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We describe a novel expression cloning method based on screening yeast surface-displayed human cDNA libraries by direct affinity interaction to identify cellular proteins binding to a broad spectrum of target molecules. Being a eukaryote, yeast protein expression pathways are similar to those found in mammalian cells, and therefore mammalian protein fragments displayed on the yeast cell wall are more likely to be properly folded and functional than proteins displayed using prokaryotic systems. Yeast surface displayed human cDNA libraries have been successfully used to screen for proteins that bind to post-translationally modified phosphorylated peptides, small signaling molecule phosphatidylinositides, and monoclonal antibodies. In this article we describe protocols for using yeast surface-displayed cDNA libraries, coupled with fluorescence-activated cell sorting (FACS), to select protein fragments with affinity for various target molecules including post-translationally modified peptides, small signaling molecules, monoclonal phage antibodies, and monoclonal IgG molecules.
The external display of heterologous proteins or protein fragments incorporated into the Saccharomyces cerevisiae cell wall, termed yeast surface display, has been successfully utilized in various applications since the initial development of the technology by Boder and Wittrup (1). Yeast surface display technology has been most extensively applied to monoclonal antibody engineering, where it has been used to affinity mature human antibody fragments and map antibody-binding epitopes (2,3).
Recently, yeast surface-displayed human cDNA libraries have been constructed and used to screen for protein fragments with affinity for various types of molecules. In this novel expression cloning system, ligands of any chemical and molecule compositions can be used as “baits” to identify binding cellular proteins providing that the bait molecules can be labeled fluorescently or immobilized to a solid matrix. Yeast surface-displayed human cDNA libraries have been successfully used to screen for proteins that bind to post-translational modifications (phosphorylated peptides) (4), small molecules (phosphatidylinositides) (5), monoclonal antibodies (6), and serum autoantibodies (7). In this article we describe protocols for using yeast surface-displayed cDNA libraries, coupled with fluorescence-activated cell sorting (FACS), to select protein fragments with affinity for various soluble molecules.
The methods below are divided into six categories: 3.1) Growth and induction of the yeast surface-displayed cDNA expression libraries. 3.2) FACS-based selection of phosphopeptide-binding protein fragments. 3.3) FACS-based selection of phosphatidylinositide-binding protein fragments. 3.4) FACS-based selection of scFv phage antibody-binding protein fragments. 3.5) FACS-based selection of mAb IgG binding protein fragments. 3.6) Plasmid recovery from individual binding clones.
Although we provide specific examples using three different targets, these protocols can serve as a general template for designing methods to select protein fragments with affinity for any soluble molecule that can be fluorescently detected. However, each unique target molecule may require modifications or optimizations of the protocol. It is critical to do a full set of controls so that the selection progress can be monitored (see Note 2).
The work is supported by grants from the National Institute of Health (R01 CA118919, R01 CA129491, R21 CA137429 and R21 CA135586).
2The controls should include incubations with secondary reagents only (previously used and used in the current round of selection) and incubations using all previous rounds so that the enrichment of target-specific binding clones can be observed from round to round. It is also important to alternate the secondary detection agent between rounds to minimize the chances of selecting clones with affinity for them. The use of proper controls and careful monitoring of the selection process is critical to maximize the chances for success and aid in troubleshooting.
3There is always a negative population after induction when analyzed by FACS and the maximum induction will vary from experiment to experiment.
4We used a BD FACSAria for sorting and a BD LSRII for analysis. The protocols are written generally and other FACS equipment can be used.
5We generally use SA-PE in the first round because it appears to give a cleaner background than SA-647 during sorting.
6At a sort rate of 50,000 events/sec, this should take about 35 min. The first round is sorted on “faith” - there should not be an obvious population of positive cells and there may not be any significant difference between the negative control and the selection incubation. It is critical to analyze enough cells to cover the full diversity of the library or else some binding clones may be lost immediately. Since the diversity is massively diminished after the first round selection, subsequent rounds take significantly less time to sort. MACS bead selection (Miltenyi Biotec) could also be used in the first round, but we prefer to use FACS.
7We recommend that the sorted cells be directly plated without spinning them down. We have observed a poor rate of recovery when we attempt to centrifuge the cells prior to plating, even when great care is taken. We sometimes dry the SD-CAA plates overnight at RT prior to plating the sort output so that a larger volume of cells can be more quickly absorbed. The efficiency of recovery of the sort output (colonies recovered vs. events sorted) should be closely monitored, especially in the first round. Any clones lost at this step are lost for good.
8The cell number should be at least 20x more than the recovered output from the first round.
9Alternation between two detection reagents is usually sufficient to prevent the enrichment of secondary reagent binding clones. However, in the absence of real target binding clones (e.g. failed sorts), you will almost always end up with secondary binders if enough rounds of sorting are carried out.
10Sorting for too many rounds will reduce the output diversity and tend to favor high affinity or high expressing clones.
11The most efficient screening protocol will depend on the equipment available to each individual researcher. We describe a protocol based on single-tube FACS analysis, which is the format likely to be available to the most researchers. It is a simple matter to adapt the protocols to a 96 well or other more high throughput format if this equipment is available. The appropriate screening method will depend on the experimental goals of each project (i.e. strong binders vs. diversity).
12Phage particles must be in a solution that does not contain free primary amine groups that will interfere with the biotinylation reaction.
13The protocol can also be scaled up to recover plasmids from a polyclonal population. These plasmids can then be sequenced as an alternative screening method. Plasmids of interest will have to be re-transformed into EBY100 and tested for target binding.