Abstract
Herein, we describe a methodology for the construction of targeted libraries intended to modify the substrate specificity of proteases expressed on the cell surface of Escherichia coli. The native outer membrane protease, OmpT, is used as a model system. The protocol relies on gene assembly using oligonucleotides and is easily adaptable to any enzyme in which information is available on the putative active site residues. Increasingly complex libraries can be generated in a systematic manner and screened using flow cytometry (fluorescence-activated cell sorting, FACS) for variants displaying altered function. Furthermore, if the substrate-binding pockets have not been elucidated, a protocol for partial multi-site saturation library construction is presented that allows for sampling a large number of residues, while maintaining an appropriate level of protein function. The entire procedure, from start to finish, should take approximately 2–3 weeks.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Yep, A., Kenyon, G.L. & McLeish, M.J. Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism. Proc. Natl. Acad. Sci. USA 105, 5733–5738 (2008).
Kaur, J. & Sharma, R. Directed evolution: an approach to engineer enzymes. Crit. Rev. Biotechnol. 26, 165–199 (2006).
Park, H.S. et al. Design and evolution of new catalytic activity with an existing protein scaffold. Science 311, 535–538 (2006).
Rothlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008).
Ballinger, M.D., Tom, J. & Wells, J.A. Furilisin: a variant of subtilisin BPN' engineered for cleaving tribasic substrates. Biochemistry 35, 13579–13585 (1996).
Bloom, J.D. et al. Evolving strategies for enzyme engineering. Curr. Opin. Struct. Biol. 15, 447–452 (2005).
Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).
Griswold, K.E. et al. Evolution of highly active enzymes by homology-independent recombination. Proc. Natl. Acad. Sci. USA 102, 10082–10087 (2005).
Martineau, P. Error-prone polymerase chain reaction for modification of scFvs. Methods Mol. Biol. 178, 287–294 (2002).
Bornscheuer, U.T. & Pohl, M. Improved biocatalysts by directed evolution and rational protein design. Curr. Opin. Chem. Biol. 5, 137–143 (2001).
Khersonsky, O., Roodveldt, C. & Tawfik, D.S. Enzyme promiscuity: evolutionary and mechanistic aspects. Curr. Opin. Chem. Biol. 10, 498–508 (2006).
Varadarajan, N., Rodriguez, S., Hwang, B.Y., Georgiou, G. & Iverson, B.L. Highly active and selective endopeptidases with programmed substrate specificities. Nat. Chem. Biol. 4, 290–294 (2008).
Morley, K.L. & Kazlauskas, R.J. Improving enzyme properties: when are closer mutations better? Trends Biotechnol. 23, 231–237 (2005).
Cobaugh, C.W., Almagro, J.C., Pogson, M., Iverson, B. & Georgiou, G. Synthetic antibody libraries focused towards peptide ligands. J. Mol. Biol. 378, 622–633 (2008).
Reetz, M.T. & Carballeira, J.D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2, 891–903 (2007).
Robertson, D.E. & Steer, B.A. Recent progress in biocatalyst discovery and optimization. Curr. Opin. Chem. Biol. 8, 141–149 (2004).
Varadarajan, N., Gam, J., Olsen, M.J., Georgiou, G. & Iverson, B.L. Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proc. Natl. Acad. Sci. USA 102, 6855–6860 (2005).
Herman, A. & Tawfik, D.S. Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries. Protein Eng. Des. Sel. 20, 219–226 (2007).
Balint, R.F. & Larrick, J.W. Antibody engineering by parsimonious mutagenesis. Gene 137, 109–118 (1993).
Santoro, S.W., Wang, L., Herberich, B., King, D.S. & Schultz, P.G. An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat. Biotechnol. 20, 1044–1048 (2002).
Vandeputte-Rutten, L. et al. Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site. EMBO J. 20, 5033–5039 (2001).
Daugherty, P.S. Protein engineering with bacterial display. Curr. Opin. Struct. Biol. 17, 474–480 (2007).
Korbie, D.J. & Mattick, J.S. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat. Protoc. 3, 1452–1456 (2008).
Abecassis, V. et al. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Res. 28, E88 (2000).
Acknowledgements
We thank Mark Pogson for assistance in preparing the figures.
Author information
Authors and Affiliations
Corresponding authors
Supplementary information
Supplementary Figure 1
List of oligonucleotides used for the gene-assembly of OmpT. Also shown are the primers used for the construction of the three-member saturation library (Glu27, Asp208 and Ser223). Restriction enzyme recognition sites are in bold and the codons that encode for Glu27, Asp208 and Ser223 are underlined. (PDF 9 kb)
Supplementary Figure 2
DNA sequence of the OmpT gene. The shaded regions indicate the breakdown of the gene into the complete forward primer set. The DNA sequence in each shaded fragment can be sequentially matched back to the forward primers (1f-24f) listed in Suppl Fig S1. (PDF 9 kb)
Supplementary Figure 3
3-D fluorescence histograms of E. coli cells through the different rounds of sorting. Cells expressing an OmpT three-member saturation library (Glu27, Asp208 & Ser223) was simultaneously labeled with 20nM selection substrate (harboring a Glu-Arg cleavage site) and 100 nM counter-selection substrate (harboring the wild-type Arg-Arg cleavage site) and subjected to five rounds of flow-cytometric sorting. Red: Pre-sort library, blue: Round-1 sort, purple: Round-2 sort, cyan: Round-3 sort, yellow: Round-4 sort. (PDF 210 kb)
Rights and permissions
About this article
Cite this article
Varadarajan, N., Cantor, J., Georgiou, G. et al. Construction and flow cytometric screening of targeted enzyme libraries. Nat Protoc 4, 893–901 (2009). https://doi.org/10.1038/nprot.2009.60
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2009.60
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.