Feb 02, 2016
From cDNA to crystal structureFrom cDNA to crystal structure
Design the constructs
Cloning
Expression of the proteins
Solubility/ stability tests
Expression optimization
Large scale expression
Purification
Crystallization screening
Crystal optimization
X-ray data collection
Phase determination
Structure determination
• BLAST-P your sequence (set PDB as database), find homologues; identify domains
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=onhttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
• predict disordered regionshttp://bioinf.cs.ucl.ac.uk/disopred/http://www.strubi.ox.ac.uk/RONN
• predict transmembrane regionshttp://www.ch.embnet.org/software/TMPRED_form.html
• predict the secondary structurehttp://bioinf.cs.ucl.ac.uk/psipred
• predict the 3D-structure http://bioinf.cs.ucl.ac.uk/psipred/http://www.bioinfo.rpi.edu/~bystrc/hmmstr/about.htmlhttp://depts.washington.edu/ventures/UW_Technology/Express_Licenses/rosetta.php
Design of the (best) constructsDesign of the (best) constructs
• from what organism does it originate?
• is your protein extracellular, intracellular or is it a membrane protein? If intracellular – where is it localized in the cell?
http://www.cbs.dtu.dk/services/TargetP/http://ihg2.helmholtz-muenchen.de/ihg/mitoprot.html
• what kind of posttranslational modification(s) does it contain?http://www.cbs.dtu.dk/services/NetNGlyc/http://mendel.imp.ac.at/sat/PrePS/index.htmlhttp://www.cbs.dtu.dk/services/NetPhos/
• what is the molecular weight (MW)?http://us.expasy.org/tools/protparam.htmlhttp://us.expasy.org/tools/pi_tool.html
• what is the isoelectric point (pI)?http://us.expasy.org/tools/protparam.htmlhttp://us.expasy.org/tools/pi_tool.html
• what is the extinction coefficient?http://us.expasy.org/tools/protparam.html
• does your protein display a measurable activity? How your protein can be assayed?
What you should know before starting to What you should know before starting to express and purify your proteinexpress and purify your protein
The three Phase Purification StrategyThe three Phase Purification Strategy
I
II
III
Purification proceduresPurification procedures
• Precipitation techniques
• Affinity chromatography
• Ion-exchange chromatography
• Hydrophobic interaction chromatography
• Gel filtration
Precipitation techniquesPrecipitation techniques
Affinity chromatographyAffinity chromatography
Makes use of specific binding interactions between molecules
1- Incubate crude sample with the immobilized ligand
2- Wash away non bound sample components from solid
support
3- Elute
• Commonly used affinity partners:– Ni2+ binds to poly-histidines (example 6xHis)– specific antibodies (anti-Flag tag)– glutathione binds to GST– Protein A or G binds antibodies
• Possible elution strategies:– pH modification– ionic strength modification– competitor ligand or analog
Affinity chromatographyAffinity chromatography
- the specificity of the interaction between histidine residues and immobilized nickel ions
Ni-NTA columnsNi-NTA columns
Affinity Separation. An exampleAffinity Separation. An example
Ion exchange chromatograpy (IEC)Ion exchange chromatograpy (IEC)
• Protein charge varies according to surrounding pH:
• When pH above pI: binding to ANION exchanger
(pH > pI Anion exchange)
• When pH below pI: binding to CATION exchanger
(pH < pI Cation exchange)
• Elution with salt concentration gradient (stepwise or continuous)
• Separation of proteins with different charge properties at different pH values
Ion-Exchange chromatographyIon-Exchange chromatography
If pH mobile phase =7.2
Then charge of the proteins: (-) (-) (+) (+)
-- + +
+
+
+
+
+
+-
--
--
-+
+
+
+
+
+
+
+
+
+
+
+
Anion exchange column = + charged
Increased salt concentration
Ion-Exchange chromatographyIon-Exchange chromatography
-- + +
+
+
+
+
+
--
Na+Na+
Na+
Na+Na+
Na+
+
+
+
+
+
+ Cl-
Cl-Cl-
Cl-
Cl-
Cl-
+
+
+
+
+
+-
-
-
--
-
Na+
Na+Na+
Typical IEX gradient elutionTypical IEX gradient elution
At low salt concentration
At high salt concentration
H
H H
H
H = hydrophobic region
HH
HH
HH
HH
HH
HH
HH
HH
Column with Resin
Hydrophobic Interaction ChromatographyHydrophobic Interaction Chromatography
HH
HH
HH
HH
HH
HH
HH
HH
Load with high salt buffer
Elute undesired molecules with decreasing salt
gradient
HH
HH
HH
HH
HH
HH
HH
HH
Elute target molecules with low salt buffer
HH
HH
HH
HH
HH
HH
HH
HH
Typical HIC gradient elutionTypical HIC gradient elution
Size-exclusion chromatographySize-exclusion chromatography
Gel filtration elutionGel filtration elution
Signs of unstable/ insoluble proteinSigns of unstable/ insoluble protein• cell-free lysate does not contain the protein
• protein is soluble but cannot be eluted from the affinity column (or, cleaved on the column protein “disappears”)
• protein precipitates during concentration
• multiple or asymmetric gel-filtration profile
• multidispersity in DLS
• multiple bands in native electrophoresis
Potential remedies:• glycerol• change the buffer• change the pH of the buffer• add a ligand• add some mild (non-ionic) detergent• clone/ express another construct
Storage of purified proteinsStorage of purified proteins
SUMMARY OF CONSENSUS PROTOCOLSUMMARY OF CONSENSUS PROTOCOL• Obtain the cDNA by amplifying genomic DNA (prokaryotic genes, or eukaryotic genes with no
introns) or sequence-verified cDNAs (eukaryotes) or by total gene synthesis.
• Use ligation-independent cloning (LIC) into an E. coli expression vector. Use T7 RNA polymerase–driven expression and an N-terminal oligohistidine tag (include a cleavage site for a protease to enable removal of the tag).
• Express the protein in a BL21(DE3) strain, with induction at low temperature (15–25 °C) in rich medium and with good aeration.
• Solubilize and purify the protein in a well-buffered solution containing an ionic strength equivalent to 300–500 mM NaCl.
• Use immobilized metal affinity chromatography (IMAC) as the initial purification step.
• If additional purification is required, use size-exclusion chromatography (gel filtration). If necessary, use ion exchange chromatography as a final ‘polishing’ step.
• The affinity tag may be removed. Use a recombinant, hexahistidine-tagged protease and reapply the sample to IMAC column to remove the protease.
From Nat. Method, 5, 138-146 (2008)
• Vapor diffusion procedures:
- hanging drop
- sitting drop• Microbatch• Dialysis• Free interface diffusion (FID)
Crystallization of proteinsCrystallization of proteins
Vapor diffusion proceduresVapor diffusion procedures
Hanging drop Sitting Drop
Cover slip(attached with grease)
Clear tape
Drop(Protein/precipitant mix)
Well(precipitant)
MicrobatchMicrobatch
• crystallization (at microscale); droplet is covered by oil.
• this prevents the
evaporation of the
very small drop.
DialysisDialysis
• sample placed in a
dialysis cell or a dialysis button• sealed with a dialysis membrane
Free interface diffusionFree interface diffusion
Protein and precipitant solutions are in contact (free interface)
Curr. Opin. Struct. Biol. 14, 577–583 (2004)
Crystallization of proteinsCrystallization of proteins
Discussion between a protein purification Discussion between a protein purification expert and a crystallographer...expert and a crystallographer...