Protein Extraction, Purification, and Quantification PDF

ra 7e če urt Protein extraction, purification, Ku ec and quantification or - L 15 C6215 ‐ Lecture 7 Igor Kučera 62 Ig C  Protein content in a cell E. coli ra...

ra 7e če urt Protein extraction, purification, Ku ec and quantification or - L 15 C6215 ‐ Lecture 7 Igor Kučera 62 Ig C  Protein content in a cell E. coli ra 7 cell density d = 1.1 g/cm3 water content w = 0.7 (70%) e protein fraction of the dry mass p = 0.55 (55%) če ur  protein concentration cm = d × (1 – w) × p = 1.1 × (1 – 0.7) × 0.55 = 0.19 g/cm3 t Ku ec average length of a protein = 300 aa average molar mass of aa = 110 g/mol or - L  molar mass of a protein M = 300 × 110 = 33 000 g/mol  protein concentration c = (0.19 g/cm3 ) × (1000 cm3/dm3) / (33 000 g/mol) = 15 = 5.76 × 10‐3 mol/dm3 62 cell volume = 1 µm3 = 10‐15 dm3 Ig  protein molecules per cell = (6 × 1023/mol ) × (5.76 × 10‐3 mol/dm3 ) × (10‐15 dm3 )= 3.5 × 106 C Compare H. sapiens HeLa 2 000 µm3 => total number is of the order of 109 Milo, Bioessays 2013, 35,1050  How many different proteins are made in a cell? ra 7 E. coli genome contains 4 240 protein e coding genes če ur Soufi et al., Front. Microbiol. 2015, 6, 103 t Ku ec 2 303 proteins identified 1 587 proteins absolutely quantified or - L Protein copy number estimates span across less than six orders of magnitude from approximately 1–300 000 protein 15 copies per cell. 62 Human genome – about 20 000 protein coding genes. Protein products for about 18 000 of Ig these genes have been detected in at least one human tissue, about 10 000 of these C proteins are present in all cells. Each gene can produce multiple forms of a protein, and these in turn can undergo several post‐translational modifications. Wilhelm et al., Nature 2014, 509, 582; Salzberg, BMC Biology 2018, 16, 94  Subcellular locations of proteins ra 7e če urt Escherichia coli Ku ec or - L 15 62 Ig C http://www.stepdb.eu/info.php https://www.rostlab.org/services/locDB/statistics.php C 62 15 Ig or - L Ku ec t Why purify proteins? če ur ra 7e  Molecular interactions and variables that affect them ra 7 Important with regard to protein structure and stability; they also take place between an individual protein and other proteins, DNA, or materials used in protein purification. e če ur Water t Ku ec molecules form a ice‐like cage structure or - L around the hydrophobe. 15 62 ΔS > 0 Ig C z1 × z2 W (kJ/mol) = 138.94 × εr × r (in nm) z1, z2 charge numbers, εr relative permittivity (dielectric constant): H2O εr = 80, hexane εr = 2  Properties of proteins that enable purification ra 7 Size Shape Proteins can vary in size from tens Protein shapes range from approximately e to several tens of hundreds aa. spherical (globular) to quite asymmetric. če ur Most proteins have Mr in the range 10 000 – 150 000. t => Fractionation on the basis of Ku ec effective (Stokes´) radius. or - L 15 Mr = 5 818 Mr = 29 891 Mr = 68 563 Mr = 103 895 Han et al., PLoS Comput. Biol. 2019, 15, e1006969 62 The shape of a protein influences its Ig effective radius. C Two protein of the same Mr: Mr = 148 970 Mr = 541 468 sediments slower => appears smaller less enters the pores => appears larger  Commonly used gel filtration media ra 7e če urt Ku ec or - L 15 62 Ig C D‐galactose 3,6‐anhydro‐ L‐galactose https://www.ucl.ac.uk/~ucbcdab/enzpur/gelexcl.htm Charge The net charge of a protein is determined by the sum of the positively and negatively charged amino acid residues. If a protein has a net positive (negative) charge at pH 7, ra 7 it is termed basic (acidic). The charge of an ionizable group is a function of pH. e če urt Ku ec or - L 15 => Fractionation on anion and cation pI by electrophoretic light scattering exchangers (anexes and catexes). (When the charged residues are not evenly 62 distributed on the surface of the protein, binding to both types of ion exchangers is Ig possible.) C Isoelectric point pI corresponds to the pH in solution at which the net surface charge, and thus the electrophoretic mobility, of https://www.entegris.com/content/dam/product‐ assets/nicompnanodlszlssystems/appnote‐isoelectric‐ a protein equals zero. point‐iep‐determination‐10528.pdf  Different cation and anion exchangers with their ionizable groups and features ra 7e če urt Ku ec or - L 15 62 Ig C Bhatt, Enzymology and Enzyme Technology 2011  Preparative isoelectric focusing (IEF) When a protein is electrophoresed through an established pH gradient, it will migrate until it reaches the pH where the net charge on the protein is zero; at that point it will stop ra 7 migrating and is said to be focused at its isoelectric point or pI. e če urt Ku ec or - L Ampholytes which are small, charged buffer molecules are used to establish the pH gradients increasing in pH from anode to cathode. When voltage is applied to a system of 15 ampholytes and proteins, all the components migrate to their respective pIs. Ampholytes rapidly establish the pH gradient and maintain it for long periods allowing the slower moving proteins to focus. 62 Rotofor system. The separation column is divided into Ig compartments by by means of polyester screens that offer C resistance to fluid convection, but do not hinder the flow of current or the transport of proteins. Gravitationally induced convection is inhibited by rotating about the horizontal axis. Rotofor® System Instruction Manual (BioRad)  Chromatofocusing ra 7e če urt Ku ec or - L 15 62 Ig C http://macromol.sbcs.qmul.ac.uk/oldsite/expertise/CF3.jpg Frey et al., Encyclopedia of Life Sciences 2001 Hydrophobicity Most hydrophobic amino acid residues are buried on the inside of a protein, but some are found on the surface (hydrophobic patches). ra 7 Hydrophobins – amphiphilic fungal proteins e če urt Ku ec or - L Lienemann et al., Appl. Environ. Microbiol. 2013, 79, 5533 15  Fractionation on hydrophobic column materials 62 Ig C Density The density of most proteins is between 1.3 and 1.4 g/cm3. However, some proteins substantially differ, e.g. phosvitin, a phosphoprotein from the egg yolk (density = 1.8 g/cm3) and β‐lipoprotein (density = 1.03 g/cm3). Solubility Proteins vary dramatically in their solubility from being essentially insoluble (300 mg/ml). Key factors affecting the solubility of a protein include pH, ra 7 ionic strength, the nature of the ions, temperature, and the polarity of the solvent. Most proteins show minimum solubility at their isoelectric point where there is less charge e repulsion. Kosmotropic ions če ur like sulfate bind water more tightly t than water binds Ku ec itself so that less water becomes or - L available to hydrate the protein surface. Cohn equation: 15 log S = log S0 – Ks I Ks constant I ionic strength 62 Ig C Solubility S (mg/ml) of RNase Sa as a Ammonium sulfate solubility curve for a function of pH. hypothetical protein (100% saturation = 4.1 M) Shaw et al., Protein Sci. 2001, 10, 1206 Burgess, Meth. Enzymol. 2009, 463, 331 Addition of miscible solvents such as ethanol or acetone causes proteins to precipitate. This is due to decrease of the relative permitivity (and thus the polarity) and dehydration of protein surface. With smaller hydratation layer, the proteins can aggregate by attractive ra 7 electrostatic and dipol forces. An empirical correlation between the the solubility and relative permitivity is log S = log S0 – (Ks/εr2). e če ur The size of protein molecule is an important factor for precipitation; the larger the t molecule, the lower the percentage of organic Ku ec solvent required to precipitate it. Kumar et al., in: Isolation and purification of or - L proteins (Hatti‐Kaul, Matthiasson Eds.), Taylor & Francis, 2005. Thermal stability 15 Proteins differ in their thermal stability and ability to renature after thermal denaturation. In general, smaller, 62 highly charged proteins are stable to Ig higher temperatures than large, more C hydrophobic proteins. Thermal denaturation can be used to precipitate the unwanted proteins while the desired protein remains Muallen & Karlish, FEBS Lett. 1979, 107, 209 unaffected.  Ligand binding Many proteins tightly and specifically bind substrates, effector molecules, cofactors, or nucleic acid sequences. ra 7e če urt Ku ec or - L Azoreductase AzrC in complex with Cibacron Blue, a Lac repressor complexed to DNA Thrombin aptamer* bound to 15 biomimetic dye Lewis, C.R. Biologies 2005, 328, exosite 1 (magenta) of thrombin (PDB: 3W78) 521 Ruigrok, Int. J. Mol. Sci. 2012, 62 13, 10537 Ig => Fractionation on a support to which the appropriate ligand has been immobilized. C *Aptamers (from the Latin aptus – fit, and Greek meros – part) are oligonucleotide (ssDNA or RNA) or peptide molecules that specifically bind to a predefined target. Usually they are created by selecting them from a large random sequence pool. Reviewed in Mascini, Angew. Chem. Int. Ed. 2012, 51, 1316  An example of preparation of an affinity matrix Lactoperoxidase is an oxidoreductase secreted into milk. It catalyses the oxidation of halides ra 7 and pseudohalides, such as thiocyanate, by H2O2 to form potent oxidant and bactericidal agents. Sulphanilamide was found to be an inhibitor of lactoperoxidase, which made it a e suitable ligand for constructing a Sepharose 4B‐L‐tyrosine affinity matrix for LPO purification. če urt Ku ec or - L 15 62 Ig C Atasever et al., Food Chemistry 2013, 136, 864  Aptamer‐facilitated protein purification ra 7e če urt Ku ec or - L Streptavidin is a homotetrameric protein from the bacterium Streptomyces avidinii. It has an extraordinarily high affinity for biotin (Kd on the order 10−14 mol/L). Streptavidin‐coupled magnetic beads are supplied, e.g., by Thermo Scientific (MagnaBind 15 Streptavidin Beads). 62 Ig C 5′‐/5BioTEG/CTC CTC TGA CTG TAA CCA CGT Beloborodov et al., J. Chromatogr. B GCC TAG CGT TTC ATT GTC CCT TCT TAT TAG 2018, 1073, 201 GTG ATA ATA GCA TAG GTA GTC CAG AAG CC‐3′ Specific sequence or structure ra 7 The precise geometric presentation of amino acid residues on the surface of a protein can be used as the basis of a separation procedure. It can serve as an epitope (antigenic e determinant), which is recognized by monospecific antibodies. če ur  Selective separation on a resin with attached monospecific antibody (immunoaffinity t chromatography) Ku ec A protein can also be immobilized and used to specifically bind another protein out of a or - L complex protein extract ‐ a subunit of an oligomeric protein (subunit exchange chromatography) 15 ‐ a protein interacting with another protein (substrate‐channeling enzymes, structural proteins …) ‐ denatured protein (isolation of chaperons) 62 Ig C Moser, Bioanalysis 2010, 2, 769 Muronetz, J. Biochem. Biophys. Methods 2001, 49, 29  Covalent immobilization of antibodies and other proteins through their amino groups ra 7e če urt Ku ec or - L 15 NHS = N‐hydroxysuccinimide 62 Ig C https://www.thermofisher.com/cz/en/home/life‐science/protein‐biology/protein‐biology‐ learning‐center/protein‐biology‐resource‐library/pierce‐protein‐methods/covalent‐ immobilization‐affinity‐ligands.html Posttranslational modifications After protein synthesis, many proteins are modified by the addition of oligosaccharides, ra 7 acyl groups, phosphate groups, or a variety of other moieties. In many cases, these modifications provide handles that can be used in fractionation. e če urt Ku ec or - L 15 Boronic acid ionisation and interaction with cis‐1,2‐diols 62 Wu et al., Chem. Soc. Rev. 2013, 42, 8032 The trimannoside binding site of Ig Concanavalin A, a lectin from jackbean Phosphoproteins Machida et al., FEBS J. C (Canavalia ensiformis) 2007, 274, 1576 Naismith and Field, J. Biol. Chem. https://www.biovision. 1996, 271, 972 com/phosphoseektm‐ phosphoprotein‐ enrichment‐kit.html  Separation of glycated and non‐glycated hemoglobins When a solution of proteins (hemolysate of red blood cells) is passed through the column, ra 7 the glycated component is retained by the complexing of its diol groups with the bound phenylboronic acid. e če urt Ku ec or - L 15 62 Ig C https://www.trinitybiotech.com/haemoglobins/boronate‐affinity‐chromatography/  Preparation of Ti‐IMAC (Immobilized Metal Affinity Chromatography) magnetic nanoparticles for phosphopeptide enrichment ra 7e če urt Ku ec or - L 15 62 Ig C GMA = glycidyl methacrylate Capriotti et al., IDA = iminodiacetic acid Talanta 2018, 178, 274 (binding site for Ti4+) Genetically engineered peptide/protein tags DNA encoding a given protein is altered to add extra amino acids on the N‐terminus or the C‐terminus of the protein being expressed. This added ‘‘tag’’ can be used as an effective ra 7 purification handle. Tags also can increase protein stability and solubility. e če urt Ku ec or - L 15 62 Ig C Terpe, Appl. Microbiol. Biotechnol. 2003, 60, 523  ra 7e če urt Ku ec or - L 15 62 Ig C https://www.labome.com/method/Protein‐Peptide‐Tags.html  Enzymatic reagents for the removal of affinity tags ra 7e če urt Ku ec or - L 15 62 Ig C Waugh, Protein Express. Purif. 2011, 80, 283  The use of an affinity tagged endoprotease The use of a self‐cleaving tag (POI = protein of interest) (CPD = Cysteine Protein Domain of Vibrio ra 7 cholerae, InsP6 inositol hexakisphosphate) e če urt Ku ec or - L 15 Waugh, Protein Express. Purif. 2011, 80, 283 Biancucci et al., BMC Biotechnology 2017, 17,1 The use of a self‐cleaving aggregation tag 62 Ig C Xing et al., Microbial Cell Factories 2011, 10, 42  Separation Process Basis of Separation Precipitation Ammonium sulfate Solubility Acetone Solubility ra 7 Polyethyleneimine Charge, size e Isoelectric Solubility, pI če ur Phase partitioning (e.g., with polyethylene glycol) Solubility Chromatography Gel filtration/size exclusion (SEC) Size, shape t Ion exchange (IEX) Charge, charge distribution Ku ec Hydrophobic interaction (HIC) Hydrophobicity Affinity Ligand‐binding site or - L DNA affinity DNA binding site Lectin affinity Carbohydrate content and type 15 Immobilized metal affinity (IMAC) Metal binding Immunoaffinity (IAC) Specific antigenic site Chromatofocusing pI 62 Electrophoresis Gel electrophoresis (PAGE) Charge, size, shape Ig Isoelectric focusing (IEF) pI C Centrifugation Size, shape, density Ultrafiltration Size, shape Burgess, in: Proteomics of the Nervous System (Nothwang and Pfeiffer Eds.) WILEY‐VCH Verlag GmbH & Co., 2008  ra 7e če urt Ku ec or - L 15 62 Ig C Protein Purification Handbook, Amersham Biosciences  Suitability of chromatographic techniques ra 7e če urt Ku ec or - L 15 62 Ig C Protein Purification Handbook, Amersham Biosciences  Soluble proteins present in Membrane‐associated and poorly soluble their natural host cells proteins (nonrecombinant) ra 7e če urt Ku ec or - L 15 62 Ig C Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons 2003  Soluble recombinant proteins Insoluble recombinant proteins (inclusion bodies) ra 7e če urt Ku ec or - L 15 62 Ig C Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons 2003  ra 7 REFOLD database, developed by S. Bottomley and his e colleagues at Monash University če ur (Australia), contains proteins that have been successfully t refolded and presents protocols Ku ec and statistics on the frequency of use of various refolding or - L techniques, disruption methods, fusion proteins, and preparation prior to refolding. 15 62 Chow et al., Nucleic Acids Res. Ig C 2006, 34, D207 http://pford.info/refolddb/  A hypothetical purification scheme ra 7 for an enzyme e purity factor = 75/15 = 5 če ur 5‐fold purification 75% recovery t Ku ec or - L 15 62 Ig Burgess, in: Proteomics of C the Nervous System (Nothwang and Pfeiffer Eds.) WILEY‐VCH Verlag GmbH & Co., 2008  Total protein assays ra 7e če urt Ku ec or - L 15 https://www.labome.com/method/Protein‐Quantitation.html 62 Ig C NanoOrange® reagent, a merocyanine dye, produces a large increase in fluorescence quantum yield upon interaction with detergent‐coated proteins. The NanoOrange assay allows for the detection of 0.01 to 10 µg/mL protein with a standard fluorometer. Jones et al., BioTechniques 2003, 34, 850  Protein inactivation and ways of preventing it ra 7e če urt Ku ec or - L 15 62 Ig C Burgess, in: Proteomics of the Nervous System (Nothwang and Pfeiffer Eds.) WILEY‐VCH Verlag GmbH & Co., 2008