title: Mammalian cell/vaccinia virus expression vectors with increased stability of retroviral sequences in Escherichia coli: production of feline immunodeficiency virus envelope protein.
abstract: Many eukaryotic DNA sequences, especially lenti-retrovirus proviral genomes and their env genes, are unstable when cloned in high-copy-number plasmids in Escherichia coli. Stability can be increased by the use of low-copy-number vectors, although plasmid yields are low. Vectors are described here that contain the intermediate-copy-number P15A ori for cloning, stable propagation and higher-yield production of plasmid DNA in E. coli, and the f1 ori for propagation as single-stranded phage. These vectors also have the capacity to direct high-yield production of protein in mammalian cells, and the option of incorporation into and expression via a T7 promoter in vaccinia virus. The SR alpha promoter, encephalomyocarditis (EMC) virus untranslated leader sequence, and poly(A) signal sequence serve as a high-yield mammalian cell expression cassette without the requirement for mRNA capping. A polyhistidine sequence is available at the 3' end of the cassette to facilitate chromatographic purification of protein. neo and gpt genes were included in some vectors to serve as selectable markers, and the dhfr gene was included in one to achieve gene amplification in mammalian cells. Dicistronic mRNAs can be generated by insertion of coding sequences up and downstream from the EMC leader. The utility of these vectors was shown through expression of feline immunodeficiency virus (FIV) Env protein, in conjunction with the tissue plasminogen activator (tPA) leader sequence.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5402.
authors.forename: R F
authors.initials: RF
authors.lastname: Wang
authors.forename: J I
authors.initials: JI
authors.lastname: Mullins
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015686:Gene Products, env / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016089:Immunodeficiency Virus, Feline / Q000235:genetics*; D008322:Mammals; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014616:Vaccinia virus / Q000235:genetics*
chemical_list: D015686:Gene Products, env; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins