Protein Purification

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Information about Protein Purification
Science

Published on April 27, 2014

Author: angelsalaman

Source: slideshare.net

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Protein Purification

1 Protein Purification Fraction Characterization ANGEL L SALAMAN-BAYRON, PhD angelsalaman@yahoo.com

2 PRESENTATION OUTLINE •Protein Purification Scheme •Protein fractionation •Chromatography techniques • Affinity Chromatography (AC) • Hydrophobic Interaction Chromatography (HIC) • Ion Exchange Chromatography (IEC) • Gel Filtration (GF) • Capillary electrochromatography (CEC) • Strategies for Protein Purification Solubility, Aggregation and Re-folding of Proteins

3 Sample Separation technique Fractionation Purification is a Multi-Step Procedure. Is there activity?Set aside N o Combine Fractionsyes Monitor purity Assay total protein Assay enzyme activity Pure? Prepare for analytical technique yes N o Repeat with another separation technique until pure

4 General Protein Purification Scheme • Grow cells in media (vector+tag) •Bacteria Suspension •Bioreactor Purification Strategy Expression SDS PAGE Assay Solubility Aggregation Recombination Characterization Mass Spectroscopy X-ray Crystallography Functional Assay Lyse the cells (appropriate buffer) Centrifuge Collect the pellet

5 1. Evaluate an assay for the protein of interest 2. Shortlist a method to have a reasonable source for that activity Set Protein Purification Strategy

6 Preparing the sample (Crude Extract) Protein from cells or tissue Microbial cells or tissue Break cells, Blender, homogenizer, sonication, pressure osmotic Pellet with intact cells, organelles, membranes and membrane proteins Supernatant with Soluble protein

7 • As the column separates the proteins in the mixture, the “effluent” drips into a series of fraction tubes that are moving at a specific rate of speed. These tubes are called fractions. • Here we are showing 20 tubes. Fraction collectors in most labs have about 75- 200 tubes. • How do we know which fractions contain protein? Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. Aromatic amino acids absorb light at this wavelength causing all proteins to have absorbance at 280nm. Many fraction collectors measure the A280 as the column is running. Collect fractions.

8 A280 Plot valuesPlot values 0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # Question 1. How do we know which fractions contain protein?

9 • Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. • The values can be plotted against the fraction number in is what is called an elution profile. • Notice the peaks on the graph. These indicate where the fractions are that contain protein. Question 1. How do we know which fractions contain protein? A280 0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fraction # A280 Fraction # Peaks

10 • Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein. Which fractions contained the desired protein? A280 0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A280 Fraction # Enz. Assay.Enz. Assay. Fraction #

11 • Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein. • Notice the results of the enzyme assay. The highest activity corresponds to one of the peaks. • Now we can have them discard tubes that don’t have enzyme activity. Which fractions contained the desired enzyme? A280 0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A280 Fraction # EnzAssay Results

12 The Way to Chromatography • In order to isolate sufficient quantities of protein, you may need to start with kilogram quantities of source (i.e. bacteria, tissues, etc.) These amounts can best be handled using precipitation methods (e.g. ammonium sulfate precipitation). Later in the purification, large columns can be used to handle gram to milligram quantities. Amounts handled on gels are typically in microgram quantities.

13 Property Methods Solubility Precipitation with ammonium sulfate (salting out)* Size / shape Size-exclusion chromotography Isoelectricpoint (charge) Ion exhange chromatography binding to small molecules Affinity chromatography Common methods of protein purification *Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme. • Purification procedures attempt to maintain the protein in native form. Although some proteins can be re-natured, most cannot! • To purify a protein from a mixture, biochemists exploit the ways that individual proteins differ from one another. They differ in: • Thermal stability: For most protein purifications, all steps are carried out at ~5°C to slow down degradation processes.

14 Picture of protein gel with lanes showing sequential purification steps Procedure Fraction vol (ml) Total Prot (mg) Activity (units) Specific activity Units/mg Crude cellular extract 1400 10000 100,000 10 Size- exclusion 90 400 80,000 200 Ion exchange 80 100 60,000 600 Note: The type and order of steps are customized for each protein to be purified. An effective purification step results in a high yield (minimal loss of enzyme activity) and large purification factor (large increase in specific activity). Purification Yield

15 Chromatographic Mode Acronym Separation Principle Non-interactive modes of liquid chromatography Size-exclusion chromatography SEC Differences in molecular size Agarose chromatography (for DNA) for DNA binding proteins - Diff. in length and flexibility Interactive modes of liquid chromatography Ion-exchange chromatography IEC Electrostatic interactions Normal-phase chromatography NPC Polar interactions Reversed-phase chromtography RPC Dispersive interactions* Hydrophobic interaction chromatography HIC Dispersive interactions* Affinity chromatography AC Biospecific interaction Metal interaction chromatography MIC Complex w/ an immobilized metal Chromatographic Modes of Protein Purification * Induced dipole – induced dipole interactions

16 Column Selection

17 Affinity Chromatography Surface bound with Epoxy, aldehyde or aryl ester groups Metal Interaction Chromatography Surface bound with Iminodiacetic acid + Ni2+ /Zn2+ /Co2+ Affinity Chromatography

18 Metal Interaction Chromatography (AC) Points to Note: 1. Avoid chelating agents 2. Increasing incubation time 3. Slow gradient elution

19 Affinity Chromatography Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa) Depends on Molecular weight Degree of substitution /ml medium ~15mmol Ni2+ Backpressure ~43psi Change the guard column filter

20 Biopolymer (phenyl agarose - Binding Surface) Driving force for hydrophobic adsorption Water molecules surround the analyte and the binding surface. When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy. Temperature plays a strong role Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer Hydrophobic Interaction Chromatography Hydrophobic region

21 ION –EXCHANGE 1 • First, to determine the charge on a protein, given its pI and the pH. • Ion-exchange column chromatography separates proteins on the basis of charge. • We will start with 4 proteins. • pH 7.2 • Positive charged column 60 Kd Low pI (6) 20 Kd Low pI (7) 20 Kd Medium pI (7) 5 Kd Hi pI (8)

22 pos • The matrix of an ion exchange is positively charged. • What do you think will happen?pos pos pos pos pos pos Run columnRun column pos pos pos pos pos pos

23 • The matrix of an ion exchange is positively charged. • Only the pos charged proteins run through the pos charged column. The others “stick” to the column. pos pos pos pos pos pos pos pos pos pos pos pos pos

24 Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen) Ion Exchange Chromatography Globular Protein Deformation due to interaction with conventional ion exchanger Maintenance of conformation while interacting with tentacle ion exchanger

25 Gel Filtration

26 • Gel filtration column chromatography separates proteins on the basis of size. • We will start with 4 proteins. • You will want to purify the “yellow one” 60 Kd Low pI (6) 20 Kd Low pI (7) 20 Kd Medium pI (7) 5 Kd Hi pI (8) Gel Filtration

27 • The matrix of a size-exclusion chromatography column is porous beads. Run columnRun column

28 • The matrix of a gel filtration column are beads with pores. • The large gray proteins can’t fit in pores so flows faster. • The red / yellow medium sized proteins get trapped in the pores. • The black small proteins stay trapped in pores longer.

29 Immune Affinity Chromatography

30 ATP immobilized on polyacrylamide resin DNA Binding Proteins Heparin Sepharose Negatively charged proteins (pI >7) are not captured/separated effectively.

31 Capillary Electrochromatography • CEC is an electrokinetic separation technique • Fused-silica capillaries packed with stationary phase • Separation based on electro-osmotically driven flow • Higher selectivity due to the combination of chromatography and electrophoresis Fused silica tube filled with porous methacrylamide-stearyl methacrylate-dimethyldiallyl ammonium chloride monolithic polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000 Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N number of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline

33 CEC columns AC, IEC columns CEC column NP, RP columns

34 Schematic of a Multi-dimensional Separation System

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

salamaa@wyeth.com 36 Fast Protein Liquid Chromatograph (FPLC) 1 2 3 5 4 • No air bubbles (Priming) • Use degassed buffers Injector Module Column Inlet Detector Fraction Collector

37 Protein Analysis

38 Reagent Derivatization Detection o-Phthaldialdeyhde Precolumn/ Postcolumn FL, Ex 340nm/Em 400nm Fluorescamine Precolumn/ Postcolumn FL, Ex 390nm/Em 490nm Indocyanine greeen Precolumn FL, Ex 765nm/ Em 820-840nm Detection of Proteins by Derivatization with Higher Sensitivity 1000 times more sensitive than UV-Vis detection

39 Solubility of a Protein Membrane proteins 1. Removal of unbroken cells from the cell lysate by low speed centrifugation (20 min at 10,000 g). 2. Isolation of the membrane particles from the supernatant by ultracentrifugation (60 min at >100 000 g). 3. Washing of the membrane particle to remove all soluble proteins. 4. Solubilization of protein from the membrane particles by a mild detergent. (detergent: protein ratio = 1:10) 5. Phosphate buffers(0.1M-0.5M), 5-50% glycerol helps. • Depends strongly on the composition of the lysis buffer. • Salt concentration Freeze-thaw protocol * Freeze quickly on dry ice and leave for 3 min. * Thaw immediately at 42 °C. Vortex vigorously to mix well. * Repeat the two previous steps three more times (4 cycles in all).

40 Protein Aggregation • Numerous physicochemical stresses can induce protein aggregation: • Heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals, phenolic compounds, and denaturants.

41 Denaturation and Renaturation Variables Good starting point Buffer composition (pH, ionic strength) 50 mM Tris-HCl, pH 7.5 Incubation temperature 30°C Incubation time 60 min Concentration of solubilizing agent 6 M guanidine-HCl or 8 M urea Total protein concentration 1-2 mg/ml Re-folding of Proteins The addition of a mixture of reduced and oxidized forms of low molecular weight thiol reagent usually provides the appropriate redox potential to allow formation and reshuffling of disulfide bonds (1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol) The most commonly used are glutathione, cysteine and cysteamine. Solubilization of Aggregated Proteins

42 Polyethylene glycol (PEG 3350) 0.1-0.4 g/L  L-Arginine hydrochloride 0.4-0.8M Nondenaturating concentrations of Urea < 2.0 M K-Glutamate  ~5M Nondenaturating concentrations Gdm/ClH < 1.0 M Proline  ~1M Methylurea  1.5-2.5 M Glycerol  20-40 % Ethylurea  1.0-2.0 M Sorbitol 20-30 % Formamide 2.5-4.0 M Sucrose  ~1M Methylfomamide 2.0-4.0 M Trehalose  ~1M Acetamide 1.5-2.5 M TMAO (trimethylamine N-oxide)  ~1M Ethanol  Up to 25% Sulfo Betaine ~1M Reagents used for Re-folding of proteins

43 n-Penthanol 1.0-10.0 mM Lauryl Maltoside 0.06 mg/ml n-Hexanol 0.1-10.0 mM CETAB  0.6 mg/ml Cyclohexanol  0.01-10.0 mM CHAPS  10-60 mM Tris   > 0.4 M Triton X-100 10 mM Na2SO4 or K2SO4 0.4-0.6 M Dodecyl Maltoside 2.0-5.0 mM Cyclodextrin  20-100 mM Sarkosyl 0.05-0.5 % Reagents used for Re-folding of proteins Cont.

44 6xHis Tagged Protein Detection Directly on the Gel (from Pierce) E. coli lysates expressing 6xHis-tagged proteins, stained with the Pierce 6xHis Protein Tag Staining Kit 

45 GST•Bind™ Purification Kits His•Bind® Purification Kits Magnetight™ Oligo d(T) Beads MagPrep® Streptavidin Beads Protein A and Protein G Plus Agaroses S•Tag™ Purification Kits Streptavidin Agarose T7•Tag™ Affinity Purification Kit ProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Maxi Kit — Effectively desalts and concentrates up to 8 mg of protein with an efficient, easy-to-use protocol.(Norgen Biotek Corporation) ProteoSpin™ Detergent Clean-up Micro Kit — Provides a fast and effective procedure to remove detergents including SDS, Triton® X-100, CHAPS, NP-40 and Tween 20. Commercially available protein purification kits

46 REFERENCES • Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000 • www.qiagen.com • www.novagen.com • http://lsvl.la.asu.edu/resources/mamajis/chromatography/chrom atography.html • http://www.cellmigration.org/resource/discovery/discovery_prote omics_approaches.html • http://www.capital-hplc.co.uk • http://www.ls.huji.ac.il/~purification • www.biovectra.com • http://www.ls.huji.ac.il/~purification

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