General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase.
A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.
Kolmar
H
H
Max-Planck-Institut für Biochemie, Abteilung Zellbiologie Martinsried bei München, F.R.G.
Ferrando
E
E
Hennecke
F
F
Wippler
J
J
Fritz
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001536:Bence Jones Protein / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D001618:beta-Lactamases / Q000235:genetics
D004274:DNA, Recombinant; D007135:Immunoglobulin Variable Region; D001536:Bence Jones Protein; D001618:beta-Lactamases