24. 重组GPCR的生产及其结构和功能分析（三）
细说实验方案，本篇原文摘录，不做翻译。
5.1 Preparation of the Expression Plasmid
Equipment and Reagents
• DNA Engine (PTC-200) Peltier ThermoCycler (Bio-Rad Laboratories)
• PCR clean-up kit (Qiagen)
• Gel extraction kit (Qiagen)
• Pfu DNA
polymerase (Promega) and 10× buffer
provided with the Pfu polymerase
• Oligonucleotide primers (Invitrogen)
• 10 mM dNTP mixture (Sigma–Aldrich): a mixture of all four dNTPs (dATP,dCTP, dGTP,
dTTP)
• DNA template, typically a plasmid at 1µg µl-1 containing the gene of interest
• DNA ligase (Invitrogen)
• 0.2 ml sterile PCR tubes (Sarstedt)
• 0.5 ml microcentrifuge tubes(Sarstedt)
• 0.8–1.0% agarose gel
• Agarose gel tank (Bio-RadLaboratories)
• 240 V power pack (Bio-RadLaboratories)
• Restriction enzymes (New EnglandBiolabs).
Method
1 Prepare a ‘master mix’ on ice as follows:
(a) 1 µl forward primer (50 pmol)
(b) 1 µl reverse primer (50 pmol)
(c) 1 µl template DNA (0.1–1 ng)
(d) 12 µl 10× buffer provided with the polymerase
(e) 2 µl 10 mM dNTPs
(f) 1 µl polymerase enzyme (e.g. Pfu from Promega)
(g) 102 µl sterile water.
2 Aliquot 10 µl volumes of the master mix intothin-walled 0.2 ml sterile PCR tubes.
3 Place the 0.2 ml PCR tubes in a thermocycler and run the following
program:
Denature/94 C; 1 min
Anneal/45–65C;1 min (gradient PCR)
Extension/72C; (500 base pairs/min)
Cycles/35
4 Run 2 µl from each tube on a 0.8–1% agarose gel at 80 V until adequately separated to confirm product size. Purify the remaining PCR product using a commercially available kit.
a
5 Digest both the PCR product and vector with the appropriate restriction enzymes. Purify the digested vector and PCR product with a commercially available kit.
b
6 Ligate the PCR product into the vector of choice. Conditions will beenzyme specific. The following example is based on the T4 DNA ligase enzyme and 5 × buffer supplied by Invitrogen.
• 30–60 fmol of vector
• 90–180 fmol of insert
• 4 µl 5× buffer
• 1 µl T4 DNA ligase
• up to 20 µl water.
Perform the ligation in 0.5 ml tubes at 12
C for 20 h followed by 65 C for 20 min to inactivate the ligase.
Notes
aPurification of the PCR product can be achieved using one of two kits. If there is a single PCR product (as visualized on the agarose gel), then a PCR clean-up kit(Qiagen) is advised. If, however, there are multiple PCR products (as visualized on the agarose gel),then the remaining sample must first be separated on another agarose gel, the relevant bandexcised and purified with a gel extraction clean-up kit (Qiagen).
bPurification of the digested vector and PCR product can be achieved usingone of two kits. If the fragments excised from the vector and PCR product are smaller thanthe lower retention limit of the PCR clean-up kit, then this product may be used. If the fragments are large enough to be retained, then the samples must first be separated on anagarose gel and the relevant bands excised from the gel and purified with a gelextraction clean-up kit.
PROTOCOL 5.2 Yeast Transformations
Equipment and Reagents
• Plasmid DNA from Protocol 5.1
• Carrier DNA: 7 mg ml-1 sonicated salmon testes DNA(Sigma–Aldrich)
• Plate solution: 40% (w/v) PEG-3350, 1
mM ethylenediaminetetraacetate (EDTA), 10 mM tris(hydroxymethyl) aminomethane hydrochloride (pH 7.5), 0.1 M LiOAc
• YPD medium: 1% yeast extract, 2% bactopeptone, 2% dextrose
• BEDS solution: 10 mM bicine-NaOH, pH 8.3, 3% (v/v) ethyleneglycol, 5% (v/v) dimethyl sulfoxide (DMSO), 1M sorbitol
• YPDS agar: 1% yeast extract, 2%
peptone, 2% dextrose, 1 M sorbitol,2% agar
• Dithiothreitol (DTT)
• Eppendorf multiporator (Hamburg)
• S. cerevisiae: freshly plated
• Yeast nitrogen base (YNB) medium:
prepare 10× stock by dissolving 134 g of YNB (with ammonium sulfate or amino acids) in 1 l of water, filter sterilize and store at4C
• 1.0 M sorbitol
• YNB medium containing 2% dextrose, 1.0M sorbitol
• Zeocin (Invitrogen): when using zeocinplasmids.
Methods
S. cerevisiae Transformation
1 Inoculate 10 ml of YPD medium with a loop full of freshly plated S. cerevisiaeand culture overnight at 30 C at 230 rpm.
2 Centrifuge 0.5 ml of the culture in a 1.5 ml microcentrifuge tube at 5000g for 2 min at room temperature and discard the supernatant.
3 Add 2–5 µg of plasmid DNA and 10 µl carrier DNA to the S. cerevisiae cells. Add 0.5 ml plate solution and vortex to resuspend.
4 Incubate the tube stationary at room temperature overnight (approximately 14h).
5 Withdraw the bottom 100–200 µl from the tube andplate on selective agar. Incubate the plate at 30C for 2–3 days or until individual colonies are distinguishable.
Making Competent P. pastoris cellsa
1 Prepare a 5 ml overnight culture of P. pastoriscells in YPD medium at 30 C and agitate at 250 rpm.
2 Dilute the overnight culture to an OD
600 of 0.15–0.20 in a volume of 50 ml YPD medium
in a flask large enough to provide good aeration.
3 Grow the yeast culture to an OD
600 of 0.8–1.0 at 30 C with 250 rpm agitation.
4 Centrifuge the culture at 500g for 5 min at room temperature and pour off the supernatant.
5 Resuspend the pellet in 9 ml of ice-cold BEDS solution supplemented with 1 ml 1.0 M DTT.
6 Incubate the cell suspension for 5 min at 100 rpm in a 30C shaking incubator.
7 Centrifuge the culture at 500g for 5 min at room temperature and resuspend the cells in 1 ml (0.02 volumes) of BEDS solution without DTT. Aliquot into 40 µl volumes.
8 The competent cells are now ready for transformation. Alternatively, thealiquots may be stored at -80 C for up to 6 months (do not snap-freeze in liquidnitrogen).
P. pastoris Transformation
1 Add approximately 4 µg of linearized plasmid DNA (usually linearized with SacI, PmeIor BstXI) to a 40 µl aliquot of competent cells in an electroporation cuvette.Incubate for 2 min on ice.
2 Electroporate samples using the following parameters: Eppendorf multiporator 1700 V, 15 ms pulse length.
3 Immediately after electroporation, resuspend the samples in 1 ml cold 1.0M sorbitol and then plate on selective media (YNB containing 2% dextrose and 1.0
M sorbitol) for auxotrophic strains. Alternatively, if using zeocin-based plasmids, resuspend the samples in 0.5 ml 1.0M sorbitol and 0.5 ml YPD, incubate in a 30 C shaker for 1 hb and then plate on media containing increasing concentrations of zeocin (100, 250,500 or
1000 µg ml-1) for the selection of multicopy integrants.
4 Incubate the plates at 30 C for 3–4 days or until colonies are distinguishable.
5 Pick individual colonies and re-plate on YPDS agar with the same zeocin concentration as the initial selection plate and repeat the incubation in step 5.
Notes
aProtocol adapted from [18].
bNote that increased numbers oftransformants can be achieved for both types of selectable
marker by incubating the resuspended cells in a 30C shaker for longer periods of time(1–3 h).
However, this is partly due to replication of transformants.
PROTOCOL 5.3 Screening for GPCR
Production in P. pastoris ClonesEquipment and Reagents
• Uniplate 24 deep-well plates (Whatman)
• Bugstopper siliconized rubber cap withErlenmeyer vent (Whatman)
• 20 ml sterile universal (Sarstedt)
• BMGY (1% yeast extract, 2% peptone,
100 mM potassium phosphate, pH 6.0, 1.34%YNB,
4× 10-5% biotin, 1% glycerol). Prepare 1 l ofmedium by dissolving 10 g yeast extract and 20 g peptone in 700 ml of water and autoclaving for 20 min. Cool the solution to room temperature and add 100 ml 10 × YNB, 100 ml 1 M potassium phosphate buffer, pH 6.0, 2 ml 500 × biotin and 100 ml 10× glycerol. The shelf life of the medium is approximately 2 months when stored at 4C.
• BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34%YNB, 4 × 10-5% biotin, 0.5% methanol). Prepare thismedium as described above for BMGY but using 10 × methanol instead of 10× glycerol.
• Stock solutions for BMGY and BMMY: for 10× glycerol (10%), combine 900 ml of water with 100 ml of glycerol, sterilize by autoclaving and store at roomtemperature. To make 10 × methanol (5%), add 95 ml of water to 5ml of methanol, filter sterilize and maintain at 4C.
To prepare 500× biotin (0.02%) dissolve 20 mg biotin in100 ml of water and filter sterilize (store at 4 C). For 1 M potassiumphosphate buffer, pH 6.0, mix 868 ml of 1 M KH2PO4 and 132 ml of 1 M K2HPO4 (adjustpH with KOH or phosphoric acid to 6.0). Autoclave and store at room temperature.
• Protran nitrocellulose transfermembrane (Whatman)
• Tween-20 (Sigma–Aldrich)
• Marvel (Premier Foods Ltd)
• 6× His monoclonal antibody; albumin free (Clontech)
• Goat anti-mouse IgG (Fab Specific)peroxidase conjugate (Sigma–Aldrich)
• Phosphate-buffered saline (PBS)
• Western transfer buffer (3.3 gTrizma-base, 14.4 g glycine, 20% methanol in 1 l water)
• EZ-ECL chemiluminescence solution(Geneflow)
• Sodium dodecyl sulfate(SDS)–polyacrylamide gel electrophoresis (PAGE) sample buffer (see [21]), SDS–PAGE gels and electrophoresis buffers
• Blocking buffer (PBS, 5% Marvel).
Method
1 Select 10 P. pastoris clones for expression screening a(see Protocol 5.2).
2 Inoculate 5 ml of BMGY media in a 20 ml sterile universal with a singlecolony of P.pastoris and incubate overnight at 30 Cwith shaking at 220 rpm.
3 Determine the OD600 ofthe overnight 3 ml culture in the morning.
4 Inoculate 3 ml BMMY with appropriate volume of the BMGY culture to achieve OD600 = 1 in the 24 deep-well plates.
b
5 Cap the plates with the siliconized rubber caps and incubatefor approximately 53 h at 30 Cwith shaking at 220 rpm (add methanol to 1% final concentration at 24 and 48 h post induction).
6 Harvest 100 µl samples at 24 and 53 h and centrifuge at 10 000g for 5 min, snap-freeze the pellets in liquid nitrogen and store at -80 C until required.
7 Resuspend the 100 µl cell culture pellets in 60 µl of SDS–PAGE sample buffer. Heat the samples for 10 min at 98 C and centrifuge for 1 min at 13 000 rpm.
8 Load 15 µl of the supernatant for SDS–PAGE andimmunoblotting. Immunoblotting can be carried out using a standard procedure cwith the following changes. Carry out blocking in PBS + 5% Marvel for 1 h at roomtemperature. Add primary antibody to blocking buffer at a dilution of 1 : 5000 and incubate at room temperature for1 h with moderate rocking. Wash the membrane in PBS + 0.2% Tween-20 twice for 5 min after primary and secondary antibody incubations. Add secondary antibody in blockingbuffer at a dilution of 1 : 5000 and incubate at room temperature for 1 h withmoderate rocking. Visualize bound proteins using EZ-ECL chemiluminescence solutionaccording to manufacturer’s instructions.
Notes
aIf the GPCR is not detected afterscreening 40 colonies, then go back, reassess and modify other factors important for producing recombinant clones during earlier steps (seeSection 5.2.2).
bMethod adapted from Boettner et al. [19].
cSeehttp://tools.invitrogen.com/content/sfs/manuals/PI96-9045 Western%20Blot%20Kit%20Rev%201008.pdf
PROTOCOL 5.4 Preparation of Yeast
Membranes
Equipment and Reagents
• Acid-washed glass beads (0.3–0.5 µm diameter)
• Phenylmethylsulfonyl fluoride (PMSF)
• 1.5–2 ml screw-cap tube
• FastPrep FP120 (Thermo ElectronCorporation): for small-scale preparations
• EmulsiFlex-C3 (Avestin): forlarge-scale preparations
• Breaking buffer (50 mM sodium phosphate, pH 7.4, 5% glycerol, 2 mM EDTA, 100 mM NaCl), store at 4C
• Buffer A (20 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) pH5.5, 50 mM NaCl, 10% glycerol), store at 4 C
• Homogenizer PTFE pestle/stainlesssteel rod 15 ml (Scientific Laboratory Supplies)
• Yeast cells, from Protocol 5.3 forexample.
Methods
Unless stated otherwise, carry out all the procedures below onice.
Small Scale (<0.5 g Cells)
1 Mix approximately 100–300 mg of wet yeast cells with 300–500 µl of acid-washed glass beads and 300–500 µl ice-cold breaking bufferin a 1.5–2 ml screw-cap tube. Add PMSF to a final concentration of 2 mM (otherprotease inhibitors may be added according to
the manufacturer’s instructions).
2 Agitate the tubes in a FastPrep FP120 set at level 6 for a period of 40 s.Place the tubes
on ice for 1–2 min to dissipate any residual heat.
3 Repeat step 2 at least five times.
4 Following disruption, observe the cells under a light microscope to ascertainthe extent
of cell breakage (>70% should be disrupted). Repeat steps 2 and 3 if necessary.
5 Remove the supernatant from the glass beads and place in a fresh tube. Wash the beads with an equal volume of ice-cold breaking buffer and add to the recovered
supernatant.
a
6 Clarify the supernatant at 10 000gfor 30 min at 4 C.
7 Isolate membranes from the clarified supernatant at 100 000g for 60 min at 4 C and resuspend the pellet in 50 µl ice-cold buffer A.
Medium Scale (5–10 g Cells)
1 To 5 g of wet cells add 7.5 ml of acid-washed glass beads, 7.5ml of ice-cold breaking
buffer and PMSF to final concentration of 2 mm. Vortex the suspension for 1–2min and
subsequently cool on ice for a period of 1–2 min.
2 Repeat the vortex steps for 10–20 cycles.
3 Determine the extent of cell breakage and repeat disruption if necessary,clarify the
samples and recover the membranes as stated above.
Large Scale (>10 g)
1 Resuspend the cell pellet in ice-cold breaking buffer at aratio of 2 : 1 buffer to pellet(v/w).
2 Pass the cell suspension through an Emulsiflex-C3 cell disrupter fitted witha chilled heat exchanger (Avestin) four times according to the manufacturer’s instructions.
Observe the cells under a light microscope to check the extent of cellbreakage. The breaking efficiency should be >90%at a homogenizing pressure of 30 000 psi.
3 Remove the unbroken cells and cellular debris by centrifugation at 10 000
g for 30 min at 4 C.
4 Centrifuge the clarified supernatant at 100 000 gfor 90 min at 4 C to collect the
membrane fraction.
5 Resuspend the membrane pellet in ice-cold buffer A using a glass homogenizerat a ratio of 10 ml buffer per gram of pellet.
Notes
aThe amount of breaking buffer used towash the glass beads can be adjusted so as not to exceed the combined filling volume of the tubes.
PROTOCOL 5.5 GPCR Solubilization and
Purification
Equipment and Reagents
• Fixed-speed roller mixer (Sturat SRT9)
• Reagents for immunoblotting (seeProtocol 5.3)
• Solubilization buffer (20 mM HEPES pH 7.0, 100 mM NaCl, 10% glycerol)
• Homogenizer (homogenizer PTFEpestle/stainless steel rod 15 ml (SLS))
• DC protein assay (Bio-RadLaboratories)
• Fixed-speed flatbed roller (Stuart)
• Glass homogenizer (SLS)
• BioRad DC protein assay kit
• SDS–PAGE sample buffer [21], SDS–PAGEgels and electrophoresis buffers
• Breaking buffer (50 mM sodium phosphate, pH 7.4, 5% glycerol, 2 mM EDTA, 100 mM NaCl), store at 4 C
• 1 ml HisTrap HP column (GEHealthcare).
Method
1 Prepare the membranes as outlined in the large-scale sectionof Protocol 5.4 but omitting the resuspension step 5.
2 Resuspend the membrane pellet in solubilization buffer using a glass homogenizer and add 10 ml of buffer per gram of pellet. Determine the protein concentrationusing a BioRad DC protein assay kit according to the manufacturer’s instructions andresuspend the membrane pellet to a final concentration of 10 mg protein per millilitre.
3 Proceed to step 4 or freeze in liquid nitrogen and store at -80 C until required.
4 For small-scale solubilisation, add 50 µl of the membrane fraction to 950 µl of solubilization buffer containing the required detergent concentration andincubate at room temperature for 1 h on a fixed-speed roller mixer.
5 Pellet the nonsolubilized material at 100 000 g, 1 h at 4 C and resuspend any visible
pellet in 200 µl breaking buffer.
6 Load the supernatant and the resuspended pellet (mixed 1 : 1 in 2 × SDS–PAGE sample buffer) for SDS–PAGE and immunoblot analysis.
a
7 For a large-scale solubilization (50–100 ml), add thepreferred detergent to final working concentration (1–5%), as determined from the small-scale screen,
b incubate and process as above.
8 Use the supernatant for purification of the tagged receptor with IMAC using a1 ml His
Trap column according to manufacturer’s instructions (see Section 5.2.6.1).
Notes
aRefer to Protocol 5.3, step 8, for instructions on immunoblotting.
bA panel of detergents and working concentrations should be screened for effective solubilization of the recombinant protein using the following panel of detergents at 2% w/v on membrane suspensions: DDM, DPC, CYFOS-4,β-OG, foscholineiso9 (FC109), LDAO, pentaethyleneglycol-n-octylether (C8E5), DPC–cholesterolhemisuccinate anddocosaethyleneglycol monohexadecylether (Brij 58; all purchased from AnatraceInc). Analyse the solubilized (supernatant) and nonsolubilized (membrane pellet) material by immunoblotanalysis for solubilization efficiency using a primary his monoclonal antibody and ananti-mouse IgG HRP-conjugated secondary antibody (Sigma).
