Abstract
Directed evolution has proven to be a powerful route to enhance the stability, catalytic efficiency, or substrate specificity of enzymes. Large repertoires of enzyme mutants are generated, followed by the isolation of those enzyme variants with the desired catalytic properties. In this chapter, we outline two general methods for the efficient isolation of enzymatic activities from very large repertoires of protein variants.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Atwell, S., and Wells, J. A. (1999) Selection for improved subtiligases by phage display. Proc. Natl. Acad. Sci. USA 96, 9497–9502.
Corey, D. R., Shiau, A. K., Yang, Q., Janowski, B. A., and Craik, C. S. (1993) Trypsin display on the surface of bacteriophage. Gene 128, 129–134.
Jestin, J. L., Kristensen, P., and Winter, G. (1999) A method for the selection of catalytic activity using phage display and proximity coupling. Angew. Chem. Int. Ed. Engl. 38, 1124–1127.
Pedersen, H., Holder, S., Sutherlin, D. P., Schwitter, U., King, D. S., and Schultz, P. G. (1998) A method for directed evolution and functional cloning of enzymes. Proc. Natl. Acad. Sci. USA 95, 10,523–10,528.
Olsen, M. J., Stephens, D., Griffiths, D., Daugherty, P., Georgiou, G., and Iverson, B. L. (2000) Function-based isolation of novel enzymes from a large library. Nat. Biotechnol. 18, 1071–1074.
Vanwetswinkel, S., Avalle, B., and Fastrez, J. (2000) Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop. J. Mol. Biol. 295, 527–540.
Soumillion, P., Jespers, L., Bouchet, M., Marchand-Brynaert, J., Sartiaux, P., and Fastrez, J. (1994) Phage display of enzymes and in vitro selection for catalytic activity. Appl. Biochem. Biotechnol. 47, 175–189.
Widersten, M., and Mannervik, B. (1995) Glutathione transferases with novel active sites isolated by phage display from a library of random mutants. J. Mol. Biol. 250, 115–122.
Heinis, C., Huber, A., Demartis, S., et al. (2001) Selection of catalytically active biotin ligase and trypsin mutants by phage display. Protein Eng. 14, 1043–1052.
Demartis, S., Huber, A., Viti, F., et al. (1999) A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage. J. Mol. Biol. 286, 617–633.
de Wildt, R. M., Mundy, C. R., Gorick, B. D., and Tomlinson, I. M. (2000) Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat. Biotechnol. 18, 989–994.
Giovannoni, L., Viti, F., Zardi, L., and Neri, D. (2001) Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening. Nucl. Acid Res. 29, E27.
Dreher, M. L., Gherardi, E., Skerra, A., and Milstein, C. (1991) Colony assays for antibody fragments expressed in bacteria. J. Immunol. Meth. 139, 197–205.
Gherardi, E., Pannell, R., and Milstein, C. (1990) A single-step procedure for cloning and selection of antibody-secreting hybridomas. J. Immunol. Meth. 126, 61–68.
Skerra, A., Dreher, M. L., and Winter, G. (1991) Filter screening of antibody Fab fragments secreted from individual bacterial colonies: specific detection of antigen binding with a two-membrane system. Anal. Biochem. 196, 151–155.
Heinis, C., Melkko, S., Demartis, S., and Neri, D. (2002) Two general methods for the isolation of enzyme activities by colony filter screening. Chem. Biol. 9, 383–390.
Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J., Hudson, P., and Winter, G. (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucl. Acid Res. 19, 4133–4137.
Clackson, T., Hoogenboom, H. R., Griffiths, A. D. and Winter, G. (1991) Making antibody fragments using phage display libraries. Nature 352, 624–628.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Tesar, M., Beckmann, C., Rottgen, P., Haase, B., Faude, U., and Timmis, K. N. (1995) Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunotechnology 1, 53–64.
Montigiani, S., Neri, G., Neri, P., and Neri, D. (1996) Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement. J. Mol. Biol. 258, 6–13.
Saviranta, P., Haavisto, T., Rappu, P., Karp, M., and Lovgren, T. (1998) In vitro enzymatic biotinylation of recombinant fab fragments through a peptide acceptor tail. Bioconj. Chem. 9, 725–735.
Schatz, P. J. (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology 11, 1138–1143.
Grob, P., Baumann, S., Ackermann, M., and Suter, M. (1998) A system for stable indirect immobilization of multimeric recombinant proteins. Immunotechnology 4, 155–163.
Baumann, S., Grob, P., Stuart, F., Pertlik, D., Ackermann, M., and Suter, M. (1998) Indirect immobilization of recombinant proteins to a solid phase using the albumin binding domain of streptococcal protein G and immobilized albumin. J. Immunol. Meth. 221, 95–106.
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
Heinis, C., Bertschinger, J., Neri, D. (2003). Calmodulin-Tagged Phage and Two-Filter Sandwich Assays for the Identification of Enzymatic Activities. In: Arnold, F.H., Georgiou, G. (eds) Directed Enzyme Evolution. Methods in Molecular Biology™, vol 230. Humana Press. https://doi.org/10.1385/1-59259-396-8:313
Download citation
DOI: https://doi.org/10.1385/1-59259-396-8:313
Publisher Name: Humana Press
Print ISBN: 978-1-58829-286-5
Online ISBN: 978-1-59259-396-5
eBook Packages: Springer Protocols