Protein Chromatography Lecture 2 - BMS2043 - Feb 2024 PDF

BMS2043- ANALYTICAL AND CLINICAL BIOCHEMISTRY CHROMATOGRAPHY & PROTEIN PURIFICATION DR PENNY LYMPANY [email protected] 28AY04 WHAT ARE WE GOING TO DISCUSS? Principles of chromatography Size-exclusion chromatography Ion-exchange chromatography Affinity chromatography Hydrophobic interaction chro...

BMS2043- ANALYTICAL AND CLINICAL BIOCHEMISTRY CHROMATOGRAPHY & PROTEIN PURIFICATION DR PENNY LYMPANY [email protected] 28AY04 WHAT ARE WE GOING TO DISCUSS? Principles of chromatography Size-exclusion chromatography Ion-exchange chromatography Affinity chromatography Hydrophobic interaction chromatography Chromatofocussing Post-chromatography processing PROTEIN PURIFICATION Characterisation of proteins Industrial applications Clinical applications HOW DOES THIS RELATE TO THE ASSESSMENTS? Practical 1 – Protein chromatography HPLC analysis of drug metabolites Drug metabolism Data analysis & statistics Practical 2 – Clinical biochemistry Creatinine clearance Data analysis & statistics WHAT IS CHROMATOGRAPHY? A technique for analyzing or separating mixtures of gases, liquids or dissolved substances Involves two distinct phases: Stationary phase (matrix) Mobile phase Oxford Dictionary of Science (1999) CHROMATOGRAPHY - HISTORY First records of chromatography date from mid 1800s Chromatography as a term – colour writing – separation of plant pigments – chlorophyll, carotenoids coined in 1900s David Talbot Day reported in 1900 on chromatography of crude petroleum as it moved through clay or limestone Mikhail Tsvet – early 1900s - used liquid adsorption column containing calcium carbonate to separate yellow, orange and green plant pigments CHROMATOGRAPHY – USES Forensics - blood, arson, post mortem Food regulation/testing – horsemeat scandal, nutritional information Athlete testing – including horses! (combined liquid chromatography and MS) Quality control – alcohol, analysis of when a food spoils, water samples, contaminents Pharmaceutical industry – purification of antibodies, creating vaccines, purity of preparations PRINCIPLES OF CHROMATOGRAPHY – MOBILE AND STATIONARY PHASES Mobile phase passed over stationary phase Sample – mobile phase Relative affinity for stationary phase allows for differentiation of sample http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/12_Chromatograp hic_and_Electrophoretic_Methods Two common methods – Column chromatography Planar chromatography PRINCIPLES OF CHROMATOGRAPHY – TYPES OF CHROMATOGRAPHY Gas Chromatography (GC) Liquid Chromatography (LC) High-Performance Liquid Chromatography (HPLC) Thin-Layer Chromatography (TLC) Other chromatography techniques (exchange chromatography, affinity chromatography) SEPARATION PRINCIPLES IN CHROMATOGRAPHIC PURIFICATION Handbooks from GE Healthcare Life Sciences: Size Exclusion Chromatography For more information refer to www.gelifesciences.com/handbooks COLUMN CHROMATOGRAPHY BASICS COLUMN CHROMATOGRAPHY - USES Separation of mixtures Purification Isolation of active components Estimation of drugs in a formulation Isolation of active constituents Separation of diastereomers COLUMN CHROMATOGRAPHY http://chemwiki.ucdavis.edu/Analytical_Chemistry/Analytical_Chemistry_2.0/12_Chromatographic_and_Electrophoretic_Methods CHROMATOGRAPHY - PREPARATION Column packing – wet/dry Principle – Adsorption Mobile phase is liquid Column equilibration Sample: Further processing Sample loading Adsorbants – Si, Al, CaCO3, starch Elution Al – less polar compounds Si – polar functional groups Column: Cleaning HOW TO SUCCEED IN COLUMN CHROMATOGRAPHY Stationary phase Removal of impurities Number of components to be separated Length of column used Affinity differences Quantity of adsorbent used Mobile phase Choice depends on solubility characteristics of mixture Low bp – recovery of eluted material Polarity – petroleum ether, CCL4, ether, acetone, benzene, water – alone or as mixture COLUMN CHROMATOGRAPHY - VARIABLES Dimension of the column: column efficiency can be improved by increasing length/width ratio of the column. Particle size of column packing: think sand vs gravel Activity of the adsorbent Temperature of the column - speed of the elution increases at higher temperatures Packing of the column Quality of solvents - solvents having low viscosities give better results PROTEIN CHROMATOGRAPHY PROTEIN PURIFICATION Purpose: Preparative vs. Analytical Source Preliminary steps Purification strategies Chromatography Protein concentration Yield & analysis PROTEIN EXTRACTION Source of protein – tissue or microbial cells We need to break open the cells – crude extract Extraction of proteins – Homogenization Sonication Freeze-thaw cycles Organic solvents PROTEIN PURIFICATION – PRELIMINARY STEPS Precipitation and differential solubilization: Salting out (Ammonium sulphate) Detergents (Triton X-100, CHAPS) – dissolve cell membrane and keep protein in solution Ultracentrifugation: Sub-cellular organelles PROTEIN PURIFICATION Need a pure preparation of a protein to determine properties or activity PROBLEM - Cells contain many proteins ?centrifugation to isolate organelles or fractions Proteins vary in size, charge, binding properties Genetic engineering – modify proteins but also function…. Source: Lehninger Principles of Biochemistry 7th Edn PROTEIN CHROMATOGRAPHY Crude extract fractionation purification of protein of interest.. Fractionation – pH, solubility, temperature … Salting out – depends on lowering solubility with salts e.g. ammonium sulphate Precipitates – centrifugation Fig. 3.16 Source: Lehninger Principles of Biochemistry 7th Edn Dialysis – remove proteins from small solutes Column chromatography – uses charge, size, binding affinity.. Differences from HPLC/GC Low pressure Low temperature Low flow rate Bigger columns/volumes http://en.wikipedia.org/wiki/Fast_prot PROTEIN PURIFICATION - CHROMATOGRAPHY Size-exclusion chromatography Ion-exchange chromatography Affinity chromatography Hydrophobic interaction chromatography Chromatofocussing http://en.wikipedia.org/wiki/Fast_prot PROTEIN PURIFICATION - CHROMATOGRAPHY ION EXCHANGE CHROMATOGRAPHY Anion-exchange chromatography Cation-exchange chromatography ION EXCHANGE CHROMATOGRAPHY Cation exchange chromatography Ion exchange chromatography ION EXCHANGE CHROMATOGRAPHY – GRADIENT ELUTION Handbooks from GE Healthcare Life Sciences: Strategies for Protein Purification For more information refer to www.gelifesciences.com/handbooks ION EXCHANGE CHROMATOGRAPHY – A QUESTION We have two peptides mixed together in solution and we need to separate them by ion exchange chromatography At the pH of the mobile phase, peptide A has a pI of 5.1 (due to more Glu and Asp residues than Arg, Lys and His) and has a net negative charge at neutral pH. Peptide B has a pI of 7.8 (more positively charged residues at neutral pH) Which would elute first from a cation – exchange resin and an anion-exchange resin and why? SIZE EXCLUSION CHROMATOGRAPHY https://www.creative-biostructure.com/custom-size-exclusion-chromatography-service-259.htm SIZE EXCLUSION CHROMATOGRAPHY Handbooks from GE Healthcare Life Sciences: Size Exclusion Chromatography For more information refer to www.gelifesciences.com/handbooks SIZE EXCLUSION CHROMATOGRAPHY Handbooks from GE Healthcare Life Sciences: Size Exclusion Chromatography For more information refer to www.gelifesciences.com/handbooks SIZE EXCLUSION CHROMATOGRAPHY – CALIBRATION CURVE http://chem.libretexts.org/Textbook_Maps/Analytical_Chemistry_Textbook_Maps/Map%3A_Analytical_Chemistry_2.0_(Harvey)/12_Chromatographic_and_El ectrophoretic_Methods/12.6%3A_Other_Forms_of_Liquid_Chromatography COMMON TERMS IN AFFINITY CHROMATOGRAPHY https://b3p.it.helsinki.fi/download/GE_Protein_Purification_Ha ndbooks/Affinity_Chromatography_Handbook.pdf AFFINITY CHROMATOGRAPHY Based on binding affinity Beads in the column - covalently attached ligand Any protein with affinity for the ligand binds to the beads - migration retarded Proteins that do not bind flow more rapidly through the column Bound proteins eluted by a solution containing either a high concentration of salt or a free ligand Free ligand competes with the ligand attached to the beads, releasing the protein from the matrix; protein product that elutes from the column is often bound to the ligand used to elute it AFFINITY CHROMATOGRAPHY Handbooks from GE Healthcare Life Sciences: Strategies for Protein Purification. For more information refer to www.gelifesciences.com/handbooks AFFINITY CHROMATOGRAPHY Handbooks from GE Healthcare Life Sciences: Strategies for Protein Purification For more information refer to www.gelifesciences.com/handbooks AFFINITY CHROMATOGRAPHY Handbooks from GE Healthcare Life Sciences: Strategies for Protein Purification For more information refer to www.gelifesciences.com/handbooks HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) [Salt] concentration is lowered gradually and sample components elute in order of hydrophobicity Handbooks from GE Healthcare Life Sciences: Strategies for Protein Purification. For more information refer to www.gelifesciences.com/handbooks HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) Binding dependent on the surface hydrophobicity of the protein Binding enhanced by high ionic strength Handbooks from GE Healthcare Life Sciences: Hydrophobic Interaction Chromatography. For more information refer to www.gelifesciences.com/handbooks PROTEIN CHROMATOGRAPHY & GENETIC ENGINEERING Many proteins do not bind a ligand that can be immobilized on a column BUT gene for almost any protein can be altered to express a fusion protein that can be purified by affinity chromatography Gene encoding the target protein is fused to a gene encoding a peptide or protein that binds a simple, stable ligand with high affinity and specificity – the tag. Tag sequences can be at amino or carboxyl terminus Source: Lehninger Principles of Biochemistry 7th Edn PROTEIN CHROMATOGRAPHY & GENETIC ENGINEERING Example - GST tag GST enzyme binds to glutathione Glutathione – immobilised on beads of agarose Retrieve target – wash with high concentration of salt or free glutathione – compete with immobilized ligand for GST binding Possible to remove the tag by protease cleavage Disadvantages? Source: Lehninger Principles of Biochemistry 7th Edn PROTEIN CHARACTERISATION PROTEIN PURIFICATION – CHECKING THE PRODUCT How pure is our protein? What is the isoelectric point (pI) Approx mw How? Electrophoresis – Polyacrylamide gel – a molecular “sieve” – slowing migration of proteins in proportion to charge to mass ratio as below Force moving macromolecule is electric potential E Electrophoretic mobility μ is ratio of velocity V to E Electrophoretic mobility also equal to net charge Z/fractional coefficient f (related to shape) Source: Fig 3.18 Lehninger Principles of Biochemistry 7th Edn SDS – PAGE Common method for estimating purity and mw Uses SDS Binding ratio 1.4x – nearly 1 molecule of SDS to each aa residue Sulfate moiety of bound SDS contributes to net negative charge – intrinsic charge of protein not significant SDS binding partially unfolds proteins – rodlike shapes Separation mainly on basis of mw Visualisation – Coomassie blue binds to proteins Source: Fig 3.19 Lehninger Principles of Biochemistry 7th Edn SDS – PAGE PROTEIN CHARACTERISATION - ISOELECTRIC POINT Source – 2D principles 2nd Edn Amersham ISO-ELECTRIC FOCUSSING To determine pI of protein Source: Fig 3.20 Lehninger Principles of Biochemistry 7th Edn CHROMATOFOCUSSING - OVERVIEW Chromatofocusing medium equilibrated with a start buffer at a pH slightly above the highest pH required. Sample is applied to the chromatographic column with the start buffer Elution buffer is passed through the column and begins to titrate the amines on the medium and the proteins – gradient pH develops Proteins in the sample that are at a pH above their pI are negatively charged and retained near the top of the column The ones having their pH below pI begin to migrate down and bind to that part of the column where the pH is above their pI CHROMATOFOCUSSING Binding dependent on the surface charge of the protein Uses ion exchange resins Uses FPLC Elutes bound species by altering the pH of the buffer Proteins elute in order of their isoelectric points. CHROMATOFOCUSSING Handbooks from GE Healthcare Life Sciences: Ion Exchange Chromatography For more information refer to www.gelifesciences.com/handbooks 2D GEL ELECTROPHORESIS So why not combine SDS page and isoelectric focussing? More sensitive than either alone.. separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different mw Source: Fig 3.21 Lehninger Principles of Biochemistry 7th Edn POST CHROMATOGRAPHY PROCESSING DESALTING Dialysis Desalting CONCENTRATION Lyophilization Ultrafiltration Chromatographic concentration Precipitation YIELD AND ANALYSIS Enzyme assay Protein assay SDS-PAGE Western Blotting Simulation: SDS-PAGE & immunoblotting WESTERN BLOTTING http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/Strategies%20for%20Protei n%20Purification.pdf READING LIST -CHROMATOGRAPHY Lehninger, chapter 3 Analytical Chemistry 2.0, chapter 12 https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Analytical_Chemistr y_2.1_(Harvey) http://www.gelifesciences.com/ This Photo by Unknown Author is licensed under CC BY-NC RECOMBINANT PROTEINS RECOMBINANT PROTEINS Manipulated form of native protein Generated in various ways in order to increase production of proteins, modify gene sequences, and manufacture useful commercial products Coding sequence for the protein of interest is isolated and cloned into an expression plasmid vector. Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture. intron-free version of the gene is often made by converting the mRNA into cDNA. But cDNA lacks regulatory regions so expression vectors provide promoter, ribosome-binding site, and terminator sequences WHAT ARE RECOMBINANT PROTEINS USED FOR Lab techniques – ELISA – matched antibody pairs – standards Western Blot – positive controls Immunohistochemistry Enzyme assays Cellular responses to stress and disease In animal models – can help with identifying therapeutic targets EXAMPLES OF RECOMBINANT PROTEINS First use – insulin (1982) Now – recombinant hormones, interferons, interleukins, growth factors, tumor necrosis factors, blood clotting factors, thrombolytic drugs, treating major diseases Enzymes – animal feed enhancement Lactic acid bacteria – used for fermenting foods, now adapted for use in human/animal digestion and nutrition ADVANTAGES OF RECOMBINANT PROTEINS Ethical considerations Quick Cost Scaling DISADVANTAGES OF RECOMBINANT PROTEINS Contamination, e.g. proteases Inactive protein, e.g. inclusion bodies Small proteins only Lack of post-translational modifications ? ANY QUESTIONS? We made it! This Photo by Unknown Author is licensed under CC BY-SA Please use the discussion board for general questions and queries: https://surreylearn.surrey.ac.uk/d2l/le/252546/discussions/List Protein chromatography - what have we discussed? Source of material Size-exclusion chromatography Ion-exchange chromatography Affinity chromatography Hydrophobic interaction chromatography Post-chromatography processing