Protein Engineering Protocols - Mycobacteriology research center

192 Bergquist and GibbsIf sequence similarity is low between the input genes, the majority of productstend to be the reassembled parental genes, and extensive searches need tobe performed to find the chimeric recombinants (4,5). Kichuchi et al. describeda method for gene shuffling that makes use of unique restriction enzyme sitesin the sequences of the parental molecules (5). The complete restriction enzymedigestion of parental genes ensured that subsequent overlap extension gave riseto hybrid genes at high frequencies.We isolated a gene coding for a thermophilic β-xylanase that had superiorperformance in the bleaching of paper pulp (6). We wished to investigate thepossibility of obtaining mutant derivatives that had enhanced stability and analtered pH optimum. Experiments using error-prone PCR and misincorporationmutagenesis followed by gene shuffling allowed the identification of mutantgenes that coded for a limited sample of the variations in sequence space butrequired extensive screening for their identification. Gene shuffling after DNaseI fragmentation of related genes (family shuffling) overwhelmingly yieldedwild-type parental sequences as the major products. After several trials of methodsdesigned to reduce the background, we devised a technique that allows shufflingof genes that differ widely in sequence similarity and G and C content, andgreatly reduces the regeneration of wild-type genes. Furthermore, the primerextension conditions may be modified to bias the resulting progeny genestoward any one (or more) of the parental input genes. We term this proceduredegenerate oligonucleotide gene shuffling (DOGS; ref. 7), and note its compatibilitywith other recursive point mutation techniques.2. Materials1. Source of genes: Family 11 xylanase genes were obtained from the following bacterialstrains: Dictyoglomus thermophilum strain Rt46B.1 xynB (8); Clostridiumstercorarium xynB (9); Bacillus sp. strain V1-4 (10); Caldicellulosiruptor sp. strainRt69B.1 xynD (11); Clostridium thermocellum xynV (12); and Streptomyces roseiscleroticusxyl3 (13). Each gene was PCR amplified from genomic DNA using therespective gene-specific primers.2. Platinum Pfx polymerase (Invitrogen, Victoria, Australia).3. Plasmid pBSII KS– (Stratagene, San Diego, CA).4. Shrimp alkaline phosphatase (Roche Diagnostics Australia, NSW, Australia).5. Escherichia coli strain DH5α.6. Universal buffer: 50 mM phosphoric acid, 50 mM boric acid, 50 mM acetic acid.Adjust pH with NaOH.7. Overlay solution: 0.5% birchwood xylan (Sigma-Aldrich, Sydney, Australia),0.5% agarose in 120 mM Universal buffer, pH 6.5. Mix, then autoclave to completelysolubilize the xylan and agarose. Allow mixture to cool to approx 50°C.8. Congo Red solution: 1% Congo Red (Sigma-Aldrich, Sydney, Australia) in water.Ensure the pH is slightly alkaline by addition of NaOH to approx 5 mM.