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Large DNA library

In vitro transcription In vitro translation

Genotype-phenotype selection particles

Selection based on phenotype

Recovery of genotype

Small number of selected genotypes

FIGuRE 3.1 Outline of in vitro directed evolution. All of the cell-free display methods discussed in this chapter—ribosome display, mRNA display, covalent and noncovalent DNA display, and in vitro compartmentalization—follow this basic procedure. First, a large, diverse library of DNA sequences is transcribed and translated to form genotype-phenotype selection particles. The link between genotype (nucleic acid sequence) and phenotype (protein) may be direct, as in ribosome display, mRNA display, and DNA display, or indirect, as in many forms of in vitro compartmentalization. Next, a selection pressure is applied to enrich the library for members having a desired property (e.g., high binding affinity for a target molecule, stability under certain conditions, ability to modify DNA). The desired genotypes are recovered and then amplified by polymerase chain reaction (PCR) for another round of selection. Error-prone PCR, DNA shuffling, or staggered extension process (StEP) may be used to introduce additional diversity during the amplification step.

without introducing additional mutations at intermediate steps, is also used in protein engineering, although the extent of sequence space that can be sampled is much smaller (Pluckthun et al. 2000).

In both evolutionary and combinatorial approaches, each selection or screening round serves to enrich the library with members exhibiting the desired property. After several rounds, the level of enrichment may be high enough to sequence a subset of the library and thereby analyze the evolved (or selected) proteins. Significant enrichment is often observed after two to ten rounds, but this depends on a handful of variables, including the diversity of the initial library, the stringency of selection, and the extent of mutagenesis between rounds.

Chapters 1 and 2 describe phage display and cell surface display, respectively, which use a phage particle or whole cell to maintain genotype-phenotype linkage. While these methods are very powerful and have their own distinct advantages, there are a number of cell-free display technologies that have been adapted from cellfree protein synthesis methods and do not require whole cells at any step. Not using cells is an advantage in terms of obtainable library size, time, fewer restraints on translation and selection, reduced expression bias, and ease of introducing diversity between rounds (see the section titled "In Vitro versus In Vivo Display" in this chapter). The following cell-free methods are discussed in detail in this chapter and summarized in Table 3.1: ribosome display, mRNA display, covalent and noncovalent DNA display, and in vitro compartmentalization. Each uses a different method for linking genotype and phenotype, but their underlying principles are similar. The next section describes some general factors to consider in choosing a cell-free display platform, designing the nucleic acid template, and performing the selections or screens.

CONSIDERATIONS IN CELL-FREE DISPLAY AND SELECTION/SCREENING Type of selection or screen

Many different properties of proteins may be engineered using in vitro techniques (Table 3.1). In general, ribosome display, mRNA display, and covalent and noncova-lent display methods are equally well suited to improve protein equilibrium affinity, off-rate, stability, and folding. They all rely on a binding event between the displayed protein and an immobilized ligand to perform the selection. In vitro compartmen-talization methods, in contrast, are primarily useful for the evolution of enzymes, which bind only transiently to their substrate. Water-in-oil-in-water emulsions, in particular, are notable for their ability to be screened by fluorescence-activated cell sorting (FACS), provided that a fluorescence-based assay is available for the detection of the desired activity. Clearly, the goal of the selection or screen will narrow down the choice of display technique.

Binding-based selections are very popular in directed evolution and warrant a brief explanation. First, the displayed library must be allowed to bind to an immobilized target molecule on a solid support (typically, an assay plate or beads). Unbound or weakly bound library members are washed away, so that only firmly bound library members remain. The method by which these members are recovered depends on the cell-free display technology used, but always involves PCR or reverse transcription followed by PCR (RT-PCR) to amplify enriched sequences. The washing times are relatively short in the early rounds to avoid losing valuable yet sparse clones due to stochastic effects. As the library becomes enriched, however, redundancy within the pool allows for more stringent washing. Both high and low affinity binders can be identified by examining the pool of binders before it is dominated by only a few sequences (Huang and Liu 2007).

An important variation on this basic affinity selection is off-rate selection. Selecting for slower off-rates leads to increased binding affinity, as on-rates are relatively constant for proteins (Northrup and Erickson 1992). After the washing steps, bound library members are allowed to dissociate from the immobilized target in the presence of a large excess of soluble target. Lower-affinity binders dissociate more rapidly from the immobilized target and are far more likely to bind to target molecules in solution, thus minimizing their rebinding to the immobilized target. After a given wait time, the solid support is washed, leaving only the highest affinity library members bound. These are amplified and carried forth to the next round. As usual, the selection pressure (time allowed for dissociation, in this case) is gradually increased.

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