Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library.
Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library. Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule. This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.
Sastry
L
L
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Alting-Mees
M
M
Huse
W D
WD
Short
J M
JM
Sorge
J A
JA
Hay
B N
BN
Janda
K D
KD
Benkovic
S J
SJ
Lerner
R A
RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003198:Computer Simulation; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D005803:Genes, Immunoglobulin; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D013154:Spleen / Q000276:immunology
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA