Transcript No Slide Title

Biochemistry 412
Analytical & Preparative
Protein Chemistry II
4 February 2005
Proteins are Amphiphilic Macro-Ions
Positively-charged basic residues
(K, R, & H)
Hydrophobic “patch”
Macromolecular
dimensions:
ca. 40 Å
Ligand binding pocket
(active site)
Negatively-charged acidic residues (E & D)
>>> The charged groups, hydrophobic regions, size, and solvation affect the
biophysical properties of the protein and largely determine its purification behavior.
Chromatography
Sample containing
proteins or peptides
Liquid flow
Liquid
flow
Separation according to:
-molecular weight/ size
-charge
-hydrophobicity
-affinity
Time
4:37
990909
1
2
3
4
5
5
Three Phase Strategy: An aid in developing the
purification scheme
Achieve final purity.
Remove trace impurities,
structural variants,
aggregates, viruses, etc.
Purity
Remove bulk
impurities
Isolate product,
concentrate, stabilize
Polishing
Intermediate
purification
Capture
Step
7
Sample Preparation
General considerations:
• Select extraction procedure according to source and
location of protein
• Use gentle procedures to minimize acidification and
release of proteolytic enzymes
• Work quickly at sub-ambient temperatures
• Use buffer to maintain pH, ionic strength
Goal: To stabilize sample
8
Always Limit the Number of Steps
Maximize the Yield at Each Step
Yield (%)
100
80
95% / step
60
90% / step
40
85% / step
20
80% / step
75% / step
20% overall
yield!
0
1
2
3
4
5
6
7
8
Number of steps
9
Gel Filtration
Gel Filtration (GF) Chromatography
The principle of gel filtration -- excluded volume
[Note: gel filtration chromatography is also sometimes
called “size exclusion chromatography”]
Vo = “void volume”
Vt = “bed volume”
Ve = “elution volume”
Vi = Vt - Vo
Principles of gel chromatography (con’d)
Gel Filtration Elution Volumes as a Function of Molecular Weight
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
Ion Exchange Chromatography
Ion Exchange (IEX) Chromatography
Ion Exchange Chromatography (con’d)
Cation exchange
column
Anion exchange
column
Some other popular chromatographic methods:
• Hydrophobic interaction chromatography
• Affinity chromatography
• Reverse phase chromatography
Hydrophobic Interaction Chromatography (HIC)
Affinity Chromatography
“Reversed Phase” Chromatography (RPC)
(elution with organic solvents)
Linking Chromatography Techniques
Start conditions
Technique
End conditions
Small sample volume
GF
Diluted sample
Buffer change (if required)
Low ionic strength
IEX
High ionic strength or
pH change
High ionic strength
HIC
Low ionic strength
Specific binding conditions
AC
Specific elution conditions
23
In addition, there are non-chromatographic
protein purification techniques, e. g.:
• Ammonium sulfate precipitation
• Sedimentation (rare)
• Recombinant gene product over-expression
• Refractile body prep (see above)
• Detergent extraction
• Heat treatment (especially for recombinant
thermophile proteins expressed in E. coli)
• Etc.
Once You’ve Purified Your Protein,
How Do You Characterize It?
Some typical analytical tests:
- SDS PAGE (both reducing and non-reducing)
- Bioassay (if you have one)
- Total protein determination
- UV spectrophotometry
- CD spectrometry
- disulfides?
- amino acid analysis
- N-terminal (& C-terminal?) sequencing
- HPLC?
- metal analysis
- mass spectrometry
- NMR spectrometry & X-ray crystallography*
- other?
*Not usually used for routine analytical purposes!!
Protein Size Determination by SDS Polyacrylamide Gel Electrophoresis
- electrode
Adapted from
T. E. Creighton,
Proteins
W.H.Freeman,
1984
+ electrode
Circular Dichroism Spectroscopy of Polypeptides
Adapted from
T. E. Creighton,
Proteins
W.H.Freeman,
1984