Dimerization of Bence Jones proteins: linking the rate of transcription from an Escherichia coli promoter to the association constant of REIV.
Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops. Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV). The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering. Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV. By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E. coli can be derived from REIV homodimer formation constant. The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E. coli chromosome. There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter. Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E. coli for immunoglobulin variants with altered association constants.
Kolmar
H
H
Institut für Molekulare Genetik, Georg-August-Universität Göttingen, Germany.
Frisch
C
C
Kleemann
G
G
Götze
K
K
Stevens
F J
FJ
Fritz
H J
HJ
D001426:Bacterial Proteins; D001483:Base Sequence; D001536:Bence Jones Protein / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011108:Polymers; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C066719:toxR protein, bacteria; D001536:Bence Jones Protein