Rapid construction and characterization of synthetic antibody libraries without DNA amplification |
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| Authors: | Xin Ge Yariv Mazor Scott P. Hunicke‐Smith Andrew D. Ellington George Georgiou |
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| Affiliation: | 1. Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712;2. telephone: +1‐512‐471‐6975;3. fax: +1‐512‐471‐7963;4. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas;5. Department of Chemistry and Biochemistry, Microbiology, University of Texas at Austin, Austin, Texas;6. Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas;7. Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas |
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| Abstract: | 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 (VH) and light (Vκ) libraries. Four high quality, chemically synthesized polynucleotides (90–140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >109 transformants could be synthesized within 1 day. Fusion to β‐lactamase and selection on ampicillin resulted in 3.7 × 108 VH and 6.9 × 108 Vκ clones highly enriched for full‐length, in‐frame genes. High‐throughput 454 DNA sequencing of >250,000 VH and Vκ 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 VH/Vκ‐β‐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. Biotechnol. Bioeng. 2010; 106: 347–357. © 2010 Wiley Periodicals, Inc. |
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| Keywords: | combinatorial DNA library synthetic antibody high‐throughput sequencing gene assembly T4 DNA polymerase |
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