EXPRESSION OF THE HUMAN T-CELL RECEPTOR V-BETA-5.3 IN ESCHERICHIA-COLI BY THERMAL INDUCTION OF THE TRC PROMOTER - NUCLEOTIDE-SEQUENCE OF THE LACITS GENE

We have constructed a vector, pKBi, for the high-level expression of t he variable beta chain 5.3 (V beta 5.3) of the human T-cell receptor i n Escherichia coli. This vector incorporates the trc promoter, a polyl inker, two transcription terminators, and the tetracycline resistance gene. Furthermore, the vector contains the lacIts gene that encodes a temperature-sensitive (ts) lac repressor, thus obviating both the need to use IPTG as a transcriptional inducer, and bacterial strains that harbor either the lacI or lacI(q) genes. The sequence of the lacIts ge ne shows an open reading frame of 1,080 nucleotides encoding 360 amino acids, and differs from the lacI gene at nucleotide 559 (with referen ce to the first nucleotide of the start codon). This nucleotide change s from G to A, causing amino acid residue 187 to change from glycine ( GGC) to serine (AGC). This mutation imparts thermal sensitivity to the lac repressor protein. This is the first time that a TCR V beta regio n has been expressed at high levels (up to 28 mg/liter of culture) wit hout fusion partners. The availability of the lacIts gene for thermal induction of the trc promoter, and the presence of the tetracycline re sistance gene should make the expression vector pKBi particularly attr active for the efficient production of human therapeutic proteins in b acteria.