Library Construction by Yeast Display Protocol-by Lukasz
Modified 5-7-10
Buffers/Reagents:
? 1M DTT (dithiothreitol), 2.5mL per 500mL YPD culture
o 0.771g DTT, water to 5mL
o Filter sterilize
? 1M Sorbitol, 1mL per electroporation, 50mL per 500mL YPD
o Autoclave
? 1 L sterile water
? 10x TE, 10mL per 500mL YPD
o 40mL 1M tris-HCl pH 7.5
o 8mL 0.5M EDTA
Water to 400mL, autoclave
? 10x lithium acetate, 10mL per 500 mL YPD
o Filter sterilize
? 500mL YPD(autoclaved)
o 5g yeast extract
o 10g Bacto-peptone(Fisher 211677)
o 10g dextrose(Sigma 9434)
? 500mL SD media(Filter to be sterilized)
o 7.4g sodium citrate
o 2.1g citric acid monohydrate
o 2.5g casamino acids
o 10g dextrose
o 3.35g yeast nitrogen base
? 5-6 SD-CAA plates (recipe for 500mL)
o 91.085g sorbitol(Fisher S459-500)
o 7.5g agar
o 7.4g Sodium citrate(Fisher, S279)
o 2.1g Citric acid monohydrate (Sigma,C7129)
Fill up to 400mL, autoclave
o 2.5g casamino acids
o 10g dextrose
o 3.35g yeast nitrogen base
Fill to 100mL, filter sterilize, add to above mix and pour plates in the cell culture hood.
? 2mm gap cuvettes, Fisher # FB102
? EBY-100 Yeast (Invitrogen)
? 10X PBS(Biocore receipe): yeast are happier for prolonged sorting in this buffer.
1L 2L
1.8mM NaH2PO4.H20 2.57 g 5.14 g
8.38mM Na2HPO4.7H20 22.49 g 44.98 g
150mM NaCL 87.65 g 175.3 g
Adjust PH to 7.4
Add dH20 to final volume
FACS sorting buffer: 1x PBS above +1%BSA-- filter to be sterilized.
? 500mL SG-CAA induction media for protein expression:
o 7.4g sodium citrate
o 2.1g citric acid monohydrate
o 2.5g casamino acids
o 40g D(+)Galactose(Acros 150615000)
o 3.35g yeast nitrogen base
Cloning strategies and library design general notes
Most inserts are placed into the yeast display vector (T7pCT302) using NheI at the amino-terminus and either BglII, XhoI or BamHI at the carboxy-terminus, depending on your vector version. Your insert needs to be in frame with the NheI restriction site since your insert will be fused to HA and Aga-2 which occur 5? to your start site. If your vector contains a c-myc tag, don?t forget to clone your insert also in frame with the c-myc tag. If your vector does not contain a c-myc tag (ie the old version pct302 or T7), then you need to design the c-myc tag into your reverse primers. The following directions assume that you will be adding a c-myc tag into your primer. Check your insert or ORF for signal sequences and transmembrane domains, these should not be cloned into the yeast-display system since they can cause problems when expressed on the surface of yeast. The Expasy tools website has many tools for predicting transmembrane domains of proteins if they are not currently known. If in doubt, ask for assistance. Furthermore a c-myc tag has before shown to cause problems with certain proteins. For example with the TNF family receptor LIGHT, a direct myc fusion prevents binding to the ligands of LIGHT, and in this case a short 6 amino acid linker SSGSGG before the myc alleviates this problem. Shorter linkers have also been used with success, including SSG or simply a glycine or two.
Cloning strategy for wild-type proteins (with addition of c-myc)
1. Design forward primer including the 6 nucleotides upstream of the NheI R.E. site (4-6 nt needed for efficient digestion by enzyme), the NheI site and then the beginning of your sequence of interest. The recognition site for NheI is GCT AGC. A start codon is not necessary after the NheI since this is a fusion protein, but can be included if desired. Your forward primer should look like this, the NheI site is bold and underlined:
5? GGT TCT GCT AGC TAC CAG TGG ATC CTG TGC CAG GGC TC 3?
It is always preferable to end a primer on a G or a C since this allows for better primer binding. If you will be inserting or mutating any pieces, you should plan to have an additional 9-12 nt after the mis-match (please see SOE protocol for more details about mismatches). The length of the primer is usually between 30-60nt depending on the Tm and properties of the reverse primer. It is always preferable to have a similar Tm for both forward and reverse primers
Upstream of Nhe I site is HA tag:
CCA TAC GAC GTT CCA GAC TAC GCT CTG CAG GCT AGT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TCT GCT AGC
Nhe I
The sequence of interest (extra cellular domain of light , for example) will be inframed with HA.
2. Design reverse primer to incorporate a c-myc tag (used to evaluate the stable of protein interested in yeast library). Since the tag causes a large overhang that does not match anything on the template, either a long primer should be ordered to at least match the overhang with a sequence on the template or perform a two-step PCR with shorter primers. We will perform a two-step PCR using BglII as our restriction site, a stop codon will be inserted between the end of the c-myc and the BglII site. Be sure that the reverse primer is the reverse complement of the top strand. The BglII recognition site is AGA TCT. The first primer will be approximately a 48nt primer that recognizes the end of your gene and some of the c-myc tag. The second primer will match half of the end of the first primer along with the rest of c-myc tag, the stop codon, the BglII site and 6 random nucleotides. BglII is bold and underlined, the matching sequence is bold.
Rev primer 1
5? TTC AGA AAT AAG CTT TTG TTC CAC CAT GAA AGC CCC GAA GTA AGA CCG
Rev primer 2 BglII *
5? GGT TCT AGA TCT TTA CAA GTC CTC TTC AGA AAT AAG CTT TTG TTC CAC
Myc Tag: EQKLISEEDL *
5? ?gaa caa aag ctt att tct gaa gag gac ttg taa -3?
3? ?ctt gtt ttc gaa taa aga ctt ctc ctg aac att -5?
3. Perform 1st PCR using forward primer and Rev primer 1 using a proofreading polymerase (Novagen/EMD biosci Cat# 710863 KOD hotstart) according to manufacturer?s recommendations.
4. Check for PCR product and either gel purify fragment (if main species is not more than 90% of products) or simply perform a PCR cleanup.
GeneJet gel extraction kit (Fermentas, #K0691):
Cheaper, faster and get better yield than Qiagen kit.
5. Use 1uL (10-50ng) as template for 2nd PCR (50ul/tube x 2 should be enough) using forward primer and Rev primer 2 BglII.
6. Run fragments on gel with previous fragments to be sure additional segment has been correctly added on.
7. Gel purify or PCR clean up the new fragment.
8. Digest PCR fragment using NheI and BglII, fermentas fast digest enzymes are compatible in the same buffer, however NheI requires at least 20 minutes for complete digestion using 1uL per every ug. To be on the safe side, digest ~2 hours.
9. Vector T7pCT302(Amp resistant)can be simultaneously digested with Nhe I & Bgl II and alkaline phosphatase treated using fermentas fastAP(#EF0651).
10. Gel purify vector, and either gel purify or PCR clean up PCR fragment.
11. Ligate fragments, usually about a 6:1 or 8:1 insert to vector (by moles not ug) works well for most inserts. Very large (>2kb) and very small fragments (<200bp) may have to be empirically tested.
12. Transform ligation using method of choice, screen positive colonies for insert, and sequence using either cloning primers or T7 promoter short primer (which binds ~100bp downstream of insert and reads towards the ORF as a reverse complement).
13. Transform plasmid into yeast using either Lithium acetate method or by electroporation (see section in library construction protocol) and plate on SD-CAA plates. Grow plates at 20-30 degrees Celsius until colonies appear.
14. Pick several colonies into ~2mL of SD-CAA media (+fresh added P/S) and grow overnight at 30 degrees.
15. When media is very turbid, spec at 600nm and induce into 2mL SG-CAA (+fresh added P/S). Final OD of SG-CAA should be about 1.0.
16. Induce protein expression for 16-48 hours at 20oC. Typically 36-48 hours is optimal and this length of time should be used for sorting purposes, however, a shorter time is fine for simple and quick analysis.
17. Take final OD at 600nm of SG media clones and determine quantity to use for approximately 1-3x10^5 yeast cells per tube. It is always easiest to first add PBS-BSA into a tube and then add your volume of yeast (typically 5-20uL).
18. Spin down tubes at ~2-3000 RPM for 1-2 minutes.
19. Aspirate PBS-BSA and stain at appropriate concentrations of ligands at room temperature for ~1-1.5 hours.
20. Wash in ice-cold PBS-BSA, spin down and aspirate.
21. Repeat steps 18-19 for secondary or add PBS-BSA (300-500uL) and analyze.
22. Determine binding curve and approximate equilibrium binding constant in yeast for wild-type protein.
23. Proceed to library construction.
It is always best to perform titrations of your ligands on the yeast cells to determine approximate binding before proceeding with sorts and library design. Directly labeled ligands are best, however chemically or enzymatically biotinylated proteins with a Streptavidin-PE secondary also work well. Avoid the use of FITC labeled reagents since this fluorophore is far dimmer than PE or APC. Typically primary staining should be performed at room temperature (1~2hr), secondary is usually performed on ice to help slow down the dissociation of primary (45min). If using very high concentrations of primary you will likewise either have to wash multiple times or use very high concentrations of secondary. High concentrations of primary when not washed away properly and left unbound to yeast-displayed proteins will lower the effective concentration of secondary reagents. This can easily be demonstrated by labeling antibody and staining at very high concentrations and washing only once. However, multiple washes will also wash away your ligand quicker as well, therefore the best recommendation is to use directly labeled primary for initial wild-type proteins. Some proteins require far higher concentrations of ligands in yeast than they would for mammalian cells.
Some proteins will not be displayed properly on the surface of yeast and no staining will be observed using your ligand of interest. Staining for c-myc should be mandatory at this point and HA is optional. A positive result for myc and negative result for your ligand indicates that your protein is on the surface of yeast but is not stable enough to bind your ligand, however this result is promising and indicates an easier road to obtaining your desired result compared to no myc expression. Lack of myc expression can also indicate truncations, which happens somewhat frequently with yeast displayed proteins. You can sequence your insert using either T7proshort of the surfSeqR primer:
SurfSeqR
5? GTT ACA TCT ACA CTG TTG TTA T
Library design strategy
Depending on your gene you need to determine whether a site-directed or random-error mutagenesis strategy would be preferable for you. Great luck has been experienced for TCRs and scFVs using site directed mutagenesis for increasing affinity. This strategy will not be discussed in this version of the protocol. For most genes, including TCRs and scFVs of known specificity, a random error mutagenesis strategy is preferable since oftentimes regions outside of the antigen binding sites can be manipulated to improve the properties of your protein. A simple random-error PCR mutagenesis strategy will be outlined here, however, multiple methods exist and it is up to the end-user to determine which is best to use. The strategy outlined here has been used successfully for 2C, Ly49C, 2B4, CD48 and KLRG1 and we recommend its use for first time users. The yeast display vector has two promoter binding sites that we will use for this purpose, Splice4/L and T7pro short. These regions are at least 100bp upstream and downstream of your insert which generates a rather large overlap on both sides of your insert that will be used for subsequent homologous recombination into yeast. The yeast machinery ligates your insert using homologous recombination. The indicated random error PCR protocol uses MnCl along with MgCl to have Taq misincorporate nucleotides. A typical Mg:Mn ratio is roughly 6.67:1, this can be scaled up or down to increase or decrease mutation rates. However larger mutation rates will also decrease yield. This mutation strategy will not change the length of your gene, except for the occasional stop codon.
Splice4/L primer sequence
5? GGC AGC CCC ATA AAC ACA CAG TAT 3?
T7proshort
5? TAA TAC GAC TCA CTA TAG GG
1. Using the wild-type version of your gene of interest as the template, set up an error prone PCR using the following recipe. Be sure to use regular Taq polymerase that DOES NOT has a proofreading ability.
dNTP mix (100uL)
12.5uL 10mM dATP
12.5uL 10mM dTTP
12.5uL 10mM dCTP
12.5uL 10mM dGTP
50uL ddH20
PCR recipe:
27uL ddH20
8uL dNTP mix
5uL Taq buffer (fermentas)
4uL 25mM MgCl2
3uL 5mM MnCl2
0.5uL 20uM T7pro primer
0.5uL 20uM Splice4/L primer
1uL Template DNA (usually 1:10 of miniprep or 10-40ng)
1uL Taq (Fermentas Taq works well, Cat# ??)
PCR program
1 cycle Hotstart
94 for 5 minutes
80 for 5 minutes
35 cycles of the following:
94 for 1 minute
53 for 1 minute (usually 50-55 degrees is used)
72 for 1 minute per kb
1 cycle of the following
72 for 10 minutes
4 degrees until done
2. Check PCR products (~30 x 50ul of PCR reactions to make library) on gel to be sure that product remains relatively clear and defined, if not, tweak the PCR protocol to achieve desired results.
3. Clean up PCR using PCR clean up kit (Cat#???).
4. While PCR is started, digest a large enough quantity of vector (~200ug to yield 50ug purified digested vector) with NheI and BglII. If using the fermentas fast digest, alkaline phosphatases can also be added simultaneously. Digest at least 1-2 hours at 37, then gel purify the vector(K0691, K0692).
5. Determine DNA conc by spec(20ul-> 500ul). You will need approximately 1ug of vector and 5ug of insert per electroporation(x30 for making library). DNA should be precipitated and pelleted using a standard ethanol precipitation or vacuum dry for maximum concentration. DNA can be left as a pellet. (see precipitation protocol).
Site Directed Mutagenesis: Site-directed mutagenesis may be used instead of random error (error-prone) mutagenesis when a target region of a particular protein is known to be important for protein-protein interactions. Examples of this include but are not limited to: CDR regions of antibodies and TCRs, specific active sites of enzymes or regions shown to be important from crystal structures. While error-prone PCR can also provide mutations in these regions, site directed mutagenesis may allow multiple mutations in one target region which may not be possible using error-prone. Site directed may also be helpful in changing binding selectivity of TCRs or scFVs. It is important to identify these regions first using either an algorithm or by going to
http://www.bioinf.org.uk/abs/ for a tool in helping identify CDRs in scFVs. An example will be shown using the CDR3 of the Vlight chain of the 7.16.4 anti-neu scFV. As yuo increase the amount of amino acids to use in a site-directed approach, the library size to cover all possibilities increases dramatically. The library size would normally be figured as 64n, where n is the number of residues and 64 is the total codon possibilities in eukaryotic cells. Since there are multiple codons for many amino acids, especially when considering the third position, we can use degenerative NNS primers. Where N stands for any amino acid and S stands for either a C or a G. This allows then the use of only 20 codons to cover all the amino acids, therefore our library size now can be calculated by 20n. You can order NNS primers by simply typing in NNS for a codon instead of the usual ATG for example when ordering from places like IDT. In essence your library will be synthesized randomly by the machine rather than by yourself. The PCR is a two-step SOE PCR.
1. The Vlight CDR3 of the 7.16.4 scFV was found flanked by C88 and F98 and consists of this amino acid sequence: Q89 Q90 S91 N92 N93 W94 P95 L96 T97. The DNA sequence is: caa cag agt aac aac tgg ccg ctc acg.
2. Start with the wt or other template in the yeast-display plasmid already. You will be using T7 and splice4/L as your flanking primers.
3. You need to order two primers. These primers will be longer than normal since the large mismatched regions in the CDR require at least 15-21 bases downstream at the 3' end for proper annealing and also ~35-50 bp upstream at the 5' end of the primer to overlap with the internal forward primer. For NNS to work properly the forward primer should be the one that has the NNS in it. I start designing my primer upstream of my CDR by ~42 bp or 14 amino acids and then have 21bp after it so that the primer will anneal properly to the template. In this case primer1 starts at:
atc aac agt gtg gag act gaa gat ttt gga atg tat ttc tgt nns nns nns nns nns nns nns nns nns ttc ggt gct ggg acc aag ctg
4. Normally however it is not advised to mutate that many amino acids at a time since the library size needs to be astronomical and also that it requires very long primers. I have ordered primers as long as 110 bp with not that many problems, so it is doable.
5. You will now need a reverse primer. This primer SHOULD NOT overlap at the NNS region with the 1st primer, but should contain all of the overlap of the first primer (ie the 42 bp upstream of the NNS). Our primer will now be the reverse complement of the 1st primers' 42 bp, but moving in the opposite or reverse direction. This primer can be short and consist of just the 42bp or you can make it longer if you wish. The only important variable is the overlap, longer overlaps between the 2 primers gives more efficient SOEing on the 2nd PCR, while longer primers increase the risk of error and also make PCRs a little more difficult.
6. You will perform 2 PCRs. The first will have two tubes both using the same template. In tube 1 you will have the forward Splice4/L primer and the reverse primer 2 with the 42bp overlap to amplify the region upstream of the mutations. Tube 2 will have the forward primer with the overlap followed by NNS, followed by 21 bp to anneal properly and then the T7 reverse primer to amplify the second part of the gene and make your NNS library.
7. The conditions of the PCR will have to be determined by the end user, but the standard conditions should first be tried. If one wants, you can order new primers for the T7 and Splice4/L by extending this sequence to match the Tms a little better. You are aiming for PCRs that yield as bright and clean a product as possible. It is not recommended to gel clean this PCR since a lot of product is lost in the purification and will affect library size yield. If however you have multiple dominant bands, either perform more PCRs or adjust your conditions. This is the most time-consuming part of the whole process.
8. Use your cleaned PCR products (that now have 42bp overlaps with each other) as templates for a second PCR. You will use T7 and splice4/L as the flanking primers or your custom designed new T7 and splice4/L. You need the large vector overlap from these primers for the subsequent homologous recombination that will be used. I typically use ~1 uL of each as a template for the 2nd PCR. Unless you will DpnI digest or gel purify after your 1st PCR, you will still have some wt template in this mix. However, I have found this to not be that large of a concern since I gel clean my library template. Do your second SOE PCR. Either gel clean your PCR (for multiple bands) or gel clean followed by DpnI digest then PCR clean your products. Since the purifications decrese yield, you will be typically using far more reactions than with the error prone step. DpnI digesting cuts bacterially produced DNA since it is methylated and will eliminate your template DNA.
9. Use your purified product with its vector overlap sections in a homologous recombination electroporations as with the error-prone method.
Some considerations: DNA should be as pure as possible. Although the standard phenol:chloroform extractions do work in library production, the use of a purification column (ie Qiagen) gives a better library yield. Vector should be cut and phosphatase treated. Insert does not need any manipulation since we are relying on homologous recombination during library construction using large overlapping fragments between vector and insert. Gel purification is usually preferred for all DNA segments when library generation is being used.
Yeast Growth and electroporation
1. Innoculate 3-6 mL YPD starter cultures with EBY-100 strain yeast (Invitrogen)+ P/S, shake overnight at 30o C at 175-200 RPMs
2. At end of day use 3-5mL starter culture to inoculate 500mL YPD+ 6ml of P/S, shake overnight at 30o C at 175-200 RPMs
3. Harvest yeast by centrifugation, 4000-5000 RPMs, 15 minutes at 4 C.
4. Re-suspend yeast in 80mL sterile deioned water (per 500mL YPD), RT or warmed to 30o C
5. Add 10mL 10X TE buffer dropwise while swirling
6. Add 10mL 10X lithium acetate dropwise while swirling
7. Incubate 45 minutes at 30o C with gentle shaking (80-100 RPMs)
8. Add 2.5mL 1M DTT while swirling
9. Shake for additional 15 minutes
10. Dilute in 500mL ice cold sterile deionized water
11. Centrifuge 4000 RPMs 10-15 minutes, aspirate supernatant
12. Resuspend pellet in 250mL ice cold sterile deionized water
13. Centrifuge 4000 RPMs 10-15 minutes, aspirate supernatant
14. Resuspend pellet in 30mL ice-cold sorbitol
15. Centrifuge 3000 RPMs 10-15 minutes, aspirate supernatant
16. Resuspend pellet in 10mL ice-cold sorbitol
17. Centrifuge 3000 RPMs 10-15 minutes, aspirate supernatant, do not aspirate yeast
18. Re-suspend in lowest volume possible, generally 1-2 mL per 500 mL YPD
19. Roll tube around and flick to resuspend, It is helpful to swirl around with pipette tip first
20. Make master mix and incubate 50 uL of yeast per electroporation with 6ug total DNA (1ug yeast display vector & 5ug insert), vector alone & insert alone.
21. Incubate on ice 30 minutes.
22. Aliquot equally to 2mm gap cuvettes also on ice.
23. Pulse at 1.5kV at 25uF and 200 ohms, time constant should be between 4-5.
24. Rescue immediately with 1mL YPD, plus 1mL of YPD to wash the cuvettes. Pool the washes to a 50mL cornical tube or flask, shake at 30 degrees 1 hour to recover the yeast.
To store the cuvettes for reuse: wash cuvettes with dH2O, soaked and stored in 95% alcohol (storing in higher% water causes pitting an corroding of cuvettes)
To reuse the cuvettes: take out from 95% ethanol solution, flick dry, wash with water, then flick dry, UV inside the cell culture hood for 30min.
25. Pellet yeast and resuspend in 1mL SD-CAA(+10ul of 100x P/S) per electroporation.
26. Take a 20 uL aliquot of theSD-CAA culture and perform 10-fold serial dilutions with 1x TE, and plate on SD plates, having 1 plate for 10uL vector alone and 1 plate for 10uL insert alone and also one plate for 10uL library electroporations. Typically I have a plate for 10, 1, 0.1, 0.01 to estimate library size.
Estimate library size by counting colonies on plates and subtracting vector background. Library size is # of colonies per mL times # of electroporations.
(colony count x dilution from 1mL x # of electroporations)
27. Dilute 10-fold with SD media+ P/S.
28. Shake at 180-200 RPMs for 48 hours at 30 C, incubate plates at RT or 30 C
29. Split 10-fold and grow additional 24 hours at 30oC.
30. Induce library in SG-CAA media to final OD 600nm of 1.0 and induce 36-48 hours.
31. Calculate amount of cells to be used so as to at least cover the library 5-10 fold. (For 10^7 size use 10^8 cells)
Some groups double stain yeast for c-myc and the ligand of choice. Although this may be done, sorting for c-myc simply selects for clones that bind c-myc at higher levels and may have absolutely nothing to do with increasing the stability of your protein. Since no library (unless only mutating 2 or 3 residues) could ever be created to cover all possible combinations, it does not make much sense to put additional selection pressure on the relatively small libraries that are generated, especially since the library will already be barely positive on the first two sorts when selecting for one ligand. Prior experience has shown that this type of selection scheme requires either; already high affinity of the ligand in question, a small number of residues mutated or an enormous library. More often than not, you will just end up resorting the library anyways. Since some libraries require increasing surface expression or stability first using a clonotypic antibody, double staining does not provide fruitful yields. Sometimes (rare instances) truncations occur for proteins selected for a clonotypic antibody. If this is a problem, go back one sort (usually using sort 3 positive library) and then sort for myc positive clones to obtain only full-length proteins. In some instances myc has even been shown to interfere with protein-protein interactions (ie LIGHT).
We recommend for all proteins that are myc positive, but that do not stain positive for your ligand or antibody, to first perform 1 round (3-4 sorts) of selection for the clonotypic antibody and obtain positive clones that have an increased surface expression (gauged by both increased myc and increased antibody staining). Then one can simply pool all these cDNAs (described later) and perform error-prone PCRs on these pooled clones. Then one can generate a library again based off these improved templates. This strategy has been required for most proteins and has lead to great success. For proteins that don?t make it to the surface of yeast (myc negative), two rounds of selection may have to be performed before stable clones are isolated in order then to be able to select for your ligand. This strategy was used for the 2C TCR.
Library screening
1. Incubate predetermined concentration of ligand with library for 1-1.5 hours at room temperature in the dark. Since large libraries will require prolonged sorting times by FACS, it may be necessary to break the staining up into several tubes and leave all but the sample that is being sorted pelleted until ready to sort. Usually the first selection uses a higher concentration of ligand to isolate ALL mutants that bind to your ligand of choice. This eliminates all negatives.
2. Wash sample 1 or 2 times depending on amount of primary used.
3. End user determines concentration of secondary, if any, to use. Secondary should be incubated on ice.
4. After a final washing and aspiration, sample may be sorted. MACS selection is not necessary if the library is ~or<2.0x10^7. The initial library screening will be split to 2 stainning/ sortings to prevent long time sitting time during sorting. Larger libraries may require MACS selection first. It is not abnormal for the library to appear to be almost completely negative on the first and sometimes second sorting. Very often the library will barely be higher than background analysis, this is normal. By the third sort you will have a good idea if your selection scheme is working. Care should be taken to not include too many negatives, but to also not be too stringent since clumping yeast may show up as false positives. Avoid having selection window be too close to the negative population. Take no more than about the top 0.5% on the first selection (unless the population is very positive), this may take some practice initially.
5. Sort sample into 3ml of SD-CAA media containing PEN/Strep and try to collect at least 20~ 40,000 cells, since low cell numbers may be detrimental to yeast growth.
6. After first sort incubate tube at 30 degrees until media is turbid (depends on amount of cells collected but is usually ~24-48 hours). Repeat this process for all sorts.
7. Induce sample and sort again. For every sort after the 1st, one should incubate several dilutions of ligand with library. For example if the first sort used a 1:10 dilution of ligand, for the 2nd sort bring along 1:10, 1:30 and 1:100. Analyze all samples and compare to background. Sort sample that gives an improvement over background. It is not uncommon for the 2nd sort to use the same concentration as the 1st sort.
8. For sort 3 perform the same titrations. Hopefully by this time a slightly lower concentration should yield a population of cells that is far more positive than sort 1. Do not use so low a concentration that is barely positive than control, since this may increase the chance of obtaining non-specific binders.
9. After sort 3, plate approximately 2000 cells on an SD-CAA plate.
10. By sort 4 you should be obtaining a definitive positive population that has an increase in binding to lower concentrations of ligand over wild-type. Plate ~2000 of sort 4 clones as well.
1) Induce yeast in 3ml of SG-CAA solution:
Measure yeast conc by spec: dilute yeast at 1/10 in dH20, measure OD600
hM: 0.431x10=4.31, 3ml/4.41=700ul- spun down at 2000rpmx2min
hH: 0.353x10=3.53, 3ml/3.52=850ul- spun down at 2000 rpmx2min
Note: hM: yeast expressing human light were sorted with mLTbR
hH: yeast expressing human light were sorted with huLTbR
Discard sup and resuspend in 3ml of SG-CAA(OD600=1), kept in 4oC for weekend and induce 24hr at 20oC, shaking.
7/28/09 Sorting#4:
2) As yeast in SG solution are difficult to be spun down, dilute 900ul of hM yeast &hH yeast in 5ml PBS/BSA, spun at 2500 rpm x3min. Aspirate sup. Add 300ul of diluted
1st ab in PBS/BSA solution at different dilution.
Sorting# Yeast 1st ab (1.5hr at RT, vortexing every 15min)
hM#1 900ul hM yeast mLTbR-hIgG1(042402), 1:100
hM#2 900 ul hM yeast mLTbR-hIgG1(042402), 1:300
hM#3 900 ul hM yeast mLTbR-hIgG1(042402), 1:1000
hH#1 900ul hH yeast huLTbR-huIgG(040997), 1:1000
hH#2 900ul hH yeast huLTbR-huIgG(040997), 1:3000
hH#3 900ul hH yeast huLTbR-huIgG(040997), 1:10000
PE 300ulhM+300ulhH yeast
3) Wash 1x with 5ml of PBS/BSA solution
4) Dilute 2nd ab 1/100 in PBS/BSA solution: PE conjugated goat anti-human IgG (Fc fragnment specific), Jackson Immu#109-115-098. Add 300ul to each tube, and incubate for 45 min on ice in dark.
5) Wash 1x with 5ml of PBS/BSA solution
6) Spun 2500x3min
7) Aspirate supernatant, re-suspend in 500ul of SD solution
Cell sorting very high binding clones , collecting into 3ml of SD+PS
hM, 19000 yeast/3ml, 2plts
hH: 22000 yeast/3ml, 1plt
9) Plating ~2000 yeast on SD plate, incubate 30oC for 2 days
10) Incubate the rest of yeast cells at 30oC ever night with shaking at 200 rpm
Analysis of clones
Clones isolated from sorting should be grown and induced just like the library. It is important to use induced wild-type as a measure of improvement and for comparison. Initially several dilutions of ligands along with negatives and anti-myc should be performed. After identifying candidate clones, these should then be screened for thorough titrations. We usually perform 2-fold serial dilutions to determine an equilibrium binding constant at room temperature. Non-linear regression curve fits should be performed using appropriate software to determine this binding constant. Thermal stability analysis should then be performed using this half-maximal binding constant for the highest signal to noise ratio. Induced yeast can usually be used for up to a weak before a new batch should be re-induced from the SD-CAA cultures. The following protocol can be used for thermal stability, however multiple different assay formats can be used from the literature.
A. thermal stability
1. Set-up water baths to measure enough temperature points. Usually this includes 4 degrees, RT, 30, 37, 42, 50, 60, 72 and close to 90 degrees. Yeast tend to behave funny at or above 72 degrees, but often clones are isolated that still retain binding at this temperature.
2. Using half-maximal binding concentration of ligand, incubate samples at room temperature for 1 hour for all temperature points to reach equilibrium.
3. Place tubes into appropriate water baths for 15-30 minutes.
4. Wash samples with ice-cold PBS-BSA.
5. Spin down and aspirate, yeast may not pellet as efficiently from higher temperature tubes.
6. Add secondary and incubate on ice or analyze samples.
7. Draw thermal stability curve.
B. Analysis binding specificity of yeast clone with mutated hLIGHT to (m&h)LTbR and (m&h)HVEM
1. Pick up 12 single colonies of hM and 8 single colonies of hH into 2ml of SD+P/S
2. Culture at 30oC O/N with shaking
3.
DNA isolation from yeast clones
After determining which clones meet your criteria, it is important to identify the mutations that have led to those properties. Yeast plasmid minipreps can be performed using either the Zymoprep I or Zymoprep II protocols according to manufacturers suggestions. Yeast plasmid preps are not very clean and need to be transferred to E. coli for amplification and submission for sequencing. The DNA yield is not very high from the Zymoprep kits, therefore higher quality competent E. coli should be used. Submit DNA from E. coli for sequencing using any of the three primers T7pro short, Splice4/L or SurfSeqR described earlier.
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