Protein Purification. Keeping the Protein Native 10/1/18. Protein Purification and Characterization. Protein Purification Source Protein Diversity

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1 Protein Purification Protein Purification Source Protein Diversity Why study Proteins? Identify a source of the target protein. How much protein is required for the study? Is it naturally abundant or is its source readily available? Protein abundance across organisms: paxdb.org Protein Purification and Characterization Why Study proteins? IMPORTANT FACTORS IN PROTEIN PURIFICATION Starting materials tissues, cells or clones expressed in E. Coli or animal cells Decisions quantity of protein, protein modification availability of samples, is it cloned yet, expense Keeping the Protein Native Stabilization of protein is key proteins are not meant to be purified, so you need to keep them alive and happy (active / native) ph both activity and structure are ph dependent Temperature most stabile at low temperature reduces energy in the system for unfolding and reduces the protease kinetics. Few proteins are unstable at low temps ATPase in mitochondria Protease inhibitors several classes of proteins catalyze the hydrolysis of peptide bonds (called proteases). Usually need to add several "suicide" inhibitors and reduce free metals which are used by the proteases 1

2 Keeping the Protein Native Protein purification Accessing the protein Freeze/Thaw Step 1. Solubilize the target. Sonication Homogenize Stabilization of protein is key proteins are not meant to be purified, so you need to keep them alive and happy (active / native) Reducing agents beta mercaptoethanol and dithiolthreitol both act as reducing agents. Prevent the oxidation of amino acids Detergents Membrane bound proteins often need added detergents (soaps) to mimic the ampipathic nature of the membrane you so cruelly ripped it from need to be above the concentration at which micelles are formed the critical micellular concentration (CMC) Isoelectric Point Proteins tend to be less soluble at the isoelectric point Enzymatic Buffer Mild detergent Cell/Tissue Lysis Homogenization breaking the cell apart Mechanical Shearing Warring blender, glass or plastic pestle like homogenizer Freeze Thaw cycles under hypnotic conditions, ice crystals disrupts membranes Enzymatic degradation of the cell membrane mostly for bacterial preparation Sonication high energy sounds to disrupt membrane Detergent disruption of memb Protein purification Recombinant protein Alternative approach: Recombinant protein Recombinant protein is derived from a recombinant DNA sequence. Recombinant DNA is a DNA molecule created from more than one strand of DNA, usually from different organisms. For instance, if the gene for human insulin were inserted into a bacterial piece of DNA called a plasmid (thus making recombinant DNA) and the bacterium began manufacturing insulin from this new plasmid it would be recombinant insulin. Anatomy of a generic plasmid Sequence that initiates Promoter Your Gene Sequence that initiates replication of DNA Origin of replication Antibiotic resistance marker Select host that carries plasmid transcription Plasmid ① Small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. ② In bacteria, a plasmid is a circular piece of double stranded DNA. ③ Provides a mechanism for carrying extragenomic sequences that can be beneficial to the organism. ④ Artificial plasmids have been developed to drive the replication of recombinant DNA sequences within a host. 2

3 Protein purification Recombinant protein Choosing a cloning vector: Anatomy of a generic plasmid Promoter Promoter Your Gene Bacteria: T7, T7lac, Trc Yeast: GAL4, AOX1 Insect: polyhedrin, p10, basic Mammalian: CMV Origin Untagged of Tagged replication Antibiotic resistance Affinity: 6XHis, FLAG, Myc, Strep marker Fusion protein GlutathioneSTransferase, Maltose binding protein Protein purification Recombinant protein 3 Phase strategy: Capture Isolate, concentrate & stabilize the target protein Intermediate purification Remove most of the bulk impurities Polishing Achieve high purity Polishing Purity Achieve final high level purity Intermediate purification Remove bulk impurities Capture Preparation, extraction, clarification Isolate, concentrate and stabilize Step Protein purification Recombinant protein Expression systems bacterial yeast insect human Ammonium sulfate precipitation salting out proteins At high concentrations of this strong salt, water is highly ordered High concentration of strong chaotropic salts strips water away from protein Lower availability of solvent (water) This favors protein interactions rather than protein solvent interactions causes aggregation of proteins (they become insoluble) Each protein has a different solubility so this is a method to isolate groups of protein Precipitation is reversible and usually non damaging to structure of the enzyme Ammonium Sulfate is most commonly used. Urea is also used but is usually is harder for the protein to refold. 3

4 Affinity chromatography Column Chromatography Separation based the interactions between a mobile phase and the chromatographic media (stationary phase) Used to separate any of the big four biomolecules 1) Chromatography is a set of laboratory techniques used for separation of mixtures. 2) The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. 3) The various constituents of the mixture travel at different speeds, causing them to separate. 4) The separation is based on differential partitioning between the mobile and stationary phases. purification based on a natural interactions for a protein and a substrate or chemical group (ligand) only proteins which recognize the molecule on the stationary phase will bind Elute by competition with the bound ligand generally a good method but it doesn t always work Some non specific interactions can occur Spacer arm may be needed to make the compound available to the protein Examples of ligand protein affinity matrix Protein purification Affinity Chromatography Affinity chromatography capitalizes on a protein s ability to interact with a specific molecule: 1) ligand 2) antibodybased Origin 3) engineered of replication HisHisHisHisHisHis Promoter Your Gene ATP. Glutathione, nickel small molecules attached to a ligand Fusion proteins can take advantage of affinity by acting as a tag: Antibiotic resistance marker glutathione S transferase (GST) binds to glutathione histidine6 binds to a nickel column Power of biochemistry and molecular biology an example of affinity chromatography ligand NAD+ Ras small protein involved in several cancers Low concentration in cells, so it difficult to purify and study create a fusion protein 1/2 Ras 1/2 Glutathione Stransferase (GST) and produce large amounts of it. lead to discovery of additional proteins involved in Ras regulation resin AMP sepharose 4

5 Ion Exchange Chromatography Protein mixture in buffer above target protein s pi and low salt concentration Flow of solution Resin with anion surface Cation Exchange Binding of proteins with an overall positive charge to anion resin Separation of proteins with an overall positive charge NaCl Ion exchange chromatography Separation of proteins based on net charge of protein exchange of ions for proteins Anion Exchanger weak exchanger diethylaminoethyl (DEAE) strong exchanger quatenaryaminoethyl (QAE) This type of resin is positively charged The resin binds negative proteins Proteins are eluted by NaCl or altering ph how does this work? Cation exchanger weak exchanger carboxymethy (CM) weak exchanger sulfipropyl (SP) protein eluted by the same means as Anion Exchange Size Exclusion Chromatography Size exclusion chromatography (SEC), aka gel filtration, separates a mixture of proteins based upon their size. Not exactly Separates based upon the frictional coefficient, f, that is expressed as the Stokes radius: radius of a smooth sphere that would have the actual f of the protein Same MW, but very different radii. Exemplifies limitation of this technique for determine MW. 5

6 Size exclusion (SEC) or gel filtration chromatography Media (solid phase) is a defined pore sizes in polymer beads, large molecules go around small molecules go through and around the beads Smaller sized proteins are retained and come out last Range of types of beads and chemistry resin can be made of agarose, acrylamide or other polymers Also used to determine the molecular weight of a protein use protein standards with known molecular weights, prepare a standard curve of these known proteins and compare the elution volumes of the knowns to the unknowns Hydrophobic Interaction Chromatography NaCl Kyte J, Doolittle RF. J Mol Biol Hydrophobic character of amino acids Kyte & Doolittle Residue type Hydrophobicity Ile 4.5 Val 4.2 Leu 3.8 Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly 0.4 Thr 0.7 Ser 0.8 Trp 0.9 Tyr 1.3 Pro 1.6 His 3.2 Glu 3.5 Gln 3.5 Asp 3.5 Asn 3.5 Lys 3.9 Arg 4.5 Example Sephacryl S200 has a fractionation range of 5 kda to 250 kda What is the exclusion limit? Would this be appropriate for a set of proteins with molecular weights of 8 kda, 15 kda, 200 kda and 500 kda? What about 15, 250, 310, 405 kda if you wanted the 15 kda protein? What about if you wanted to purify the 310 kda protein? Protein Characterization Electrophoresis The transport of particles by an electrical field through a solid media a good method for determining the purity of a protein and analyze a mixture of proteins Separation of charged compounds based on an applied electrical field, net charge and frictional coefficient (mass and shape of molecule) Similar to DNA gels proteins and very small DNA (oligonucleotides) use acrylamide 6

7 Denatured Electrophoresis SDS PAGE Separation of proteins based on size not charge Denature by chemical and thermal means Heat defeats tertiary and quaternary features partially denatures protein Reducing agent ßmercaptoethanol or dithiothreitol Disulfide bonds Detergent sodium dodecyl sulfate (SDS) boil to unravel the protein and solvate protein with ampipathic SDS each SDS has 2 negative charges SDSProtein bound is proportional to the MW Each protein molecule will be sufficiently negative Therefore each protein will be very negativity charge regardless of the amino acid composition, The size of protein influences the migration separation is based on size only not charge. Native Gel Electrophoresis Separations based on native size and charge Two proteins of a similar size but different charge will migrate differently Protein interactions can influence the migration of protein Isoelectric Focusing Electrophoresis Separation of proteins based on isoelectric point Native or denatured electrophoresis in a ph gradient of polyampholytes ph gradient is formed when electrical field is applie Proteins will migrate, depending on net charge, until there is no longer a charge on the protein. How does this happen? 2DElectrophoresis 2Delectrophoresis allows separation of proteins by both size and isoelectric point. Each spot represents a different protein. The horizontal represents the isoelectric focusing direction, while the veritcal represents the SDS PAGE direction. 2 Dimensional Electrophoresis Combination of native or denatured PAGE and IEF Run in two directions 1 PAGE to separate by size 2 IEF to separate by charge alone Good to separate very crude mixtures or determine the difference between two proteins that are the same size but with a different pi 7

8 Immuno Analysis Immunoglobins 5 major classes main antibody in sera is IgG antigen foreign substance that triggers antibody formation epitope section of antigen that antibody recognizes Antibodies consist of heavy and light chains Fab region highly variable recognize target (antigen) FC heavy chain interacts with other proteins polyclonal vs. monoclonal antibodies Antibodies polyclonal from sera of an animal several epitopes to the same antigen some may cross react with other proteins in a nonspecific manner produce lots of antibodies al long as the animal lives and you continue to boost monoclonal derived from single cell hybrid of mouse spleen and a immortal cell line (lymphocyte and myeloma) inject mice then can grow cell in a dish antibodies purified from cell culture media single epitope, very specific unlimited production of antibodies Antibodies in specific analysis ELISA (Enzyme Linked ImmunoAssay) most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) 4 Secondary Antibody Conjugated to an enzyme 3 Primary Antibody Recognizes Antigen Plastic Dish 5 Enzyme reacts with substrate producing colored product 2 Unreacted binding sites are Covered with a nonreactive protein 1 Protein of interest is Bound to plastic Western blot good for mixtures of proteins, identifying size and characteristics transfer proteins form SDS PAGE to paper for antibody analysis. Primary antibody recognizes protein antigen a secondary antibody recognizes the Fc region and is conjugated to a second molecule to act as a signal 8