Abstract
Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled (“parental”) molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation before shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of β-xylanase genes that possess widely different G and C contents.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Crameri, A., Whitehorn, E. A., Tate, E., and Stemmer, W. P. C. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319.
Stemmer, W. P. C. (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391.
Stemmer, W. P. C. (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. USA 91, 10,747–10,751.
Schmidt-Dannert, C., Umeno, D., and Arnold, F. H. (2000) Molecular breeding of carotenoid biosynthetic pathways. Nature Biotechnol. 18, 750–753.
Kikuchi, M., Ohnishi, K., and Harayama, S. (1999) Novel family shuffling methods for the in vitro evolution of enzymes. Gene 236, 159–167.
Morris, D. D., Gibbs, D. D., Chin, C. W., et al. (1998) Cloning of the xynB gene from Dictyoglomus thermophilum strain Rt46B.1 and action of the gene-product on kraft pulp. Appl. Environ. Microbiol. 64, 1759–1765.
Gibbs, M. D., Nevalainen, K. M. H., and Bergquist, P. L. (2001) Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling. Gene 271, 13–20.
Sakka, K., Kojima, Y., Kondo, T., Karita, S., Ohmiya, K., and Shimada, K. (1993) Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A: identification of catalytic and cellulose binding domains. Biosci. Biotechnol. Biochem. 57, 273–277.
Yang, V. W., Zhuang, Z., Elegir, G., and Jeffries, T. W. (1995) Alkaline-active xylanase produced by an alkaliphilic Bacillus sp isolated from kraft pulp. J. Ind. Microbiol. 15, 434–441.
Morris, D. D., Gibbs, M. D., Ford, M., Thomas, J., and Bergquist, P. L. (1999) Family 10 and 11 xylanase genes from Caldicellulosiruptor isolate Rt69B.1. Extremophiles 3, 103–111.
Fernandes, A. C., Fontes, C. M., Gilbert, H. J., Hazlewood, G. P., Fernandes, T. H., and Ferreira, L. M. (1999) Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes. Biochem. J. 342, 105–110.
Elegir, G., Szakacs, G., and Jeffries, T. W. (1994) Purification, characterization and substrate specificity of multiple xylanases from Streptomyces sp strain B-12-2. Appl. Environ. Microbiol. 60, 2609–2615.
Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., McCallum, C. M., and Henikoff, S. (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26, 1628–1635.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882.
Rice, P., Longden, I., and Bleasby, A. (2000) EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.
Teather, R. M. and Wood, P. J. (1982) Use of Congo Red polysaccharide interaction in enumeration and characterisation of cellulolytic bacteria from bovine rumen. Appl. Environ. Microbiol. 43, 777–780.
Lever, M. (1973) Colorimetric and fluorometric carbohydrate determination with p-hydroxybenzoic acid hydrazide. Biochem. Med. 7, 274–281.
Britton, H. T. S. and Robinson, R. A. (1931) Universal buffer solutions and the dissociation constant of veronal. J. Chem. Soc. 1, 1456–1462.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Bergquist, P.L., Gibbs, M.D. (2007). Degenerate Oligonucleotide Gene Shuffling. In: Arndt, K.M., Müller, K.M. (eds) Protein Engineering Protocols. Methods in Molecular Biology™, vol 352. Humana Press. https://doi.org/10.1385/1-59745-187-8:191
Download citation
DOI: https://doi.org/10.1385/1-59745-187-8:191
Publisher Name: Humana Press
Print ISBN: 978-1-58829-072-4
Online ISBN: 978-1-59745-187-1
eBook Packages: Springer Protocols