Proteins Purification BIOC2069 #1 PDF

Proteins #3 Working With Proteins  Protein Purification Every protein has different properties therefore every protein will require a unique purification protocol to be developed. Before starting to develop a purification protocol some questions need to be asked. The answer...

Proteins #3 Working With Proteins  Protein Purification Every protein has different properties therefore every protein will require a unique purification protocol to be developed. Before starting to develop a purification protocol some questions need to be asked. The answers to these questions will shape the approach to be taken.  A. QUANTITY Dependent upon what the purpose of obtaining the protein is for i.e. qualitative, quantitative or commercial production. Th e answer to this question will determine the source and the approach  QUANTITY Full chemical and physical analysis of the protein will require several hundred mgs of the protein Large quantities requires high capacity and low resolving power early in the purification protocol.  QUANTITY Kinetic analysis of an enzyme requires a few mgs of protein < 100 mg Techniques would include medium to high resolution such as ion-exchange chromatography to affinity chromatography  QUANTITY The generation of polyclonal antibodies requires approximately 5 mgs of antigen Small quantities required – omit bulk methods and start with medium to high resolution Limited N-terminal sequencing data requires mg of protein (IEF and SDS-PAGE) B. Do we need to retain biological activity? If the answer is YES then this limits the techniques we can pursue. Any techniques that result in denaturation through the presence of high levels of detergent cannot be used e..g. SDS-PAGE Techniques that use extreme pH such as immunoaffinity chromatography may disrupt biological activity. Techniques such as reverse phase HPLC use organic solvents result in the loss of activity.  B. Do we need to retain biological activity? Techniques such as ion exchange chromatography and size exclusion are most appropriate as they use conditions under which the majority of proteins retain their activity. The purification will have to be carried out at low temperatures i.e. 4oC. For labile molecules the purification will require few steps and few buffer changes to maintain biological activity.  B. Do we need to retain biological activity? If the answer to the question is NO Then there are no limits to the purification techniques that we can use.  C. Level of Purity Required In the majority of cases a 100% pure preparation is unlikely, but in many cases we need to get as near to this value as possible i.e. crystallization. The level of purity required has to be determined i.e. the level of contamination that will not confuse or invalidate the chemical, physical or kinetic results needs to be determined.  D. Source of Protein In many cases the source of the protein is determined by the nature of the project e.g. working to isolate flavonoid biosynthetic enzymes from anthurium requires anthurium tissue as the starting point. However even amongst such preparations the strain/cultivar may be varied to produce an optimum preparation.  D. Source of Protein However if there is some flexibility in the choice of starting material then a number of issues need to be considered. The chosen tissue should have a high availability/ high content of the protein of interest and preferably a low content of interfering compounds.  D. Source of Protein (Plants) Plants contain a rigid cell wall which requires a more vigorous disruption than mammalian cells. The majority of the plant cell is taken up by the vacuole which results in a greatly reduced level of protein per cell than found in mammalian tissue.  D. Source of Protein (Plants) Plants also contain high levels of compounds such a phenolics which damage proteins during isolation. Large levels of starch are also found in plant cells which can interfere with protein purification. Plants contain high levels of proteases which can lead to the degradation of proteins through the purification scheme.  D. Source of Protein (Plants) All of these problems can be overcome e.g. use of proteases inhibitors, PVP, BSA etc. Large amount of starting material But if the protein of interest is in general occurrence then plant tissue would NOT be the choice for the protein purification  D. Source of Protein (Microbial/ Fungal) One advantage of microbial tissue is that growth conditions can be optimzed for optimal preparation. However in some cases equipment for growth can be specialized as well as apparatus for the disruption of the cells (e.g. French Press)  D. Source of Protein (Animal Tissue) The most convenient tissue for a purification protocol Tissue can be easily obtained from the abattoir or from lab animals such as rodents  D. Source of Protein (Cloning) Proteins of low abundance in other tissues can be over expressed in E. coli And other systems. Other advantages include the ability to introduce a tag into the protein (His, S etc) to allow ease of purification.  D. Source of Protein (Cloning) There are a number of disadvantages to this approach If a multimeric protein is required it is difficult to express a number of genes in the bacteria Assembly of proteins may not occur properly Often bacterial synthesis results in the protein of interest being incorporated into inclusion bodies in a insoluble manner. Strategy – Exploiting Differences (Location, solubility, size, charge, hydrophobicity, special binding or interactions) A. Location Often the first step in a purification process is the isolation of specific tissue and organelles that contain the protein of interest. By employing differential centrifugation cells can be subfractionated into their various compartments.  B. Solubility Proteins vary with regard to the number and types of various amino acids they contain. This gives rise to variations in hydrophobicity, charge and solubility in organic solvents or neutral salts. Different proteins will precipitate from solution at differencing concentrations of organic solvents and neutral salts. Precipitation from solution is often one of the earliest steps in a purification strategy.  C. Size Separation on the basis of size occurs under both denaturing and non-denaturing conditions e.g. Gel filtration Ultrafiltration SDS-PAGE Native PAGE  D. Charge Different proteins contain differing amounts and types of charged amino acid side chains. This results in proteins possessing different net charges at different pHs. A number of purification techniques take advantage of this IEF (isoelectric focusing) Ion exchange chromatography  E. Specific Binding Proteins (especially enzymes) bind specific molecules during their functions and this binding can be exploited for purification Affinity chromatography Immunoaffinity chromatography Immobilized metal ion affinity chromatography Dye ligand affinity chromatography Lectin affinity chromatography  E. Special Properties Different stabilities to temperature and pH can be exploited E.g. enzymes isolated from thermophylic organisms  Documenting the Purification At each stage of the purification process the level of purity/activity needs to be documented An inventory needs to be kept with regards to fractions, total protein content and amount of protein of interest. If a protein is an enzyme then its activity should be monitored If the protein of interest has no enzymatic activity then the purification can be monitored through SDS-PAGE and/or Western analysis  Documenting the Purification Total Protein The quantity of protein present in a fraction is obtained by determining the protein concentration of a part of each fraction and multiplying by the fractions total volume DECREASES THROUGH THE PURIFICATION  Documenting the Purification Total Activity The enzyme activity for the fraction is obtained by measuring the enzyme activity in the volume of the fraction used in the assay and multiplying by the fractions total volume DECREASES THROUGH THE PURIFICATION  Documenting the Purification Specific Activity Obtained by dividing the total activity by total protein INCREASES THROUGH THE PURIFICATION  Documenting the Purification Yield A measure of the activity retained after each purification step as a % of the activity of the crude extract. The amount of activity in the crude extract is taken to be 100% DECREASES THROUGH THE PURIFICATION  Documenting the Purification Purification Level A measure of the increase in purity and is obtained by dividing the specific activity, calculated after each purification step, by the specific activity of the initial extract INCREASES THROUGH THE PURIFICATION Step Total Total activity Specific Yield (%) Purification protein (units) activity, (units level (mg) mg-1 Homogenization 15,000 150,000 10 100 1 Salt fractionation 4,600 138,000 30 92 3 Ion-exchange 1,278 115,500 90 77 9 chromatography Molecular 68.8 75,000 1,100 50 110 exclusion chromatography Affinity 1.75 52,500 30,000 35 3,000 chromatography  An example of a purification scheme Purification of isocitrate dehydrogenase from pig hearts 1. Obtain 20 pig heats from the local abattoir (keep on ice) 2. All subsequent steps carried out at 4oC 3. Cut up hearts into small chunks and place in blender – add suitable buffer. 4. Blend hearts and strain through several layers of muslin to remove non disrupted tissue 4. Differential centrifugation to isolate mitochondria. Break open mitochondria and ultracentrifuge to remove membranes 5. Sulphate cut to 30% - discard pellet, cut to 40% retain pellet 6. Dialysis to remove salt 7. Apply to anion exchange column 8. Apply to sizing column 9. Concentrate in a C10 centricon  At each stage monitor the activity of the protein by examining the production of NADH (abs 340nm) by providing isocitrate and NAD+ Measure the total amount of protein at each stage to generate a specific activity. Visualize the procedure by running SDS- PAGE