Protein Purification Principles and Practice PDF

“Principles of purification of Proteins” Dr. Manal Abouelwafa Badr University in Assiut Biotechnology Department 29 Feb, 2024 [email protected] This course will give an introduction to the field of proteomic and Genomics and the ava...

“Principles of purification of Proteins” Dr. Manal Abouelwafa Badr University in Assiut Biotechnology Department 29 Feb, 2024 [email protected] This course will give an introduction to the field of proteomic and Genomics and the available proteomic technologies and the data mining tools. Lecture 3  QS Q1: What is the difference between Bioinformatics and Computational biology? Q2: what is the difference between Genomics, transcriptomics, proteomics, metabolomics, and metagenomics? Q3: Write on central dogma stages? Q4: What is the reason for protein-protein interaction? Q5: Name some protein-protein interaction techniques? Q6: what is the idea of Y2H? Q7: Van del waals forces is a part from protein-protein interaction mechanism (True or False)? Q8: What is the main idea of Gel filtration chromatography? Q9: Name protein-protein interaction database?  BLOTTING ▪ Blotting provides a means of identifying specific molecules out of a mixture. It employs three main steps. First, the mixture of molecules is separated by gel electrophoresis. ▪ The mixture could be DNA (Southern Blot), RNA (Nothern Blot), or protein (Western Blot) and the gel could be agarose (for DNA/RNA) or polyacrylamide (for protein).  BLOTTING ▪ After the gel run is complete, the proteins or nucleic acids in the gel are transferred out of the gel onto a membrane/paper that physically binds to the molecules. ▪ This “blot", as it is called, has an imprint of the bands of nucleic acid or protein that were in the gel. ▪ The transfer can be accomplished by diffusion or by using an electrical current to move the molecules from the gel onto the membrane.  BLOTTING ▪ The membrane may be treated to covalently link the bands to the surface of the blot. ▪ A visualizing agent specific for the molecule of interest in the mixture is added to the membrane. ▪ For DNA/RNA, that might be a complementary nucleic acid sequence that is labeled in some fashion (radioactivity or dye). ▪ For a protein, it would typically involve an antibody that specifically binds to the protein of interest.  BLOTTING ▪ The bound antibody can then be targeted by another antibody specific for the first antibody. ▪ The secondary antibody is usually linked to an enzyme which, in the presence of the right reagent, catalyzes a reaction that produces a signal (color or light) indicating where the antibody is bound. ▪ If the molecule of interest is in the original mixture, it will “light" up and reveal itself. NORTHERN BLOTTING CONTENTS Protein purification Methods for protein purification Chromatography Types of Chromatography Adsorption Chromatography Thin Layer Chromatography Column Chromatography Partition chromatography Separation principles  PROBLEM Often need one component in a cell in purified form why purified? what does "pure" mean? sometimes, partial enrichment is enough Task is to separate desired component from a complex mixture It's usually important to maintain the activity of the component throughout the process yield is important but so is intactness, activity achieving one is often at the expense of the other Components can be: nucleic acids (DNA, RNA) proteins protein/nucleic acid complexes (snp's, transcription complexes) large cellular complexes (ribosomes, spliceosomes)  PROTEIN PURIFICATION Proteins huge number of proteins (> than the10,000of genes encoded in a genome) most cell types in multicellular organisms express tens of thousands of different proteins relative abundances of various proteins vary widely Goals of protein purification obtain a particular protein free of others and other cell components obtain a good yield (absolute amount and proportion of starting amount) maintain the activity of the protein Problems: denaturation proteolysis in vitro mixing a measurement of how well the process worked some characterization of the purified protein  METHODS FOR PROTEIN PURIFICATION 1. Develop a quantitative assay quantitative measurement of activity quantitative immunoassay gel electrophoresis assay (immunoblot or gel activity assay) generally want to increase Specific Activity at each step Definition of Specific Activity for proteins: units per milligram of total protein "unit" is defined by the researcher, and can be different when described by different people or different venders a unit is a quantitative measure of activity, usually associated with a turnover rate (for enzymes) or amount needed for stoichiometric binding (for receptors, ligands, DNA binding proteins) theoretical maximum for any given protein METHODS FOR PROTEIN PURIFICATION 2. Obtain source of material whole organisms organs or tissues embryos tissue culture cells microorganisms need to understand sample size +'s and -'s of any particular source, e.g.: how hard is it to obtain, grow, handle amount of proteolytic activity may sometimes be better to use a lower producing source that is cleaner is the protein active in a particular source? are inhibitors present in a particular source? some organs and tissues have connective tissues that are hard to remove overproduction in a heterologous system (basis for much of biotechnology industry) Protein purification Low abundance  Analytical purification produces a relatively small amount of a protein for a variety of research or analytical purposes, including identification, structural characterization, and studies of the protein's structure, post-translational modifications, and function. Protein purification High abundance  Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin). Many steps and much quality control is required to remove other host proteins and other biomolecules, which pose a potential threat to the patient's health.  METHODS FOR PROTEIN PURIFICATION 3. Make an extract from source almost always want to keep it very cold (just above freezing) gentle breaking in some cases can obtain big purification in one step by separating cellular compartments (e.g., purify nuclei from cytoplasm before extracting nuclear proteins) 4. Begin separating components remove nucleic acids, polysaccharides, cell membrane debris ammonium sulfate precipitations, other crude fractionations (pH or other salt precipitations, antibody clearing, "autolysis") these crude steps are often needed to avoid ruining or overloading chromatography agents  METHODS FOR PROTEIN PURIFICATION 5. Fractionate by chromatography several steps are almost always needed need to assay for amount and purity at each step need a way to decide when you're finished  What is Chromatography? Principles of Chromatography Chromatography is a separation method where the analyte is combined within a liquid or gaseous mobile phase., which is pumped through a stationary phase. Usually one phase is hydrophilic and the other is lipophilic. The components of the analyte interact differently with these two phases. Depending on their polarity they spend more or less time interacting with the stationary phase and are thus retarded to a greater or lesser extent. This leads to the separation of the different components present in the sample. Each sample component elutes from the stationary phase at a specific time called as retention time. As the components pass through the detector their signal is recorded and plotted in the form of a chromatogram.  Types of Chromatography The four main types of chromatography are: 1. Adsorption Chromatography Different compounds are adsorbed on the adsorbent to different degrees based on the absorptivity of the component. Mobile phase is made to move over a stationary phase, thus carrying the components with higher absorptivity to a lower distance than that with lower absorptivity. 2. Thin Layer Chromatography (TLC), the mixture of substances is separated into its components with the help of a glass plate coated with a very thin layer of adsorbent, such as silica gel and alumina, The plate used for this process is known as chrome plate. The solution of the mixture to be separated is applied as a small spot at a distance of 2 cm above one end of the plate. The plate is then placed in a closed jar containing a fluid termed as an eluant, which then rises up the plate carrying different components of the mixture to different heights. 3. Column Chromatography Using a column of suitable adsorbent packed in a glass tube, The mixture is placed on the top of the column, and an appropriate eluant is made to flow down the column slowly. Depending upon the degree of adsorption of the components on the wall adsorbent column, the separation of the components takes place. The component with the highest absorptivity is retained at the top, while the other flow down to different heights accordingly. 4. Partition chromatography A continuous differential partitioning of components of a mixture into a stationary phase and mobile phase takes place. The example of partition chromatography can be seen in paper chromatography. paper is used as a stationary phase which is suspended in a mixture of solvents that act as a mobile phase. spots at the base of the chromatographic paper with the mixture to be separated and as the solvent rises up this paper, the components are carried to different degrees depending upon their retention on the paper. The components are thus separated at different heights.  Separation principles Sizing principle is based on exclusion of larger molecules from pores in resin; smaller molecules require longer times of transit sizing resins = "gel filtration" different matrices have different size ranges examples: Sephadex, Sephacryl, Ultrogel, some HPLC resins Ion exchange separate on the basis of net charge wash and elute with higher salt concentrations determine highest [salt] that your protein binds to the resin, and use this to load or to pre-wash before eluting your protein examples: DEAE cellulose, phosphocellulose, some HPLC resins Affinity chromatography separate based on binding to residues specific or semi-specific to the protein you are purifying easy to overload with non-specific proteins examples: sequence-specific DNA sites, antibody resin 1. Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. 2. Ion exchange chromatography The molecules separated on the basis of their charge are eluted using a solution of varying ionic strength. By passing such a solution through the column, highly selective separation of molecules according to their different charges takes place. Cation Exchange chro. Anion Exchange chro. 3. Affinity chromatography is a method for selective purification of a molecule or group of molecules from complex mixtures based on highly specific biological interaction between the two molecules.  Fast Protein Liquid Chromatography FPLC chromatogram  Qs Q1 What is the basic principle of chromatography? Chromatography is based on the concept of separating molecules in a mixture added to the ground or solid and liquid stationary state (stable phase) when travelling with the aid of a mobile phase. Q2 What is the Rf value in chromatography? RF stands for retention factor in paper chromatography, or the distance a fluid compound moves up a plate of chromatography. For each particular solvent, all compounds have a common RF value, and RF values are used to equate unknown samples with known compounds. Q3 How is RF value useful? The distance travelled by sample divided by the distance travelled by the solvent. It is a component characteristic and can be used to classify components for a given system at a known temperature. Q4 Where is chromatography used? Chromatography is used in industrial processes to purify materials, test trace amounts of contaminants, isolate chiral compounds and quality control test products. Chromatography is the physical process of separating or analyzing complex mixtures.  Reference Protein Purification: Principles and Practice Robert K. Scopes Third Edition Springer-Verlag 1994