Abstract
In vitro polymerase chain reaction (PCR)-based recombination methods are used to shuffle segments from various homologous DNA sequences to produce highly mosaic chimeric sequences. Genetic variations created in the laboratory or existing in nature can be recombined to generate libraries of molecules containing novel combinations of sequence information from any or all of the parent template sequences. Evolutionary protein design approaches, in which libraries created by in vitro recombination methods are coupled with screening (or selection) strategies, have successfully produced variant proteins with a wide array of modified properties including increased drug resistance (1,2), stability (3–6), binding affinity (6), improved folding and solubility (7), altered or expanded substrate specificity (8,9), and new catalytic activity (10).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Stemmer, W. P. (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 81, 10,747–10,751.
Crameri, A., Raillard, S. A., Bermudez, E., and Stemmer, W. P. C. (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291.
Giver, L., Gershenson, A., Freskgard, P. O., and Arnold, F. A. (1998) Directed evolution of a thermostable esterase. Proc. Natl. Acad. Sci. USA 95, 12,809–12,813.
Zhao, H. M. and Arnold, F. H. (1999) Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng. 12, 47–53.
Miyazaki, K., Wintrode, P. L., Grayling, R. A., Rubingh, D. N., and Arnold, F. H. (2000) Directed evolution study of temperature adaptation in a psychrophilic enzyme. J. Mol. Biol. 297, 1015–1026.
Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J., and Pluckthun, A. (2001) Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. USA 98, 75–80.
Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999) Rapid protein-folding assay using green fluorescent protein. Nat. Biotech. 17, 691–695.
Zhang, J. H., Dawes, G., and Stemmer, W. P. C. (1997) Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94, 4504–4509.
Kumamaru, T., Suenaga, H., Mitsuoka, M., Watanabe, T., and Furukawa, K. (1998) Enhanced degradation of polychlorinated biphenyls by directed evolution. Nat. Biotech. 16, 663–666.
Altamirano, M. M., Blackburn, J. M., Aguayo, C., and Fersht, A. R. (2000) Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold. Nature 403, 617–622.
Stemmer, W. P. C. (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391.
Zhao, H., Giver, L., Shao, Z., Affholter, J. A., and Arnold, F. H. (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261.
Shao, Z., Zhao, H., Giver, L., and Arnold, F. H. (1997) Random-priming in vitro recombination: an effective tool for directed evolution. Nucl. Acids Res. 26, 681–683.
Volkov, A. A. and Arnold, F. H. (2000) Methods for in vitro DNA recombination and random chimeragenesis. Meth. Enzymol. 28, 447–456.
Ninkovic, M., Dietrich, R., Aral, G., and Schwienhorst, A. (2001) High-fidelity in vitro recombination using a proofreading polymerase. BioTechniques 30, 530–536.
Cline, J., Braman, J. C., and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551.
Judo, M. S. B., Wedel, A. B., and Wilson, C. (1998) Stimulation and suppression of PCR-mediated recombination. Nucleic Acids Res. 26, 1819–1825.
Kong, H., Kucera, R. B., and Jack, W. E. (1993) Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J. Biol. Chem. 268, 1965–1975.
Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. D. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 436–9440.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Aguinaldo, A.M., Arnold, F.H. (2003). Staggered Extension Process (StEP) In Vitro Recombination. In: Arnold, F.H., Georgiou, G. (eds) Directed Evolution Library Creation. Methods in Molecular Biology™, vol 231. Humana Press. https://doi.org/10.1385/1-59259-395-X:105
Download citation
DOI: https://doi.org/10.1385/1-59259-395-X:105
Publisher Name: Humana Press
Print ISBN: 978-1-58829-285-8
Online ISBN: 978-1-59259-395-8
eBook Packages: Springer Protocols