Domain libraries: synthetic diversity for de novo design of antibody V-regions.
A completely synthetic gene library encoding the variable light (VL) immunoglobulin domains has been constructed in vitro. The library was constructed by assembling a set of six oligodeoxyribonucleotides (oligos) using the polymerase chain reaction (PCR). Three out of the six overlapping oligonucleotides were synthesized with randomized complementarity determining regions (CDR) with the codon pattern, (NNS)n, where N is any of the four nucleotides (nt) and n is the number of codons with variation in the CDR. The framework regions, taken from the D1.3 anti-lysozyme antibody (Ab), were kept intact. Overlapping regions of approx. 20 nt, together with two additional flanking primers carrying the desired restriction sites, allowed the construction of a library in one single PCR reaction. The VL library was cloned into the phage display vector pEXmide3, and ten randomly picked clones were sequenced. These sequences exhibited complete diversity in all the three CDR and the codons for five canonical amino acid (aa) residues were kept intact and identified. Seven clones contained the full-length gene for the VL domain while deletions were observed in three clones. The restricted use of nt at the third position successfully avoided the stop codons TGA and TAA, whereas the stop codon TAG is read as Gln in an amber suppressor strain. We call this synthetic Ab diversity Domain Library, and it represents an example of synthetic libraries with extensive, multiple randomized sequences. The use of Domain Libraries opens up the possibility for design in Ab engineering, e.g., additional CDR regions can be added or their length varied.(ABSTRACT TRUNCATED AT 250 WORDS)
Söderlind
E
E
Department of Immunotechnology, Lund University, Sweden.
Vergeles
M
M
Borrebaeck
C A
CA
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000916:Antibody Diversity / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D015723:Gene Library; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011897:Random Allocation
D000906:Antibodies; D003062:Codon; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009113:Muramidase