Lecture 10 PROTEIN ISOLATION

Lecture 10 PROTEIN ISOLATION 1 Protein isolation Outlines: assay; homogenization; fractionation; centrifugation; quantitation; chromatography, electrophoresis; sequencing; people. 2 Nobel Prize Winners on chromatography and electrophoresis 3 Nobel Prize Winners on mass spectroscopy and sedimentation 4 Nobel Prize Winners on organelles 5 Nobel Prize Winners on sequencing 6 Assay Determining an effective assay can be difficult, but he more specific the assay, the more effective the purification. (i) Lactate dehydrogenase (spectroscopic method, NADH production, 340 nm absorption). (ii) Deoxyhypusine synthase (Radioactive labeling method). (iii) Specific activity 7 Homogenization and Fractionation (i) Proteins have to be released from cells to be purified; hence homogenization. (ii) Different homogenization methods, depending on the nature of starting materials and the property of desired protein. (iii) The homogenate will have to be fractionated to determine the enriched fraction. Fractionation can be achieved by a number of methods such as fractional precipitation (heat, acid, solute competition, such as ammonium sulfate precipitation (salting out)) and differential centrifugation (for organelles and soluble proteins). (iv) The enzyme is membrane associated (e.g. RuBISCO), or it is an integral membrane protein (e.g. Succinate dehyrogenase), or it is located inside an organelle or vesicle (e.g. MDH), or it is insoluble under the extraction conditions (inclusion body). 8 Centrifugation (i) differential centrifugation. (ii) Rate-zonal centrifugatopn. (iii) Ultracentrifugation. 9 Centrifugation Differential centrifugation used to separate protein fractions 10 Sedimentation-equilibrium technique The method was used to accurately estimate the mass of a protein under non-denaturing condition, using the Svedberg equation. 11 Sedimentation-equilibrium technique Sedimentation coefficient of different organelles. 12 Sedimentation-equilibrium technique Correspondence of S value and molecular mass. 13 Protein Quantitation (i) Lowry assay: The sensitivity of the procedure of Lowry is moderately constant from protein to protein, and it has been so widely used that Lowry protein estimations are a completely acceptable alternative to a rigorous absolute determination in almost all circumstances where protein mixtures or crude extracts are involved. The method is based on both the Biuret reaction, where the peptide bonds of proteins react with copper under

alkaline conditions producing Cu+, which reacts with the Folin reagent, and the Folin- Ciocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. The method is sensitive down to about 0.01 mg of protein/ml, and is best used on solutions with concentrations in the range 0.01-1.0 mg/ml of protein. (ii) Bradford Assay: Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (1976) 72, 248-254. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Within the linear range of the assay (~5-25 mcg/ml), the more protein present, the more Coomassie binds. 14 Protein Quantitation NanoOrange protein quantitation reagent becomes fluorescent upon binding to a detergent coat around the protein. Because proteins bind comparable amounts of detergent on a mass basis, there is little protein-to-protein variation, allowing you to accurately quantitate protein mixtures of unknown composition. By contrast, UV absorbance measurements are much less sensitive, vary widely among proteins due to differences in the percentages of UV-absorbing amino acids and are inaccurate in the presence of UV-absorbing contaminants. 15 Protein Quantitation Experimental results using the NanoOrange kit. 16 Dialysis This involves putting a protein solution in a sac composed of a semi-permeable membrane (dialysis tubing). The pores in the membrane allow the passage of small molecules (<5000 Da) but proteins are retained. 17 Dialysis On an industrial scale, a related method diafiltration is employed. In this case a rigid nitrocellulose membrane of defined pore size is used that allows the passage of small solutes. Various pore sizes giving different molecular weight cut-offs can be selected. The solution is pushed through the membrane under pressure and blockage is prevented by paddles. Fresh (washing) buffer is fed in at the top of the device. The diafiltration set-up can also then be used to concentrate the protein solution by stopping the buffer flow (in this mode the technique is termed "ultrafiltration". Small versions of these devices are now manufactured for research scale separations using centrifugal force to drive the passage of small solutes. 18 Liquid Chromatography 19 Gel Filtration chromatography In this method large molecules tend to be excluded from the pores that are contained in the beads whilst small molecules can penetrate into them and effectively pass through a larger volume than the larger molecules. Although the small molecules can be thought of as having a greater affinity for the stationary phase, the method is really based on "exclusion volumes" and moreover, for the method to be quantitative, interactions with the hydrophilic bead matrix must be minimised. Typical matrices are agarose, dextran, silica etc. 20 Gel Filtration chromatography Different view of gel filtration chromatography. 21 Gel Filtration chromatography

Gel filtration chromatography is used to separate large molecules on the basis of size. Two columns are run simultaneously. The first column contains Sephadex G-75, which separates blue dextran and hemoglobin. The second column contain Sephadex G-10, which separates hemoglobin and riboflavin. Because there is a difference in the two packing materials, the hemoglobin molecule runs very differently in the two columns. 22 Gel Filtration chromatography Commonly used resins for gel filtration chromatography. 23 Ion Exchange Chromatography In this method, protein in a solution that has a low ionic strength is added to the column. The protein interacts with the beads (the column matrix) via electrostatic interactions. In anion exchange chromatography, the matrix carries a net positive charge which interacts with negatively charged proteins strongly; these tend to partition almost completely into the stationary phase. The removal of the proteins from the column is achieved by washing the column with a buffer of increasing ionic strength. The ions in the buffer compete as counterions and displace the bound protein molecules. Occasionally, the same effect is produced by washing the column with a buffer of decreasing ph; as the ph lowers, the charge on the protein is reduced, leading to weaker interaction with the positively charged matrix. In cation exchange chromatography the matrix is negatively charged and it interacts with positively charged proteins (rarer). Elution is again achieved by a gradient of ionic strength, or by an increasing ph gradient. The method relies on having two pumps which can control the mixing of a low and high ionic strength buffer to form the buffer gradient that washes the column. Separation still depends on the "theoretical plate" model because the protein is bound in a sharp band at the start of the column. As it is eluted it begins to pass down the column, but its progress is still determined by its ability to re-associate with the stationary phase. Thus separation is achieved by two methods: (i) the binding strength of the protein at a given ionic strength, (ii) the partition constant as it passes down the column after its initial displacement from the start of the column. 24 Ion Exchange Chromatography In anion exchange chromatography, the matrix carries a net positive charge which interacts with negatively charged proteins strongly (Resins carrying positive charges are called anion exchanger, because anions will exchange on this type of column). In cation exchange chromatography the matrix is negatively charged and it interacts with positively charged proteins (Resins carrying negative charges are called cation exchanger, because cations will exchange on this type of column). 25 Ion Exchange Chromatography Elution is achieved by a gradient of ionic strength, or by an increasing ph gradient. The method relies on having two pumps which can control the mixing of a low and high ionic strength buffer to form the buffer gradient that washes the column. 26 Ion Exchange Chromatography Commonly used resins for ion exchange chromatography. 27 Affinity chromatography This method relies on the knowledge of the properties of the enzymes or proteins that you want to isolate. Either: (a) Antibodies must be available that have been raised against the enzyme. or (b) The enzyme must be known to bind to something such as a ligand non-covalently in a reversible way (e.g. a glycosylated enzyme will bind to lectin). In this method the antibodies or ligand are covalently attached to the column matrix by a simple chemical cross-linking reaction. The column is then washed and the protein mixture applied. It is expected that the enzyme will bind to the ligand or antibody and be retained on the column whilst the rest of the complex mixture passes through the column. Elution of the non-covalently bound enzyme is achieved by a ph or ionic

strength step in the buffer. Typically, elution by a ph drop to ph5 or by an ionic strength jump to 1M NaCl is used to remove the enzyme. In principle, this chromatography method has the greatest resolving power of any of the methods. Strictly speaking, it is not dependent on the "theoretical" chromatography principles described above, indeed the method would work with any suitable support such as a filter. Even magnetic particles to which the ligand or antibody are chemically linked have sufficed to achieve a rapid and reasonable purification. 28 Affinity chromatography The use of glucose-resin to isolate glucose binding protein Concanavalin A. 29 Affinity chromatography The use of antibody as the affinity matrix for protein isolation. 30 Affinity chromatography Drawbacks: The binding to ligand or antibody can often be so strong that very harsh conditions are needed to elute the enzyme. This leads to denaturation. Non-specific binding may also occur, especially where large ligands such as antibodies are employed. This non-specific binding can overwhelm the "correct" binding sites at very high protein concentrations, leading to poor purification. Thus there is often a limit to the protein concentration that can be applied to an affinity column. 31 HPLC There is nothing magical about HPLC. All the chromatographic methods described above can be used with HPLC columns and equipment, but with HPLC the separation of peaks (ie the resolution) is almost always better than with standard chromatography columns. The reason for this is that HPLC columns are packed with much smaller beads than those used in standard columns. This simple alteration greatly increases the NTPs and hence resolution. The reason that small beads cannot be used in conventional columns is that the much tighter packing of the beads gives rise to high back-pressures which would mean zero flow rates. With HPLC the column is in a metal or plastic container and is connected via metal tubing capable of withstanding high pressures. The column is then connected up to high pressure pumps that can push buffer solutions through the column matrix at high pressure. A special injection valve is required to inject the protein sample into the high pressure system. Flow rates are usually higher than with conventional columns and therefore with shorter separation times. Because most buffer solutions are incompatible with stainless steel, titanium columns and fittings are usually used for HPLC systems designed for protein purification. HPLC columns tend to be smaller and shorter than conventional columns because of the much greater NTPs/m. The pumps are usually controlled by a PC and the outflow from the column is connected to a spectrophotometer that continually monitors the absorbance (usually at 280nm) of the eluate. Finally the eluate is collected in a fraction collector, also linked up to the PC. 32 HPLC Liquid chromatography is a term which refers to all chromatographic methods in which the mobile phase is liquid. The stationary phase may be a liquid or a solid. There are various columns used in liquid chromatography depending on the type of separation preferred. Each column contains a small diameter packing material. The column is a large (mm id) tube containing small (µm) particles (gel beads) known as stationary phase. The chromatographic bed is composed by the gel beads alone when they are inside the column. The sample is introduced into the injector and then carried into the column by the flowing solvent. Once in the column, the sample mixture separates as a result of different components adhering to or diffusing intothe gel.as the solvents is forced into the chromatographic bed by the flow rate, the sample separates into various zones of sample components. These zones are referred to as bands.

33 HPLC HPLC chromatogram. 34 FPLC Fast performance liquid chromatography (FPLC) is a type of liquid chromatography where the solvent velocity is controlled by pumps. The pumps controls the constant flow rate of the solvents. The solvents are accessed through tubing from an outside resevoir. The flow rate of the solvent is set through computer input and controlled by pumps. 35 Electrophoresis Proteins and nucleic acids migrate in an electric field according to their net charge, and for this reason separation can be achieved. In many cases, electrophoresis is coupled to a second separation method to achieve very high resolution of different proteins. The most popular method is SDS-PAGE where electrophoresis is coupled to a seiving device (the polyacrylamide gel) that differentially retards larger proteins. 36 Electrophoresis Free-flow electrophoresis This is the simplest in concept but most difficult to achieve, requiring expensive equipment. Buffer of very low ionic strength is passed slowly between two large glass plates separated by a narrow gap, (1kV) is applied across the width of the plates, perpendicular to the buffer flow. Protein mixture (which must be in the same low ionic strength buffer) is injected in at the top of the plates and begins to flow down with the buffer flow. Proteins that carry a net -ve charge will also migrate towards the +ve electrode whilst +vely charged proteins go in the opposite direction. Buffer flow is adjusted so that protein never reaches the electrodes before it arrives at the bottom of the plates where different fractions are collected in small tubes. At the bottom of the plates, a beam of uv light at 280 nm sweeps across and the absorption due to protein is recorded. Although expensive, this method is one of the few electrophoresis methods that can be termed preparative rather than analytical. 37 Electrophoresis Gel electrophoresis, how samples are separated inside the gel matrix. 38 Electrophoresis In PAGE, a gel made by polymerising acrylamide and bisacrylamide together forms a network of pores, the sizes of which are defined by the initial acrylamide and bisacrylamide concentrations. In some cases a gel is created that contains a gradient of concentration of the monomers and hence a variation in pore size going from the top of the gel to the bottom. When the electric field is applied across the gel, protein migrates according to its charge and thus, the distance it migrates in the gel in a given time is determined by the pore size, the molecular size of the protein and its net charge. Although this method resolves proteins extremely well, its main drawback, as above, is the low capacity of the technique (sub mg quantities only). 39 Electrophoresis The key feature for SDS-PAGE is that all SDS-coated proteins have the same electrophoretic mobility. Thus molecular sieving effect is the major parameter that causes separation of proteins. 40 Electrophoresis Typical results of SDS-PAGE, protein bands are visible after Commassie Blue dye staining. 41 Electrophoresis Storage of the gel.

42 Electrophoresis Detection of the protein bands by either fluorographic, radiographic labeling method 43 Electrophoresis Mw determination based on SDS-PAGE. 44 Electrophoresis Staining and destaining kit for making protein bands clearly visible. 45 Isoelectric focusing These two methods are related. Both involve the production of a stable ph gradient in some matrix. In chromatofocussing that matrix is a column, whilst in IEF it is a polyacrylamide or agarose gel. Both methods rely on the behaviour of a group of small buffer molecules termed ampholytes. These ampholytes migrate rapidly in the presence of the electric field and distribute themselves at a certain point in the electric field. These ampholytes create a local ph gradient (because of their weak acid or base nature), and by mixing several different ampholytes with different pis, a gradient of ph can be created in the gel or column. Protein placed in the system will now migrate according to its net charge, but it will stop migrating when it reaches a ph that corresponds to its isolelectric point (pi). If the protein is unmodified (by e.g. phosphorylation), the pi will be very sharply defined and hence the "focusing" in the name. For IEF, bands must be cut out of the gel at the end of the run; for chromatofocusing, they can be eluted after or during the focusing. Drawbacks: Neither technique is very suitable for industrial scale since capacity is low. High protein concentrations leads to precipitation of material at the pi (see earlier) and this can block the matrix or gel. Under these conditions, the resistance (ie R) rises enormously at the local blockage and heating/denaturation occurs. Also many enzymes denature at their pi which is often at a low ph, and sometimes the ampholytes themselves inhibit some enzymes. Polyampholytes: small multicharged polymers. The gel contains polyampholytes that form ph gradient in the gel under the electric field. Proteins can then be separated based on their pi values. ph gradient: polyampholytes with many pi values differ by 0.01. 46 Isoelectric focusing Experimental results. 47 Two dimensional gel electrophoresis Proteins are separated based not only their sizes but also their pi values. 48 Two dimensional gel electrophoresis Experimental results of two dimensional gel electrophoresis. 49 Summary of the results of protein purification A single band on SDS-PAGE can be accepted as a criterion of pure protein (or polypeptide). 50 Summary of the results of protein purification Calculation of yield and purification of isolated proteins. 51 Determination of Amino acid Composition acid hydrolysis, ion-exchange chromatography or HPLC, ninhydrin or fluorescamine quantitation. 52 Determination of Amino acid Composition

reagents used for identification of various amino acids. 53 Determination of Amino acid Composition N-terminal amino acid determination. 54 Determination of Amino acid Composition Amino acid composition as analyzed by HPLC. 55 Edman Degradation The reaction for Edman degradation. 56 Edman Degradation Many cycles of Edman degradation reveals the peptide sequence. Peptides of 50 amino acid residues can be sequenced easily. The gas-phase sequenator can analyze picomole quantities of peptides or proteins. 57 Edman Degradation PTH-amino acids will be resolved by HPLC. 58 Cleavage of polypeptides Polypeptides are cleaved by enzymatic or chemical methods into small peptides for sequencing. 59 Cleavage of polypeptides Specific cleavage sites by various enzymes or chemicals. 60 Cleavage of polypeptides Reconstruction of polypeptides from cleaved peptides. 61 MALDI-TOF Schematic diagram illustrating the principle of MALDI-TOF. 62 MALDI-TOF MALDI-TOF mass spectrum of insulin and lactoglobulin. 63 MALDI-TOF Data from MALDI-TOF spectroscopy. Protein Purification: SUMMARY Sources: (i) wild type proteins from biological samples; (ii) recombinant proteins Techniques: (i) extraction and fractionation; (ii) crude separation (precipitation, solubilization, centrifugation); (iii) liquid chromatography (gel filtration, ion-exchange, affinity); (iv) electrophoresis (denaturing, native, 2-D, isoelectric focusing); (v) storage (dialysis, lyophilization). 1. Fractionation

Ammonium sulfate precipitation: Differential precipitation of proteins by ammonium sulfate is one of the most commonly used fractionation methods. Based on differential solubility of proteins. Isoelectric precipitation: Based on the low solubility of a protein at its isoelectric point. Solvent precipitation: Ethanol or other organic reagents and polyethylene glycol of different sizes. Heat precipitation: Based on the difference in heat stability of proteins. 2. Chromatography 1) Gel filtration chromatography: All gel filtration media are based on an exclusion limit of these media. Three basic types of gel matrix are commonly used: (i) Cross-linked dextran (e.g. Sephadex), dextrans are cross-linked with epichlorohydrin, the porosity and the fractionation range depend on the degree of cross-linking. (ii) allyl dextrans cross-linked with bisacylamide (e.g. Sephacryl), more rigid, can be used with organic solvents. (iii) various derivatives of agarose (e.g. Sepharose). Size and shape are the determining factors for separation. Other important parameters include ph, temperature, ph, ionic strength, buffer composition, column size, etc. 2) Ion-exchange chromatography: The gel matrix used above can be derivatized with many different functional groups and thus generate desired anion- or cation-exchange properties. The choice is almost limitless. Anion exchangers are positively charged and cation exchangers are negatively charged. The strngth of the exchangers depend on the pka value of the functional groups. 3) Hydrophorbic chromatography: The gel matrix is derivatized with epoxides that contain long alkyl chains (5 to 12 carbons). The proteins are eluted by decreasing-ionic-strength gradient. 4) Affinity chromatography: Immobilize suitable ligand (e.g. substrate, inhibitor, co-factor, antigen, antibody, carbohydrate, oligonucleotide, hormone, receptor etc) onto the coupling gel matrix (e.g. CNBr-activated Sepharose 4B, AH-Sepharose 4B and CH-Sepharose 4B, activated thio-sepharose 4B, thiopropyl-sepharose 6B etc). 5) Metal affinity chromatography: The most popular metal affinity chromatography uses Ni- NTA resins for isolating histidine tagged recombinant proteins. 3. Gel Electrophoresis 1) Principle of electrophoresis: There are two types of electrophoresis, moving boundary electrophoresis and zone electrophoresis. The electrophoretic mobility of the protein (or any molecules) is determined by its net charge (Ze - ), the electric field (E), and the frictional coefficient (f) of the protein. The frictional coefficient is related to viscosity coefficient and the size of the protein according to Stoke's law. The absolute value of electrophoretic mobility is difficult to measure. Fortunately, you don't have to worry about it in running gel electrophoresis. 2) SDS-PAGE: Acrylamide and bisacrylamide form the gel matrix. Sodium dodecyl sulfate (SDS), a strong detergent, binds to protein at a 0.5 SDS per residue and thus totally eliminate the concern of shape factor and charge factor. The mobility of the protein in SDS-PAGE is determined solely by the molecular sieving effect. The proteins are separated by the size and can be detected by dye staining (e.g. Commassie Blue,

detection limit ~0.1 µg) or silver staining (detection limit ~0.01 µg). SDS-PAGE, a very versatile and useful technique in modern biochemistry and molecular biology, it can be used to determine Mw, subunit composition, purity, peptide map, western blot analysis, autoradiography, and fluorography. 3) Polyacrylamide gel: Native gel electrophoresis can be used to prepare active enzyme, detection of active enzyme (in-gel enzymatic assay, e.g. kinase, acetylase, esterase, dehydrogenase etc), determine the size of multisubunit proteins. PAGE is also used in DNA sequencing and other sequencing related techniques (e.g. DNase footprinting, methylation interference, gel mobility shift, and differential display etc). 4) Agarose gel: The electrophoretic mobility for all DNA molecules on agarose (or polyacrylamide) gel are the same (why?). Agarose gel can detect DNA at about 1 ng range and is essential in modern molecular biology. 5) Isoelectric focusing gel: ph gradient on the gel is formed by the inclusion of ampholine carrier. Extremely useful in determining the isoforms of a protein. 6) Two dimensional gel: The first dimension is ph and the second dimension is Mw, thousands proteins can be resolved on a two-dimesional gel. Primary Structural Analysis: 1. Size determination: (i) traditional methods (hydrodynamic properties such as osmotic pressure, sedimentation, light scattering); (ii) gel filtration; (iii) gel electrophoresis; (iv) GC- Mass 2. Primary structure: (i) end-group analysis; (ii) sequence determination; (iii) cdna Secondary and Tertiary Structural Analysis: 1) Electronic Spectroscopy (UV-VIS, ORD, CD, fluorescence) 2) Magnetic Spectroscopy (nmr, epr) 3) X-ray Diffraction 4) Molecular modelling