A vector for the removal of deletion mutants from antibody libraries.
To reduce the number of deletion mutants from antibody (Ab) libraries that had been amplified by PCR from peripheral blood lymphocytes, we constructed the Ab expression vector, pLAB, in which DNA coding for a single-chain Ab was inserted into the gene encoding beta-lactamase (Bla) at the 3'-terminus of its signal sequence. After transforming Escherichia coli with this vector, a fusion protein with a functional Bla domain was produced that was able to protect the bacteria from the action of ampicillin (Ap). Libraries can therefore be usefully propagated with this vector, since only those clones carrying inserts that are in frame with Bla will survive Ap selection, while others that carry out-of-frame deletions or internal stop codons are eliminated.
Seehaus
T
T
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Breitling
F
F
Dübel
S
S
Klewinghaus
I
I
Little
M
M
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D004274:DNA, Recombinant; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
D000906:Antibodies; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases