The plasmid vectors developed in this work will provide a rapid screening system useful for protein engineering in animal cells.
authors.affiliation: Laboratory for Molecular Biology, Hoechst Japan Ltd., Kawagoe, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Takeshita
authors.forename: K
authors.initials: K
authors.lastname: Tezuka
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: H
authors.initials: H
authors.lastname: Honkawa
authors.forename: A
authors.initials: A
authors.lastname: Matsuo
authors.forename: T
authors.initials: T
authors.lastname: Matsuishi
authors.forename: T
authors.initials: T
authors.lastname: Hashimoto-Gotoh
mesh_terms: D000818:Animals; D001483:Base Sequence; D003360:Cosmids; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005170:Factor X / Q000235:genetics; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011486:Protein C / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping
chemical_list: D011486:Protein C; D011994:Recombinant Proteins; D005170:Factor X; D004247:DNA