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Construction and flow cytometric screening of targeted enzyme libraries

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.

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Figure 1: The concept of expanding zone to create libraries of increasing complexity.
Figure 2
Figure 3: Two-color sorting strategy.

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Acknowledgements

We thank Mark Pogson for assistance in preparing the figures.

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Correspondence to George Georgiou or Brent L Iverson.

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)

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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

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