Lecture 14 February 23, 2016 Biotech 3 Protein

Lecture 14 February 23, 2016 Biotech 3 Protein

Lecture 14 February 23, 2016 Biotech 3 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. 1. 2. 3. 4.

Physio-chemical properties charge or hydrophobicity Binding affinity Tags Size large or small Biological activity Substrate analogs Protein Purification Preparation Secreted or not secreted? Secreted Purify protein from cell growth cell media Not secreted (intracellular) Extract protein from cell culture 1. Repeated freeze thaw cycles 2. Sonication 3. Homogenization by high pressure (French press 4. Homogenization by grinding (bead mill)

5. Permeabilization by detergents (e.g. Triton X-100) and enzymes (e.g. lysozyme) Collect pellet if insoluble Centrifuge Collect supernatant if soluble 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!! Ammonium Sulfate Precipitation

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

Step 1 Cont. Protein aggregates and precipitates out from solution Ammonium Sulfate Precipitation Step 2 Ammonium Sulfate Precipitation Step 3 Ammonium Sulfate Precipitation Step 4 (repeat 1-3)

30 % Continue process: 40%, 50%, 60%...etc. Ammonium Sulfate Precipitation Example Step Crude extract 10% (NH4)2SO4 20% (NH4)2SO4 30% (NH4)2SO4 50% (NH4)2SO4

70% (NH4)2SO4 80% (NH4)2SO4 Pellet - total protein (mg) 100 1000 1000 800 800 1000

Supernatant total protein (mg) 6000 5900 4900 3900 3100 2300 1300 Pellet - total enzyme activity 0

100 500 800 500 100 Simple Pellets can be added together Removes nonprotein molecules not seen in table Supernatant total enzyme activity 2000

2000 1900 1400 600 100 0 Protein Purification Chromatography 1.Affinity 2.Ion Exchange 3.Hydrophobic interaction 4.Size exclusion 5.Reverse phase

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+) Metal Chelating NTA and 6XHis Tag Pictured: Nitrilo triacetic acid (NTA) Alternatives: nitrile triacetic acid (IDA)

Tris carboxymethyl ethylene diamine (TED) Nickel Support Polyhistidine tag Metal Chelating NTA Bound to 6XHis Tag Metal Chelating Imidazole Metal Chelating Imidazole Competes with His Tag

Chromatograph Example Types of Affinity Chromatography Additional examples: a) 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 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. 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) Strong CM-Sepharose (Carboxymethyl) Weak Cation Exchange Chromatography Step 1 - Load

Step 1. LOAD Cation Exchange Chromatography Step 2 - Binding Step 1. LOAD 2. BINDING Cation Exchange Chromatography Step 3 - Wash Step

1. LOAD 2. BINDING 3. WASH Cation Exchange Chromatography Step 4 - Elute 1. 2. 3. 4. Step LOAD

BINDING WASH ELUTE Cation Exchange Chromatography Step 4 Elute Cont. 1. 2. 3. 4. Step

LOAD BINDING WASH ELUTE 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) Strong DEAE-Sepharose (Diethylaminoethyl) Weak Anion Exchange Chromatography Step 1 - Load

Step 1. LOAD Anion Exchange Chromatography Step 2 - Binding Step 1. LOAD 2. BINDING Anion Exchange Chromatography Step 3- Wash Step 1. LOAD

2. BINDING 3. WASH Anion Exchange Chromatography Step 4 - Elute 1. 2. 3. 4. Step LOAD BINDING

WASH ELUTE Anion Exchange Chromatography Step 4 Elute Cont. 1. 2. 3. 4. Step LOAD BINDING

WASH ELUTE Isoelectric Point (pI) Isoelectric Point (pI) - is the pH point where the net chare of a protein is zero. Environment pH is lower + Environment

< Lower pH means more protons in solution Protein will be more protonated Protein will carry a greater POSITIVE charge pI < pH is greater -

Higher pH means less protons in solution Protein will be more deprotonated Protein will carry a greater NEGATIVE charge Isoelectric Point Exercise Environment Environment pH is lower + <

pI < pH is greater - Example: pH 9 7 5

pI 4 pI 7 pI 10 Isoelectric Point Exercise Answers Environment Environment

pH is lower + < pI < pH is greater - Example:

pH pI 4 pI 7 pI 10 9 - -

+ 7 - 0 + +

+ 5 Protein Sequence Analysis Protein pI How do we know the pI of a protein? MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTLV EQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEP

GLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYG MIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE ExPASy Bioinformatics Resource Portal Protein pI How do we know the pI of a protein? Check on ExPASy Bioinformatics Resource Portal http://www.expasy.org/ Sequence of interest MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINK AGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERDGQLAQL

QGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTE MRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWRQLLAMASAVSH RDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLE HKNQILSFLWQANRRLHSRAPLCERLSPVLNGLQNLTLLRDIELRVYDTD DEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHT QYGILLATLPQGRHLSHDQQQLVDTLVEQLTATLALDRHQERQQQLIVM EERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNE LNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPP RLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDN GCGVPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPE KTFTDVQGDTHE Theoretical pI = 6.27

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

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 3. Hydrophobic Interaction Role of Salt

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 3. Hydrophobic Interaction Examples Role of Column Hydrophobicity of the column ligand influences protein binding. Capacity and hydrophobicity strength

Phenyl > Butyl > Octyl 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 proteins hydrophobic patches. Protein elutes off from hydrophobic interaction column at low salt concentration. Protein Sequence Analysis Cont. Role of Protein

How do we know if protein of interest is hydrophobic? MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTLV EQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEP GLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYG MIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE 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 http://www.expasy.org/ MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTLV EQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEP GLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYG MIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE Protein Transmembrane Regions

Role of Protein How do we know if protein of interest is hydrophobic? Check on ExPASy Bioinformatics Resource Portal http://www.expasy.org/ Identified two transmembrane regions Protein Transmembrane Regions Cont. Role of Protein How do we know if protein of interest is hydrophobic? Check on ExPASy Bioinformatics Resource Portal

http://www.expasy.org/ Clone protein without hydrophobic region to obtain a soluble product (potentially). Reverse Phase Chromatography 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 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. 4. Size Exclusion Chromatography Protein Separation 4. Size Exclusion Chromatography

Chromatograph 4. Size Exclusion Chromatography - Standard 4. Size Exclusion Chromatography Why? Why do we care for the size, cant we just look up the molecular weight online? 4. Size Exclusion Chromatography FUN! Why do we care for the size, cant we just look up the molecular weight

online? Its FUN to perform protein chromatography! But thats not the only reason, theres more to it than that! 4. Size Exclusion Chromatography Analysis Method Why do we care for the size, cant we just look up the molecular weight online? Its FUN to perform protein chromatography! Can determine the Native molecular weight. Purification step. Analytical

Bound cofactors Multimeric protein Determine number of subunits (Quaternary structure) Example Protein MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERD GQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWR QLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNG LQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTLV EQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEP GLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYG MIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE

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

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

2%Glycerol 50mM NaCl 0.5M Imidazole Q Column Buffer A Tris pH 7.5 50mM NaCl 2% Glycerol 40mM DTT Buffer B Tris pH 7.5 1M NaCl

2% Glycerol 40mM DTT Butyl Column Buffer A 50mM Tris pH 7.5 2% Glycerol 40mM DTT Buffer B 50mM Tris pH 7.5 2% Glycerol 40mM DTT

1M Ammonium Sulfate Superdex 200 Superdex Buffer 50mM Tris pH 7.5 0.3M NaCl 2% Glycerol 40mM DTT

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