Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display
Homologous recombination in yeast can be exploited to reliably generate libraries of >107 transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that sha...
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Published in | Nucleic acids research Vol. 32; no. 3; p. e36 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
2004
Oxford Publishing Limited (England) |
Subjects | |
Online Access | Get full text |
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Summary: | Homologous recombination in yeast can be exploited to reliably generate libraries of >107 transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5′ homology to the cut vector and the second scFv PCR product lacked 3′ homology to the cut vector, or both PCR products had both 5′ and 3′ homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 104–105 transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast‐displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site‐specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast. |
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Bibliography: | istex:170728917F1F5B171B50E91045EA1F930ECC70EB Received December 31, 2003; Revised and Accepted January 20, 2004 local:gnh030 To whom correspondence should be addressed. Tel: +1 617 253 4578; Fax: +1 617 258 5766; Email: wittrup@mit.edu ark:/67375/HXZ-57RLDF6K-S ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0305-1048 1362-4962 1362-4962 |
DOI: | 10.1093/nar/gnh030 |