AXM mutagenesis: An efficient means for the production of libraries for directed evolution of proteins

Holland, Erika G.; Buhr, Diane L.; Acca, Felicity E.; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K.; Weiner, Michael P.; Kiss, Margaret M.

doi:10.1016/j.jim.2013.05.003

Cited by 16 publications

(25 citation statements)

References 28 publications

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“…These oligonucleotides were chemically cleaved from the microarray, but the oligonucleotide yield was not sufficient for library construction without an amplification step. The oligonucleotides were amplified and the library was generated using a modified version of our published AXM cloning method (32) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Platform for high-throughput antibody selection using synthetically-designed antibody libraries

et al. 2016

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Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>1020), the practical diversity that can be achieved by transformation of E. coli is limited to about 1010. To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.

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Section: Resultsmentioning

confidence: 99%

Platform for high-throughput antibody selection using synthetically-designed antibody libraries

et al. 2016

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“…To compare the efficiency of incorporation of a single megaprimer spanning the entire scFv gene, we have generated a library of variants by our previously described AXM mutagenesis method (Holland et al, 2013 and Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…We and others have previously shown that by incorporating restriction sites in the template plasmid used for Kunkel mutagenesis, we can eliminate parental (non-recombinant) clones from the library (Holland et al, 2013; Huang et al, 2012). However, the in vitro method requires two rounds of E. coli transformations to obtain the final library and adds labor and cost to the library construction process.…”

Section: Discussionmentioning

confidence: 99%

“…The Error-Prone PCR protocol of Cadwell and Joyce (Cadwell and Joyce, 1994) as modified by Holland et al (Holland et al, 2013) was followed. In a 0.2 ml PCR tube, 10 µl of 10× mutagenic PCR buffer (70 mM MgCl 2 , 500 mM KCl, 100 mM Tris (pH 8.3), 0.1% (wt/vol) gelatin) was combined with 10 µL of 10× dNTP mix (2 mM dGTP, 2 mM dATP, 10 mM dCTP, 10 mM dTTP), 20 fmole of input DNA, 30 pmole each of forward and reverse primers, and H 2 O for a final volume of 88 µl.…”

Section: Methodsmentioning

confidence: 99%

“…We previously developed a mutagenesis technique that combines the universal nature of error-prone PCR with the efficiency of Kunkel mutagenesis in order to generate libraries up to three orders of magnitude larger than the standard error-prone PCR and subcloning approach (Holland et al, 2013). Using our method, termed AXM mutagenesis, a large, mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA.…”

Section: Introductionmentioning

confidence: 99%

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In vivo elimination of parental clones in general and site-directed mutagenesis

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The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5’-CCGCGG-3’ and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 107 from a single transformation and with greater than 90% recombinant clones.

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Gene Mutagenesis Methods

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Directed Evolution of Selective Enzymes

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AXM mutagenesis: An efficient means for the production of libraries for directed evolution of proteins

Cited by 16 publications

References 28 publications

Platform for high-throughput antibody selection using synthetically-designed antibody libraries

Platform for high-throughput antibody selection using synthetically-designed antibody libraries

In vivo elimination of parental clones in general and site-directed mutagenesis

Gene Mutagenesis Methods

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