Construction of combinatorial antibody expression libraries in Escherichia coli.
A lambda vector system has been developed for the construction and coexpression of diverse populations of heavy and light chain cDNA sequences. Heavy and light chain sequences within the immunological repertoire are generated by the polymerase chain reaction using primers directed to conserved regions within the variable region framework. Two lambda vectors are employed for the independent construction of heavy and light chain cDNA libraries. The libraries are randomly combined to produce a population of lambda phage with each containing one heavy and one light chain cDNA sequence. The vectors direct the synthesis and secretion of functional Fab antibody fragments from a dicistronic operon. Libraries of up to 1 x 10(7) recombinants can be obtained with a diversity approaching in vivo estimates. Analysis of the heavy and light chain combinations reveals that the complete antibody repertoire can be generated by subsequent reshuffling of heavy and light chain cDNAs within an initial Fab-producing library. Inherent bias found in vivo toward certain heavy and light chain combinations can be virtually eliminated.
Huse
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Ixsys Inc, San Diego, CA 92121.
D000906:Antibodies / Q000235:genetics*; D000917:Antibody Formation / Q000235:genetics; D010582:Bacteriophage lambda; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D015698:Genomic Library; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins