Principles of Protein Isolation and Purification PDF

Protein Isolation Techniques  Protein Purification Protein purification: is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms a series of processes to Whole tissue remove other...

Protein Isolation Techniques  Protein Purification Protein purification: is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms a series of processes to Whole tissue remove other unwanted protein of interest proteins and components (protein cannot be isolated in one step) 2  Protein Purification Purification of protein is an essential first step in understanding their function Proteins must be released from the cell to be purified Based on the basic properties of protein like solubility (salt, pH, temperature), size, charge, and binding properties (ligands). 3  GENERAL STEPS Selection of a protein source Tissue homogenization and solubilization Stabilization of proteins Isolation techniques utilize different properties of protein 4  HOMOGENIZATION AND SOLUBILIZATION Homogenization - the process requires the disruption of the outer cell membrane, intracellular membranes and the surrounding extracellular structures. For proteins from muscle that would mean grinding it up, for an intercellular protein that would mean breaking the cells open, etc. This is always done in the presence of a buffer and inhibitors. 5  HOMOGENIZATION AND SOLUBILIZATION Use of detergent solution - increase the solubility of proteins (for protein membrane extraction). Help stabilize and solubilize proteins as they are released from the cell. Use of other reagents (chaotropic agents) such as urea and guanidine HCl (they breakdown the structure of the protein and dissolve well in water) – HELP BREAK APART THE LIPID CELL MEMBRANE. 6  SALTING IN AND SALTING OUT SALTING IN refers to the increase of proteins solubility in a solution with low salts concentration. Low salts concentrations --- the solubility of the protein increases. This could be explained by the following: Salt molecules stabilize protein molecules by: Decreasing the electrostatic energy between the protein molecules which increase the solubility of proteins 7  SALTING IN AND SALTING OUT SALTING OUT refers to the precipitation of the proteins at high salts concentration. It is a purification method that relies on the basis of protein solubility (reducing the solubility) High salts concentrations ------ increase the ionic strength of a protein solution ----- decreases the protein solubility thus precipitation 8  Proteins have characteristic salting out points, and these are used in protein separations in crude extracts. Salting out, is a purification method at initial molecule purification its lacks the ability for precise isolation of a specific protein. Powerful tool to separate classes of proteins that vary in size, charge, and surface area among other characteristics. 9  AMMONIUM SULFATE The salt commonly used is ammonium sulfate because: 1. Its large solubility in water. 2. Its relative freedom from temperature effects. 3. It has no harmful effects on most of the proteins. The amount of salt needed to isolate a specific protein is determined from the salt's fractionation table 10 Beyond the Hofmeister Series: Ion-Specific Effects on Proteins and Their Biological Functions | The Journal of Physical Chemistry B (acs.org) 11  CENTRIFUGATION The proteins are separated after salt addition by centrifugation Centrifuging the liquid containing the cells in a high-density medium may precipitate the desired cells depending on the density of each constituent cell. Density-gradient ultracentrifugation is additionally applicable for eliminating undesired cellular impurities or obtaining certain cell organelles. 12  DIALYSIS Removal of salt molecules from the isolated protein solution through a semi- permeable dialysis bag is called dialysis. The salt molecules move from the more concentrated solution (from inside the dialysis Buffers & salts exchange until an equilibrium is bag) to the less established between the inside & outside of concentrated solution. the membrane. However, this process could not distinguish between proteins effectively. 13 Amino Acid Separation by Chromatography Chromatography is a separation technique used to separate complex mixtures of amino acids. Notice in Figure 3 how a TLC (thin-layer chromatography) plate is used in separating a mixture containing four amino acids Proteins are separated using column chromatography (..in particular high-performance liquid chromatography (HPLC)) because proteins tend to denature when separated using TLC. An amino acid mixture is separated by spotting the mixture on slide I and then using a solvent mixture and ninhydrin to reveal each amino acid (slide IA). HPLC Advantage over TLC 1. Resolution and Sensitivity 2. Quantitative Analysis 3. Selectivity and Versatility 4. Automation and Reproducibility 5. Speed and Throughput  CHROMATOGRAPHY Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. Column chromatography is one of the most powerful fractionation methods. It can separate components of mixtures based upon: adsorption (liquid-solid) partition (liquid-solid) affinity differences among their molecular weights 17  GEL FILTRATION GFC is a form of partition chromatography used to separate molecules of different molecular size. 18 19  ION EXCHANGE (BY CHARGE) Proteins may have net positive or negative charge at different pH range. If a protein has a net positive charge, negatively charged molecules bind to positively charged solid supports and positively charged molecules bind to negatively charged supports. 20 21  Affinity Chromatography (by specific binding affinity) Is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Example enzyme/substrate, receptor/ligand, enzyme/inhibitor, antibody/antigen interactions. High selectivity, resolution, and capacity in protein purification schemes can be easily achieved with affinity chromatography. 22  AFFINITY CHROMATOGRAPHY Isolates targeted protein in three steps Attaching the chemical group which could affinity with target protein to the column. Loading the sample into the column and let the target protein affinity with chemical group. Eluting the target protein by high concentration 23 24  HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Is a process of separating components in a liquid mixture. A liquid sample is injected into a stream of solvent (mobile phase) flowing through a column packed with a separation medium (stationary phase). The flow rate of the solvent is set through computer input and controlled by pumps. The peak represent different kinds of compounds and result is high resolution and rapid separation 25 HPLC FLOW DIAGRAM 26 OVERVIEW 27 28  QUANTITY AND QUALITY ANALYSIS MASS SPECTROMETRY Is used to identify unknown compounds via molecular weight determination, to quantify known compounds, and to determine structure and chemical properties of molecules. Edman degradation classic method to sequentially removes one amino acid at a time from the N-terminus of a protein, which is then identified. 29 Gel Electrophoresis Electrophoresis is a separation technique used to separate proteins based on charge and size by applying an electric field Protein electrophoresis is typically carried out in a gel matrix. PAGE – Polyacrylamide Gel Electrophoresis (carried out in a gel which serves as a molecular sieve. Figure 4 Two forms of electrophoresis 32 Two-dimensional electrophoresis of proteins from the E. coli bacterium. Separation by mass is in the y-direction while separation by size is in the x-direction. 34 The first breakthrough in speeding up protein synthesis was the development of a device in 1960 by R. Bruce Merrifield that could automatically synthesize polypeptide chains. Pancreatic ribonuclease (124 amino acid units) and human growth hormone (188 amino acid units) have been prepared with this technique. Biological systems ( i.e. via genetic engineering) are currently being used and developed to speed up the process of protein synthesis.  Resources Used Hein, M. et al (2013) INTRODUCTION TO GENERAL, ORGANIC AND BIOCHEMISTRY, 11th Ed. John Wiley and Sons, Inc., USA. Hill, J.W., McCreary, T.W. and Kolb, D.K. (2012) CHEMISTRY FOR CHANGING TIMES, 13th Ed. Prentice Hall International, Inc. Janson, J.-C. (2011). Protein purification: High-resolution methods and applications (3rd ed.). Wiley-Blackwell. Chemistry LibreTexts. (n.d.). Salting-in and salting-out. Chemistry LibreTexts. 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