Strategies to Purify Protein Jeremy Berg of the Johns Hopkins University John Tymoczko of Carleton College, Lubert Stryer's Biochemistry Lubert Stryer, Peter Gianakopoulos ,Mitchell Guss, Rong-Huay Juang and ………. חיפשתי , מצאתי ,שיניתי וחיברתי אני יורי רזניק מקיף "ח" אשדוד . לא מאמין שכבר סיימתי. אוף......

4.הפרדה על פי זיקה ביולוגית (אנזים-מצע, אנטיגן-נוגדן, שיטות להפרדת חלבונים 1.השפעת ריכוזי מלח ( אמוניום סולפט ) על חלבונים שונים In /Salting Out Salting 2.הפרדה על פי גודל: כרומטוגרפית סינון בג'ל דיאליזה אלקטרופורזה ב- S.D.S 3.הפרדה על פי מטען (אלקטרופורזה , מחליפי יונים , השקעה ב- pI ) 4.הפרדה על פי זיקה ביולוגית (אנזים-מצע, אנטיגן-נוגדן, הורמון-קולטן) 5.הפרדה על פי קוטביות

1 1 3 2 4 2 3 4 4 4 How to Separate These Objects 6 5 6 5 6 5 5 Shape Size Density 1 3 4 6 7 8 9 10 11 12 2 5 wood stone cotton wood wood cotton stone wood stone cotton stone cotton 1 2 3 Shape 4 Size 6 cotton wood stone 4 8 5 8 4 6 7 8 5 Density 5 7 9 10 11 12 Different rolling speed Different sedimentation Sieving different sizes Juang RH (2004) BCbasics

Basic Principles of Protein Purification Cell Organelle Homogenization Macromolecule Small molecule Cell Debris Amino acid, Sugar, Nucleotides, etc Nucleic acid Protein Carbohydrate (Lipid) Ammonium sulfate fractionation Size Charge Polarity Affinity אלקטרופורזה , מחליפי יונים , השקעה ב-PI chromatography, Salting-out Gel filtration, SDS-PAGE, Dialysis Affinity chromatography Juang RH (2004) BCbasics

Strategies to Purify Protein I. Solubility Salting out Proteins are less soluble at high salt concentrations The salt concentration at which a protein precipitates differs 1.השפעת ריכוזי מלח ( אמוניום סולפט ) על חלבונים שונים In /Salting Out Salting

Salting out Ammonium sulfate (half saturated) Ethanol (90%) “salting out” adding salt eg ammonium sulfate salting-out effect as [salt]  less H2O is available for hydration of protein proteins will aggregate

Strategies to Purify Protein II. Size Dialysis Useful for removing small molecules Not an efficient means to separate proteins Gel filtration chromatography Good strategy for separating proteins of different sizes Larger molecules elute earlier 2.הפרדה על פי גודל: כרומטוגרפית סינון בג'ל ,דיאליזה , אלקטרופורזה ב- S.D.S

Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium.

molecular weight, native molecular sieve chromatography (gel filtration, molecular sizing) light scattering hydrodynamic (equilibrium centrifugation) mix large molecules first

Gel Filtration Chromatography Gel Filtration Chromatography. A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones.

Polyacrylamide Gel Electrophoresis.

Polyacrylamide Gel Electrophoresis Polyacrylamide Gel Electrophoresis. The negatively charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the anode, at the bottom of the gel. The sieving action of a porous polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.

molecular weight, denatured SDS gel electrophoresis molecular weight, denatured gel filtration in denaturing solvent

+ SDS-PAGE cont’d Dithiothreitol -reducing agent -breaks disulphide bonds between subunits -S-S- DTT -S-H HS- +

- - + detergent binds strongly to proteins approx. 1 SDS/2 a.a. SDS-PAGE cont’d detergent binds strongly to proteins approx. 1 SDS/2 a.a. Sodium dodecyl sulfate - = SDS - Protein + Proteins (subunits) become linear Gives all ptns an equal mass : charge ratio

SDS-PAGE cont’d

Strategies to Purify Protein III. Charge Ion exchange chromatography Based on a proteins charge density Elute bound proteins with an increasing concentration of a counter ion (e.g. NaCl) 3.הפרדה על פי מטען (אלקטרופורזה , מחליפי יונים , השקעה ב- pI )

אלקטרופורזה - Kabat & Tiselius (1939) + Albumin a b g + - Globulins Electrophoresis - a molecule with a net charge will move in an electric field The velocity of a molecule is described by  = Ez/f v = velocity E = electric field strength z = net charge of a molecule f = frictional coefficient (depends on both mass and shape)

Electrophoretic analysis of serum Sample application Separation by charge - - + + Anode Cathode Albumin a b g + - Globulins

מחליפי יונים Ion-Exchange Chromatography. This technique separates proteins mainly according to their net charge.

Ion Exchange - charge CATION EXCHANGE Mixture of proteins is applied to the negatively charged -ve and neutral proteins pass in the void volume, +ve proteins stick Excess of Na+ ions is added +ve proteins are eluted Could have used change in pH to elute the proteins CATION EXCHANGE

Solubility  fractional precipitation Solubility depends on temperature, pH, ionic strength Increasing temperature generally increases solubility of limited practical relevance in biology Solubility depends on the pH minimum at the pI - the isoelectric point – the pH when the net charge on the molecule is zero

Solubility with pH Minimum when pH =pI pI is the pH at which the molecules have zero net charge

Strategies to Purify Protein IV. Binding activities Affinity chromatography Covalently attach “bait” to resin Pass extract over the column Wash to remove non-specific and weak interacting proteins Elute Usually by passing “bait” molecule through column 4.הפרדה על פי זיקה ביולוגית (אנזים-מצע, אנטיגן-נוגדן, הורמון-קולטן)

Affinity Chromatography Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G). Could have used change in pH to elute the proteins

Glutathione Column Purpose: To purify GST or GST fused proteins GST = Glutathione = 3 a.a. peptide (Glu-Cys-Gly) = has a high affinity for

Glutathione Cont’d Supernatant (Day 1) Wash w/ Solution A Sepharose column Flow through

Elution Step to collect GST Glutathione Cont’d Elution Step to collect GST 10 mM Glutathione -add in 1mL fractions Look!!! MORE beer! Collect in fractions Could have used change in pH to elute the proteins

5.הפרדה על פי קוטביות Polarity :adsorption chromatography hydrophobic interaction chromatography

chromatography

chromatography

amino acid analysis, chromatography

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