Rapid construction and characterization of synthetic antibody libraries without DNA amplification.
We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.
Ge
Xin
X
Department of Chemical Engineering, University of Texas at Austin, 78712, USA.
Mazor
Yariv
Y
Hunicke-Smith
Scott P
SP
Ellington
Andrew D
AD
Georgiou
George
G
D000906:Antibodies / Q000235:genetics* / Q000276:immunology*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D015723:Gene Library; D014644:Genetic Variation; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology; D011119:Polynucleotides / Q000138:chemical synthesis / Q000235:genetics; D012641:Selection, Genetic; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology*; D014170:Transformation, Genetic
D000906:Antibodies; D050505:Mutant Proteins; D011119:Polynucleotides; D057127:Single-Chain Antibodies