The multivalency of yeast display was coupled with multivalent target presentation utilizing a variety of solid surfaces, as diagrammed in , to identify a selection method capable of isolating rare, weak interactions among large nonbinding populations. Each surface was coated with a target molecule and then incubated with a mixture of yeast, a small fraction of which expressed a protein capable of binding to the target. Unbound yeast were removed by washing the surface and the captured yeast were analyzed in order to determine the ability of the surface to enrich binding clones from the initial mixture.
Profile of multivalent selection methods
Profile of materials tested
presents the ability of each surface tested to enrich binding yeast. Nonfunctionalized surfaces were coated by passive adherence of the target protein, lysozyme, to the surface, and the lysozyme-binding fibronectin clone L3.3.1 (nanomolar affinity) was used as the binding population. Cobalt, or Talon® conjugated surfaces were simply washed and then used to select yeast with a surface displayed his6 tag. Streptavidin coated surfaces were incubated with biotinylated lysozyme and used to enrich L3.3.1-expressing yeast. The surfaces tested included nitrocellulose, several plastics, glass, agarose, and magnetic beads. Binding yeast were mixed with a 10 to 1,000-fold excess of nonbinding yeast, such as EBY100 yeast lacking any display construct plasmid, or yeast expressing a nonbinding construct. Surfaces were incubated with the mixed yeast population, washed to remove unbound yeast, and the selected yeast populations were then either plated or analyzed by FACS to determine the final prevalence of binding clones. The ability of each surface to select binding yeast was determined by evaluating the enrichment of the binding clone population (final prevalence/initial prevalence), which could then be compared to the theoretical maximum enrichment to purity. For all materials, a number of wash and incubation conditions were tested, and several materials, including each of the functionalized surfaces, had some ability to enrich binding yeast.
Interestingly, none of the passively coated materials were able to enrich the lysozyme binding clone L3.3.1. This inability may be due to our use of a relatively small protein target and a binder with a conformationally sensitive epitope. Passive adherence may have partially or completely denatured the lysozyme and destroyed the epitope recognized by L3.3.1. Although larger proteins may avoid complete denaturation when passively attached, partial unfolding is still likely to both abolish native and generate novel epitopes—a highly undesirable presentation of the target antigen in a de novo selection method. So, although a tag-free means to perform selections is desirable, the use of tag is preferable to altering the conformation of the target antigen—especially as a tag may be necessary for purification of the antigen even if unnecessary for the selection. Additionally, if the tag is present at only a single site and is used to immobilize the target on the selection material, much of its surface may be buried or occluded.
The two surfaces with the best selective profiles identified from these initial tests were agarose and magnetic beads, and these materials were characterized more extensively. First, a mixture of yeast expressing either a his6 tagged scFv or a c-myc tagged fibronectin domain at a 1:4 ratio were incubated with Talon agarose in order to test enrichment of the his6-expressing population. The yeast:bead slurry was poured into a column and washed and eluted. Fractions were then labeled for FACS analysis to determine the relative prevalence of each population in the various fractions. presents the percentage of each yeast population present in each column fraction and clearly shows the ability of agarose beads in a column format to be used in selections.
As a direct means of comparison, his6-tagged yeast were mixed with nonbinding yeast at a ratio of 1 in 1,000 and then applied to both agarose and magnetic Talon-functionalized beads. presents the profile of the initial population as well as that of the yeast eluted from each type of bead. Despite being present below the threshold of detection in the initial population (red trace), binding yeast have been enriched to approximately 1 in 4 yeast by agarose beads (blue trace), and to near purity by magnetic beads (green trace), indicating the excellent ability of these materials to isolate binders.
FACS profile of selected populations
However, since one of the criteria for the selection methodology is use of a minimum amount of antigen, agarose beads, which by design have a high binding capacity due to extensive porosity, while ideal for protein purification, are nonideal for target antigen presentation as many of these binding sites are likely to be inaccessible to the yeast. A less porous surface with fewer contours, such as magnetic beads is better suited for efficient presentation of the target antigen in this regard.
Having demonstrated the ability of several multivalent surfaces to isolate binding yeast, we next determined whether magnetic beads, the most promising of these surfaces, met the other criteria for naïve library selections.
Testing criteria for successful de novo selections
First, we desired a method capable of isolating extremely weak binders, so magnetic beads were coated with lysozyme and used to select a series of lysozyme-binding fibronectin domains with affinities spanning a million-fold range from uM to pM. The weakest clone, L0.7.1, was identified by FACS selections of soluble multivalent lysozyme,10
and has such a low affinity that it could not accurately be measured by FACS, while the tightest binder, L7.5.1 interacts with 3 pM affinity. presents the enrichment of lysozyme binding clones from nonbinding clones at a starting ratio of 1:1,000 (gray bars). The million-fold decrease in affinity has no effect on the ability of the beads to select the binding clone.
Efficiency of magnetic bead sorting
In contrast, when yeast are first incubated with soluble lysozyme and then quickly washed and allowed to bind to beads as was done previously in the protocol of Yeung and Wittrup,22
enrichment is highly dependent on affinity (black bars). In fact, this seemingly minor protocol alteration drastically reduces the ability to enrich the clone with the weakest binding, which is likely most representative of the binders present in naïve libraries. In each case, selection depends on the ability to generate and maintain an interaction between yeast and bead, and the ability of each yeast to interact multivalently with a bead is dependent on the surface density of the interacting molecules. In utilizing a soluble target incubation, multivalency is limited by affinity as only some subset of surface displayed molecules will be bound to the target antigen, and capable of interacting with the beads. Alternatively, the degree of multivalency that can be achieved using target-coated beads is set by either the display level of the yeast, or the coating density of the target, and is therefore an inherent property of system, and these parameters can be set to maximize possible valency.
As a means to estimate the lower affinity limit for capture with a target-coated surface, a rough calculation of the effective concentration of the target can be made by quantifying the amount of antigen on each bead and dividing that value by the bead volume, giving an approximate local concentration. The magnetic beads used in this study present the target antigen at an effective local concentration in the millimolar range, and therefore may be able to isolate extremely weak interactions.
Having established the ability to isolate very weak interactions, we investigated the effect of rarity on enrichment. Binding and nonbinding yeast were mixed at ratios varying from 1:10 to 1:100,000 and were bead selected, resulting in the enrichment values presented in . For both interactions between a his6 tag and chelated cobalt, and the L0.7.1 fibronectin clone and lysozyme, enrichment scaled well with the theoretical maximal enrichment for each initial prevalence (lines), with a maximal increase in prevalence from 1 in 100,000 cells to 1 in 3 (30,000-fold enrichment). Thus, magnetic bead selection easily captures rare clones.
One of the limits of FACS selections is that it restricts the size of a population that can be sorted; thus, the number of yeast that can be screened effectively by magnetic beads was investigated. Lysozyme-binding (L0.7.1) and nonbinding yeast were mixed at a ratio of 1:1000 at total population sizes varying from 5 × 107 to 4 × 109, and enrichment values after 1 and 3 washes were determined for each population (). Consistent enrichment values were found across all population sizes, and demonstrate that four billion yeast can be straightforwardly surveyed in a single micro-centrifuge tube in 1 h. Whereas scaling up FACS selections requires either multiple sorters or significantly more time, magnetic bead selections can be scaled up by simply using a larger vessel or running several microcentrifuge tube-sized selections in parallel—allowing even the largest yeast libraries to be screened to high coverage multiplicity with ease.
To evaluate the flexibility of magnetic bead selections, the ability to enrich clones displaying several different scaffolds capable of interacting with various targets was investigated. shows the enrichment values of clones expressing either fibronectin or scFv domains that recognize both small molecule and protein targets. Proteins were immobilized on streptavidin-coated beads via a biotin tag, while small molecules were chemically conjugated to amine-functionalized beads. Binding yeast were efficiently isolated from a larger population of nonbinding yeast, independently of their displayed scaffold, size of the target antigen, and whether the targets were chemically conjugated or immobilized via a tag.
Finally, we tested the yield of bead selections in order to determine the probability that each binding yeast cell would be captured. Yield is a critical selection parameter in two respects. The chances of capturing each binding yeast will first influence the coverage multiplicity used, and secondly determine the ability of the method to act as an effective negative selection, wherein yeast with undesirable binding properties are removed from the library population. shows the percent of binders present that were recovered from selections of binding yeast (L0.7.1) present at a ratio of 1 in 1,000 nonbinders. In most cases, more than three-quarters of the binders present were successfully recovered, and this fraction declined significantly only when the number of binders present was comparable to the binding capacity of the beads (several million binders) decreasing the likelihood that binding yeast would encounter available binding sites.
Similarly, shows the number of yeast isolated from repeated rounds of selection against streptavidin. After each selection, yeast in the supernatant were reapplied to fresh streptavidin beads. The number of binders isolated from each round is approximately equal to 80% of the total number present. Thus, after 4 rounds of selection, ~99.8% of streptavidin-binding clones have been captured, leaving only 0.16% in the population. The ability to thoroughly deplete the population of streptavidin binders via such negative selection serves as a highly efficient means to constrain the undesirable selection of reagent binders and steer the selective pressure of each positive selection toward the intended target. This restraint of alternative solutions to the selective pressure represents a significant advantage over negative selections by FACS, particularly as extremely weak interactions can be captured and because the yeast do not require subsequent regrowth and induction before positive selection. Therefore, negative selections using magnetic beads coated with reagents would be useful when paired with any method of positive selection in order to improve the chances of isolating the desired binding interaction.
Thus far, we have demonstrated that magnetic beads can be used to isolate highly rare and very weak binders to both protein and small molecule targets with high quantitative yield from very large populations. To further profile the factors affecting these selective abilities, we carried out a series of experiments in which parameters such as the time of incubation and surface density of interacting molecules were varied. presents the enrichment of lysozyme binding clones (L0.7.1) from nonbinding yeast after 1 wash when incubated at a starting ratio of 1:1,000 for varying periods of time. The magnetic beads rapidly isolate the binding population, showing near maximal enrichment after 1 h.
Dependencies of magenetic bead sorting
Additionally, since the binding affinities that can be captured using this method are so low, and because in most tests the nonbinding population consisted of yeast that did not express a surface-displayed construct, we undertook extensive testing of negative controls in order to ensure that only specific interactions were selected. Beads coated with a given target were used in tests against yeast expressing constructs capable of interactions with other targets. Four such mismatched pairs were tested, and all prevalences after selection were comparable to the initial prevalence of 1 in 1,000 (), demonstrating that the enrichment of binding yeast represents a specific interaction and is not an artifact of a biased comparison, nor the effect of a promiscuous system.
Lastly, as described previously, this method depends on the number of molecular interactions or degree of multivalency that can be achieved, and is therefore predicted to be impacted by both the density of the target ligand coating the beads, and the density of the construct displayed on the surface of the yeast. Accordingly, show the effect of construct display level and target presentation level on enrichment. As expected, the higher the surface density of interacting molecules—whether limited by the bead or the yeast, the more likely a multivalent interaction can be achieved resulting in higher enrichment values. Because the yeast expression level is a significant factor in determining enrichment, care should be taken to ensure use of cultures that have been induced to express as highly as possible.
Demonstrations of method utility in de novo isolation
To fully test the method, we also sought to select de novo binders from two naïve libraries, including the same fibro-nectin library utilized to generate the clones presented in Hackel et al., using lysozyme-coated beads as opposed to the FACS methodology applied previously, allowing direct comparison of the two methods. In the bead selections carried out here, each library was first depleted of streptavidin or bead binders by an initial incubation with beads as a means to avoid enriching reagent-binding clones. Nonbinding yeast were removed from the supernatant and applied to beads coated with lysozyme. These beads and the yeast bound to them were then directly transferred into growth media and subsequently induced for the next round of the process. After two such selections, a FACS sort to isolate c-myc positive clones was performed to remove truncated clones from the population. The clones were diversified as described,10
and the resulting population was bead selected twice followed by a c-myc positive FACS sort. Several clones were isolated, sequenced, and characterized.
Interestingly, L0.7.1, the initial binder isolated by FACS selections was also identified in these bead sorts. However, this clone was not the dominant member (present once in eight sequenced clones) of the selected population, and therefore it appears as though the bead selections were able to identify additional binders that were not isolated by FACS. As we cannot determine accurate affinity values by FACS for such weak binders, it is unclear whether these additional clones represent stronger binders that happened to be missed by FACS, or weaker ones that FACS could not isolate. In either case, the inclusion of additional diversity in subsequent rounds of evolution is likely to be beneficial, especially given the use of a loop shuffling protocol.
A second fibronectin library was bead sorted in parallel, and clones with nM binding affinities were isolated following the same protocol—allowing isolation of validated lysozyme-binding clones from two different libraries in 1 week using only about 100 pmol (10 µg) of target antigen. The supplemental information contains additional instructions for performing selection from naive libraries.
Additionally, as a means to demonstrate the excellent ability of the method to function in negative selections, bead selections were used to isolate clones capable of discriminating between streptavidin and biotin-streptavidin. Multiple negative selections against streptavidin with its biotin-binding site unoccupied were performed prior to each positive selection against biotin-streptavidin. After six selections and two rounds of mutagenesis, clones with nanomolar binding affinity to biotin-streptavidin, but undetectable affinity to streptavidin were isolated (data not shown). This result shows the robust nature of beads in both positive and negative selections, and the ability to isolate interactions of interest with a high degree of specificity.
The excellent capabilities of this multivalent selection method establish it as the method of choice for de novo selections. Bead-based selections followed by mutagenesis can be iterated, and the success of each round can be assessed by comparing binding to reagent-coated beads to antigen-coated beads. Once a population displays increased binding to the target antigen, it can be mutagenized and selection stringency can be increased in the following rounds. Stringency may be tuned by altering the duration and frequency of wash steps, decreasing expression of the surface-displayed construct or decreasing antigen density on the beads. Alternatively, as long as thorough negative selections are performed, a soluble antigen incubation as in the method described by Yeung and Wittrup can be utilized to increase stringency. Once FACS-detectable (nM) binders have been isolated via rounds of bead selection with increasing stringency, affinity maturation can proceed via FACS. presents a schematic diagram of the process for generating high affinity binders with high efficiency from naïve libraries.
Selection profile for successful molecular evolution
Overall, by allowing multivalent presentation of the target antigen locally on a particle surface, the bead selection method described here is able to sort large populations and quantitatively isolate even weak binders in a highly rapid and efficient manner.