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 the variable beta chain 5.3 (V beta 5.3) of the human T-cell receptor in Escherichia coli. This vector incorporates the trc promoter, a polylinker, 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 lacIq genes. The sequence of the lacIts gene shows an open reading frame of 1,080 nucleotides encoding 360 amino acids, and differs from the lacI gene at nucleotide 559 (with reference to the first nucleotide of the start codon). This nucleotide changes 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 region has been expressed at high levels (up to 28 mg/liter of culture) without fusion partners. The availability of the lacIts gene for thermal induction of the trc promoter, and the presence of the tetracycline resistance gene should make the expression vector pKBi particularly attractive for the efficient production of human therapeutic proteins in bacteria.
Adari
H
H
Department of Molecular Biology, T Cell Sciences, Inc., Needham, MA 02194, USA.
Andrews
B
B
Ford
P J
PJ
Hannig
G
G
Brosius
J
J
Makrides
S C
SC
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D056968:Lac Repressors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013753:Tetracycline Resistance / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; D012097:Repressor Proteins