title: A tightly regulated high level expression vector that utilizes a thermosensitive lac repressor: production of the human T cell receptor V beta 5.3 in Escherichia coli.
abstract: A series of vectors has been constructed to express the human T cell receptor V beta 5.3 under the control of the hybrid trc promoter in Escherichia coli. Transcriptional induction of the trc promoter was achieved chemically by using isopropyl beta-D-thiogalactopyranoside (IPTG) in a bacterial strain that harbors the lacIq gene, or thermally by using the mutant lacIts gene that encodes a temperature-sensitive lac repressor [Bukrinsky et al. (1988) Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli. Gene 70, 415-417]. Several of the plasmids tested also contain the E. coli heat-stable enterotoxin II (STII) signal sequence for protein secretion. In addition, the gene 10 leader sequence from bacteriophage T7 and a minicistron localized upstream of the V beta 5.3 coding sequence were tested for their potential effect on protein production. These elements increased protein yield two-fold when transcription was induced by IPTG, but had no detectable effect on protein yield when transcription was induced thermally. The highest protein yield was obtained when V beta 5.3 was expressed either from plasmid pKB containing the STII signal in strain LJ24, or from plasmid pKBi that lacks the signal sequence, in the protease deficient strain SG21173 (lon, htpR. clp). Both plasmids contain the lacIts gene, the trc promoter, the two transcription terminators of the rrnB operon, and a tetracycline selection marker. Production of V beta 5.3 using pKBi-V beta 5.3 in strain SG21173 in a 5-liter fermenter under controlled growth conditions yielded over 25 mg V beta 5.3/liter culture. Conversion of the lacIts to the lacIqts gene yielded vector pKBiq-V beta 5.3 which exhibits complete repression of the trc promoter at 30 degrees C. This stringent regulation of the thermally inducible trc promoter, the elimination of IPTG, the inclusion of the tetracycline resistance gene, and the high level of protein yield should render this expression system broadly useful for the high level production of heterologous proteins in E. coli, for both basic research and human therapeutic applications.
authors.affiliation: T Cell Science Inc, Needham, MA 02194, USA.
authors.forename: B
authors.initials: B
authors.lastname: Andrews
authors.forename: H
authors.initials: H
authors.lastname: Adari
authors.forename: G
authors.initials: G
authors.lastname: Hannig
authors.forename: E
authors.initials: E
authors.lastname: Lahue
authors.forename: M
authors.initials: M
authors.lastname: Gosselin
authors.forename: S
authors.initials: S
authors.lastname: Martin
authors.forename: A
authors.initials: A
authors.lastname: Ahmed
authors.forename: P J
authors.initials: PJ
authors.lastname: Ford
authors.forename: E G
authors.initials: EG
authors.lastname: Hayman
authors.forename: S C
authors.initials: SC
authors.lastname: Makrides
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D005819:Genetic Markers; D056968:Lac Repressors; D011948:Receptors, Antigen, T-Cell; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside