1. Lecture 14
February 23, 2016
Biotech 3

2. Protein Purification
A process intended to isolate one or more proteins from a mixture, usually from cells or whole tissue extracts.
Exploit protein properties for purification purposes.
Physio-chemical properties – charge or hydrophobicity
Binding affinity – Tags
Size – large or small
Biological activity – Substrate analogs

3. Protein Purification - Preparation
Secreted or not secreted?
Secreted – Purify protein from cell growth cell media
Not secreted (intracellular) – Extract protein from cell culture
Repeated freeze thaw cycles
Sonication
Homogenization by high pressure (French press
Homogenization by grinding (bead mill)
Permeabilization by detergents (e.g. Triton X-100) and enzymes (e.g. lysozyme)
Collect pellet if insoluble
Centrifuge
Collect supernatant if soluble

4. Ammonium Sulfate Precipitation
Common 1st step in protein purification.
Add increasing amounts of ammonium sulfate to protein extract.
Exposes hydrophobic groups on the protein.
Hydrophobic groups attract other protein hydrophobic groups.
Results in aggregation of protein.
Protein precipitates from solution.
Inexpensive.
Reversible aggregation!!

5. Ammonium Sulfate Precipitation Steps

6. Ammonium Sulfate Precipitation – Step 1
1. Add ammonium sulfate
Must perform on ice!
Increments of percent of saturation example)
10%, 20%, 30% etc.

7. Ammonium Sulfate Precipitation – Step 1 Cont.
Protein aggregates and precipitates out from solution

8. Ammonium Sulfate Precipitation – Step 2

9. Ammonium Sulfate Precipitation – Step 3

10. Ammonium Sulfate Precipitation – Step 4 (repeat 1-3)
30 %
Continue process: 40%, 50%, 60%...etc.

11. Ammonium Sulfate Precipitation Example
Step
Pellet - total protein (mg)
Supernatant - total protein (mg)
Pellet - total enzyme activity
Supernatant -total enzyme activity
Crude extract
6000 -
2000
10% (NH4)2SO4
100
5900
20% (NH4)2SO4
1000
4900
1900
30% (NH4)2SO4
3900
500
1400
50% (NH4)2SO4
800
3100
600
70% (NH4)2SO4
2300
80% (NH4)2SO4
1300
Simple
Pellets can be added together
Removes nonprotein molecules not seen in table

12. Protein Purification - Chromatography
Affinity
Ion Exchange
Hydrophobic interaction
Size exclusion
Reverse phase

13. 1. Affinity Chromatography
Separates proteins based on specific interaction.
Reversible interaction
High selectivity
High resolution
High capacity
Can obtain several 1000-fold purity
Example: Metal ion Affinity Chromatography
Chelates metal ions such as Ni2+ (Co2+ , Cu2+, Zn2+)

14. Metal Chelating – NTA and 6XHis Tag
Pictured:
Nitrilo triacetic acid (NTA)
Alternatives:
nitrile triacetic acid (IDA)
Tris carboxymethyl ethylene diamine (TED)
Polyhistidine tag
Nickel Support

15. Metal Chelating – NTA Bound to 6XHis Tag

16. Metal Chelating – Imidazole

17. Metal Chelating – Imidazole Competes with His Tag

18. Chromatograph Example

19. Types of Affinity Chromatography
Additional examples:
Tags
Glutathione-S-Transferase (GST) tag
Maltose binding protein (MBP) tag
b) c) Lectin affinity
Lectins are carbohydrate binding proteins
Concanavalin A is a lectin
Can purify glycosylated from non-glycosylated proteins
d) Immunoaffinity (Antibodies)
Protein A
e) Hormone, vitamin
f) Enzyme
Substrate analogue, inhibitor, cofactor
g) Nucleic acid

20. 2. Ion Exchange Chromatography
Separates molecules based on charge
Beads of resin are modified so that they contain ac cationic or anionic functional group that can be positively or negatively charged.
Beads of resin are modified so that they contain a cationic or anionic functional group that can be positively or negatively charged.
Species of interest is applied to the column and the sample either binds to the resin or passes through the column.
A gradient of salt or pH is used to elute the desired compound from the resin.

21. Cation Exchange Chromatography
Cation Exchange – “Exchanging cations”, meaning the resin on the column is anionic which will binds to cations.
Strong and weak cation exchange groups.
Strength refers to extent of variation of ionization with pH and not strength of binding.
Ionized at wide range of pH.
Influences loading capacity of column at high or low pH.
Examples:
S-Sepharose (Sulphopropyl)
CM-Sepharose (Carboxymethyl)
Strong
Weak

22. Cation Exchange Chromatography Step 1 - Load

23. Cation Exchange Chromatography Step 2 - Binding
LOAD
BINDING

24. Cation Exchange Chromatography Step 3 - Wash
LOAD
BINDING
WASH

25. Cation Exchange Chromatography Step 4 - Elute
LOAD
BINDING
WASH
ELUTE

26. Cation Exchange Chromatography Step 4 – Elute Cont.
LOAD
BINDING
WASH
ELUTE

27. Anion Exchange Chromatography
Anion Exchange – “Exchanging anions”, meaning the resin on the column is cationic which will binds to anions.
Strong and weak anion exchange groups.
Strength refers to extent of variation of ionization with pH and not strength of binding.
Ionized at wide range of pH.
Influences loading capacity of column at high or low pH.
Examples:
Q-Sepharose
(Quaternary ammonium)
DEAE-Sepharose
(Diethylaminoethyl)
Strong
Weak

28. Anion Exchange Chromatography Step 1 - Load

29. Anion Exchange Chromatography Step 2 - Binding
LOAD
BINDING

30. Anion Exchange Chromatography Step 3- Wash
LOAD
BINDING
WASH

31. Anion Exchange Chromatography Step 4 - Elute
LOAD
BINDING
WASH
ELUTE

32. Anion Exchange Chromatography Step 4 – Elute Cont.
LOAD
BINDING
WASH
ELUTE

33. Isoelectric Point (pI)
Isoelectric Point (pI) - is the pH point where the net chare of a protein is zero.
Environment
Environment
pH is lower < pI < pH is greater
Lower pH means more protons in solution
Protein will be more protonated
Protein will carry a greater POSITIVE charge
Higher pH means less protons in solution
Protein will be more deprotonated
Protein will carry a greater NEGATIVE charge

34. Isoelectric Point Exercise
Environment
Environment
pH is lower < pI < pH is greater
Example: pH
pI 4
pI 7
pI 10 9 7 5

35. Isoelectric Point Exercise Answers
Environment
Environment
pH is lower < pI < pH is greater
Example: pH
pI 4
pI 7
pI 10 9 - + 7 5

36. Protein Sequence Analysis
Protein pI
How do we know the pI of a protein?
MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTL VEQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTE PGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHY GMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

37. ExPASy – Bioinformatics Resource Portal
Protein pI
How do we know the pI of a protein?
Check on ExPASy – Bioinformatics Resource Portal
Sequence of interest
MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINK AGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERDGQLAQ LQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTE MRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWRQLLAMASAVSH RDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGL EHKNQILSFLWQANRRLHSRAPLCERLSPVLNGLQNLTLLRDIELRVYDT DDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSH TQYGILLATLPQGRHLSHDQQQLVDTLVEQLTATLALDRHQERQQQLIV MEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRN ELNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLP PRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQD NGCGVPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIP EKTFTDVQGDTHE
Theoretical pI = 6.27

38. 3. Hydrophobic Interaction
Separates proteins based on hydrophobicity.
Start with high salt concentration
End with low salt concentration
Typical elution profile of hydrophobic column

39. 3. Hydrophobic Interaction – Role of Water
Good solvent for polar substances
Poor solvent for non-polar substances
Highly ordered “water shells” surround hydrophobic surfaces of ligands and proteins
Hydrophobic substance merge to minimize exposed area.
Hydrophobic proteins bind to hydrophobic column ligands.
Interaction of protein with column matrix may depend on van der Waals forces which increase as the structured order of water increases

40. 3. Hydrophobic Interaction – Role of Salt
Principle is similar to “salting out” proteins
High salt concentrations sequester water molecules
Decrease salt concentration to elute protein.
Relative effectiveness of protein precipitation (promote hydrophobic interaction):
Na2SO4 > KSO4 > (NH4)SO4 > Na2HPO4 > NaCl > LiCl

41. 3. Hydrophobic Interaction -Examples
Role of Column
Hydrophobicity of the column ligand influences protein binding.
Capacity and hydrophobicity strength
Phenyl > Butyl > Octyl

42. Principles of Hydrophobic Interaction
Excess salt (yellow) binds to water (blue)
Less water is available to form a “shell” around the protein.
Protein hydrophobic patches become exposed
Exposed hydrophobic patches bind to hydrophobic chains bound to column resin.
Lowering salt concentration reduces exposure of a protein’s hydrophobic patches.
Protein elutes off from hydrophobic interaction column at low salt concentration.

43. Protein Sequence Analysis Cont.
Role of Protein
How do we know if protein of interest is hydrophobic?
MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTL VEQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTE PGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHY GMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

44. ExPASy – Bioinformatics Resource Portal –Transmembrane region determination
Role of Protein
How do we know if protein of interest is hydrophobic or has transmembrane regions?
Check on ExPASy – Bioinformatics Resource Portal
MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTL VEQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTE PGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHY GMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

45. Protein Transmembrane Regions
Role of Protein
How do we know if protein of interest is hydrophobic?
Check on ExPASy – Bioinformatics Resource Portal
Identified two transmembrane regions

46. Protein Transmembrane Regions Cont.
Role of Protein
How do we know if protein of interest is hydrophobic?
Check on ExPASy – Bioinformatics Resource Portal
Clone protein without hydrophobic region to obtain a soluble product (potentially).

47. Reverse Phase Chromatography
Organic solvent running buffer solutions (ex methanol, ethanol, propanol, tetrahydrofuran, acetonitrile)
Nonpolar carbon chain beads (C2 to C18 bound to silica)
Beads are not polysacharide based
Higher pressure
Beads do not collapse
Higher resolution
Smaller particle size determination

48. 4. Size Exclusion Chromatography
Also known as “Gel Filtration Chromatography”.
Separate proteins based on size.
Column with resin that has small holes.
Smaller proteins will enter holes more readily than larger proteins.
Smaller proteins will thus be retained more than larger proteins and elute at a later volume.
Larger proteins will move more readily through column and elute at an earlier volume.
There is no competitive “elution buffer”.

49. 4. Size Exclusion Chromatography – Protein Separation

50. 4. Size Exclusion Chromatography Chromatograph

51. 4. Size Exclusion Chromatography - Standard

52. 4. Size Exclusion Chromatography – Why?
Why do we care for the size, can’t we just look up the molecular weight online?

53. 4. Size Exclusion Chromatography – FUN!
Why do we care for the size, can’t we just look up the molecular weight online?
It’s FUN to perform protein chromatography!
But that’s not the only reason, there’s more to it than that!

54. 4. Size Exclusion Chromatography – Analysis Method
Why do we care for the size, can’t we just look up the molecular weight online?
It’s FUN to perform protein chromatography!
Can determine the Native molecular weight.
Purification step.
Analytical
Bound cofactors
Multimeric protein
Determine number of subunits (Quaternary structure)

55. Example Protein
MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTL VEQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTE PGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHY GMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

56. Example Protein Characteristics
Full length MW 67KDa
Truncated at Met219
Truncated MW 44.2KDa (With His Tag)
pI = 5.94
MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVL NGLQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQL VDTLVEQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTF RLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPE NAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHEHHHHHH

57. Ni Column Buffer A Buffer A.2 Buffer B Tris pH 7.5 2% Glycerol
50mM NaCl
Buffer A.2
1M NaCl
Buffer B
2%Glycerol
0.5M Imidazole

58. Q Column Buffer A Tris pH 7.5 50mM NaCl 2% Glycerol 40mM DTT Buffer B

59. Butyl Column Buffer A Buffer B 50mM Tris pH 7.5 2% Glycerol 40mM DTT
1M Ammonium Sulfate

60. Superdex 200 Superdex Buffer 50mM Tris pH 7.5 0.3M NaCl 2% Glycerol
40mM DTT