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Using yeast display, heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall. Yeast display can be used to screen large expressed protein libraries for proteins or protein fragments with specific binding properties. Recently, yeast surface displayed cDNA libraries have been constructed and used to identify proteins that bind to various target molecules such as peptides, small molecules, and antibodies. Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Coupled with fluorescence-activated cell sorting (FACS), yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface displayed cDNA libraries using pre-existing yeast two-hybrid cDNA libraries as a starting point.
Various techniques have been developed to screen large expressed protein libraries for proteins or protein fragments with specific binding properties, including the yeast two-hybrid system and phage display (1–9). However, these systems have limitations. Because it relies on the internal coexpression of “bait” and “prey” fusion proteins, the two-hybrid system cannot be used to identify proteins that bind to externally synthesized or modified proteins or compounds. Phage display is limited by potential expression bias against eukaryotic proteins expressed in a prokaryotic host and the low number of fusion proteins displayed on each phage particle (2, 6). To address these limitations, yeast surface-displayed libraries of human cDNA fragments can be utilized. Heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall (10), and yeast surface display technology has been successfully used to affinity mature human antibody fragments and map antibody-binding epitopes (11,12). Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Yeast surface displayed human cDNA libraries have been successfully used to screen for proteins that bind to various target molecules (13–16). Coupled with fluorescence-activated cell sorting (FACS), yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface displayed cDNA libraries using pre-existing yeast two-hybrid cDNA libraries as a starting point.
The methods below are divided into four categories: 3.1) Generation of frameshift variants of pYD1 vector. 3.2) Library construction. 3.3) Transformation of library into yeast. 3.4) Test library induction. The construction of yeast display libraries and the generation of frameshifted variants of the original pYD1 yeast display vector have been described previously (13,14). The pYD1 frameshift variants theoretically allow for the in-frame expression of a larger number of cDNA inserts. The cDNA in the protocol we describe comes from pre-made Invitrogen cDNA libraries designed for yeast two-hybrid experiments. We chose libraries designed for two-hybrid experiments because they have been random-primed and size-selected (0.3 – 1.5kb) and will thus display domain-sized protein fragments that may be more efficiently expressed and folded than full-length proteins. The choice of cDNA source material will depend on the final application for the library. A wide variety of pre-made cDNA libraries are available and it should be possible to adapt the presented cloning scheme to use other cDNA sources. Other protocols for the de novo generation of cDNA libraries have been described and can also be adapted for the generation of yeast surface-displayed cDNA libraries based on the protocols we describe.
|Klenow fill in for pYD(+1)||Mung bean chew back for pYD(−1)|
|3 μL 10x React 2||3 μL 10x Mung bean reaction buffer|
|2 μL 0.5 mM dNTP mix||20 μL Linearized pYD1 (≈2 μg)|
|20 μL Linearized pYD1 (≈2 μg)||1 μL Mung bean nuclease (10U/μL)|
|2 μL Klenow fragment (5U/ul)||6 μL ddH20|
|4 μL ddH20||Incubate at 30°C for 30 min|
|Incubate at RT for 30 min|
|3 μL 10x React 3||5 μL 10x React 3|
|10 μg pYD1 plasmid||20 μg cDNA library plasmid|
|2 μL EcoRI||2 μL EcoRI|
|Bring volume to 30 μL with ddH2O||Bring volume to 50 μL with ddH2O|
|Incubate at 37°C for 3 h.|
|Control ligation||Library ligation|
|2 μL 10x ligation buffer||2 μL 10x ligation buffer|
|700 ng linearized vector||700 ng linearized vector|
|1 μL T4 DNA ligase||300 ng cDNA insert|
|Bring volume to 20 μL with ddH2O||1 μL T4 DNA ligase|
|Bring volume to 20 μL with ddH2O|
|Incubate ligations for 16 h at 16°C.|
The work is supported by grants from the National Institute of Health (R01 CA118919, R01 CA129491, R21 CA137429 and R21 CA135586).
1The pYD1 yeast display vector and the EBY100 yeast strain were commercially available from Invitrogen at the time of our library construction work. Please check with the vendor for currently availability.
2We used random primed, size-selected (0.3–1.5 kb; average size, 0.75 kb) human cDNA libraries from Invitrogen as the source material for the libraries we have made so far. Other pre-made cDNA libraries can be used by modifying the cloning scheme.
3This is a standard ligation condition with 1 μg DNA in 20 μL with a 3:1 insert to vector molar ratio, but could be optimized for each individual case.
4These are the conditions suggested by Lucigen and they have worked well for us. We have generated similar results with Bio-Rad (Gene Pulser II) and Eppendorf (2510) electroporators.
5If there are too many colonies on the vector control transformation plate, the vector digestion, dephosphorylation, and purification should be repeated.
6Analyze the sequence to verify that the inserts are cDNA and diverse. Check to ensure that all three frameshift variants of pYD1 are present. Colony PCR on a larger number of colonies can be carried out to check the size distribution and percentage of empty vector (should be very low), but sequencing is the best method of quality control.
7Transformation plates can be sealed with parafilm and stored at 4°C until enough colonies have been accumulated over the course of several days. Then they can be recovered simultaneously and pooled.
8We found that a longer drying period for the plates allows cells to be more conveniently plated so that cells don’t run down the side when plates are inverted.
9We usually get > 5 × 107 transformants from ten transformations. Plates can be wrapped in parafilm and stored at 4°C until the desired number or transformants has been collected. Then the transformants can be simultaneously collected and pooled.
10If the titer is close to the desired titer, more than one aliquot can be thawed for selection experiments. If the titer is too low for some reason, the library should be re-transformed into yeast and aliquots re-frozen.
11There is always a negative population after induction when analyzed by FACS and the maximum induction will vary from experiment to experiment. This is seen even when using pYD1-transformed EBY100 as a positive control. In order for the V5 epitope to be expressed, the cDNA insert must have an open reading frame (ORF) that spans its entire length and is in frame with both the upstream AGA2 coding region and the downstream V5 coding region. Since only one-third of clones with a full length ORF in frame with the AGA2 coding region will also be in frame with the V5 epitope, the actual number of clones with a full length AGA2-fused ORF will be approximately three times the number of V5-positive clones.