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

A series of vectors has been constructed to express the human T cell r eceptor V beta 5.3 under the control of the hybrid trc promoter in Esc herichia coli. Transcriptional induction of the the promoter was achie ved chemically by using isopropyl beta-D-thiogalactopyranoside (IPTG) in a bacterial strain that harbors the lacI(q) gene, or thermally by u sing the mutant lacIts gene that encodes a temperature-sensitive lac r epressor [Bukrinsky et al. (1988) Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodefici ency 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 poten tial effect on protein production. These elements increased protein yi eld two-fold when transcription was induced by IPTG, but had no detect able 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 defi cient strain SG21173 (lon, htpR, clp). Both plasmids contain the lacIt s gene, the trc promoter, the two transcription terminators of the rrn B 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 cult ure. Conversion of the lacIts to the lacI(q)ts gene yielded vector pKB iq-V beta 5.3 which exhibits complete repression of the trc promoter a t 30 degrees C. This stringent regulation of the thermally inducible t rc promoter, the elimination of IPTG, the inclusion of the tetracyclin e resistance gene, and the high level of protein yield should render t his expression system broadly useful for the high level production of heterologous proteins in E. coli, for both basic research and human th erapeutic applications.