Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display

Abstract

Homologous recombination in yeast can be exploited to reliably generate libraries of >10(7) 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 10(4)-10(5) 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.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

“…These techniques are attractive for their simplicity, as they require only a straightforward transformation step. In vivo homologous recombination has also been used extensively in the context of library generation for directed evolution applications; the highly efficient recombination machinery of organisms such as Saccharomyces cerevisiae has frequently been employed to build single-gene libraries containing 10 4 -10 10 variants (22)(23)(24)(25)(26). However, previously reported in vivo assembly techniques for multigene constructs have been low yielding, generating only tens to hundreds of recombinants at a time and making them impractical for the construction of libraries.…”

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

“…For the insertion of a (G 4 S) 3 linker between VH and VL gene, the following sequence was added to the 5 0 end of the VH reverse primer (5 0 -CGA GCC CCC GCC ACC CGA ACC GCC CCC ACC TCT-3 0 ) and the 5 0 end of the V j and V k forward primer (5 0 -GGT TCG GGT GGC GGG GGC TCG GGC GGG GGT GGC TCA GAT CT-3 0 ). To allow homologous recombination of amplified scFv inserts with pCTCON by gap repair function [15], the following sequence was added to the 5 0 end of the VH forward primer (5 0 -AGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC-3 0 ) and the 5 0 end of the V j and V k reverse primer (5 0 -TCA GAT CTC GAG CTA TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC-3 0 ). Then each amplified VH and VL family gene was purified from 1% agarose gels and quantified by absorbance at 260 nM.…”

“…The assembled scFv gene products ($800-850 bp) were purified from 1% agarose gels and concentrated using Pellet Paintä (Novagen) [12]. The scFv antibody gene libraries (10 lg) were mixed with linearized pCTCON (1 lg) by NheI/BamHI digestion, and were transformed into the yeast EBY100 strain by homologous recombination using a Bio-Rad Gene Pulser electroporation apparatus [12,15]. A total of four transformations were performed in parallel.…”

In vivo evolution of metabolic pathways: Assembling old parts to build novel and functional structures

“…Although in vivo homeologous recombination has been described in wild type cells, [29][30][31][32] there seem to be a limit in the number and size of fragments and their nucleotide divergence preventing the correct recombination of non-identical sequences in DNA repair proficient cells. It was reported that double and single strands breaks license chromatid exchanges.…”