Contents

Overview of Protein Engineering Using Cell Surface Display 24

Bacterial Surface Display 25

Overview 25

Surface Display on E. coli Bacteria 25

Surface Display on Staphylococcus Bacteria 29

Advantages and Disadvantages of Bacterial Surface Display 29

Yeast Surface Display 30

Overview 30

Yeast Surface Display Platforms 30

Advantages and Disadvantages of Yeast Surface Display 34

Emerging Cell Surface Display Systems: Insect Cell and Mammalian Cell

Display 35

Library Screening Methods 36

Overview 36

Fluorescence-Activated Cell Sorting (FACS) 36

Magnetic Bead Sorting 38

Panning 39

Considerations for Choosing a Cell Surface Display Platform for Protein

Engineering 41

Cell Surface Display Outlook 42

Acknowledgments 42

References 42

Cell surface display systems are effective tools for protein engineering by directed evolution, and show great promise in both medical and industrial applications. With this technology, combinatorial protein libraries are expressed on the surface of host cells and screened in a high-throughput manner to identify mutants with a desired phenotype. Numerous cell surface display systems have been developed using a range of host organisms, including Gram negative bacteria (Daugherty 2007), Gram positive bacteria (Wernerus and Stahl 2004), yeast (Gai and Wittrup 2007; Pepper et al. 2008), insect cells/baculovirus (Makela and Oker-Blom 2008), and mammalian cells (Beerli et al. 2008). These systems complement phage display technology and cell-free protein engineering systems, discussed in Chapters 1 and 3 of this volume, respectively. This chapter surveys the most common cell surface display formats, describes library screening methods, and discusses issues that should be considered when initiating a protein engineering project using cell surface display technology.

Currently, bacteria and yeast are the most commonly used cell surface display platforms for protein engineering. In addition to protein engineering, cell surface-displayed proteins have been used in applications such as vaccine development (Leclerc et al. 1991), bioabsorbants (Pazirandeh et al. 1998), biocatalysts (Shiraga et al. 2005), and biosensors (Shibasaki et al. 2001). Figure 2.1 outlines how cell surface display is commonly used to engineer proteins. A typical project starts with deciding on a suitable surface display platform, based on characteristics of the protein to be displayed and access to an appropriate screening method. A mutant DNA library of the protein of interest is then generated, incorporated into a display plas-mid, and transformed into the host organism. The size and diversity of the resultant library is estimated, and protein expression on the cell surface is confirmed. Using standard techniques, library sizes of up to 1011 transformants have been reported with each cell displaying thousands of identical copies of a particular

1. Create 2. Transform into host

DNA library and induce protein display *

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