Protein Purification - Lecture notes 1 PDF

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Summary

Protein Purification The separation of a chosen protein from a mixture of proteins/proteins in solution  Proteins have different properties due to their different structural difference – can be separated on the basis of these propertiesTraditional Methods of Purification Rely on sequential applic...

Description

Protein Purification  

The separation of a chosen protein from a mixture of proteins/proteins in solution Proteins have different properties due to their different structural difference – can be separated on the basis of these properties

Traditional Methods of Purification  

Rely on sequential application of several methods that each exploit a different property First preparation of insulin were made by extracting bovine pancreas - Could not be used for long - Caused severe pain and abscess’ - Contained impurities

Separation by Solubility 

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Early purifications of insulin achieved by using different concentrations of alcohol - Insulin was soluble in 80% ethanol – other proteins are insoluble and therefore can be removed - Insulin was insoluble in 95% ethanol – insulin will precipitate and therefore can be removed Surface of soluble proteins consist of polar residues Solubility of proteins differ because their surfaces differ

Precipitation by Ammonium Sulphate (Salting Out)   

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Depends on the polarity of the protein surface This determines how soluble it is in water Dissolved salt also needs to be salivated so adding ammonium sulphate decreases the water available to solvate the protein - Water molecules interact with the charged particles t form solvation shell - Most of the proteins surface is not charged - When small charged particles are added they compete with the protein for a solvation shell - Increase in salt solution to a certain point will cause proteins to lose their solvation shell, proteins interact with each other and precipitate When high concentrations of small, highly charged ions such as ammonium sulphate are added, these groups compete with the proteins to bind to the water molecules This removes the water molecules from the protein and decreases its solubility resulting in precipitation (when there are not sufficient water molecules to interact with protein molecules) In general, higher molecular weight proteins will precipitate out at lower salt concentrations Salting out is also used for concentrating dilute solutions of proteins Add ammonium sulphate and dissolve Precipitation of some proteins that are insoluble at that [ammonium sulphate] Centrifuge – to separate the soluble (supernatant) and insoluble (pellet) Mixture of soluble proteins at [ammonium sulphate] and re-dissolved pellet (precipitated proteins) formed Take each fraction and assay to find which one contains the protein of interest

6. Each assay of soluble and re-dissolved proteins will have different solubility at specific [ammonium sulphate] – this curve is used to separate known proteins

Size Exclusion Chromatography        

Separated on the basis of size Sample is applied to the top of the column Colum contains porous beads made from porous but highly hydrated polymer (dextran or agarose) Small proteins are able to enter the pores in the beads (larger ones cannot enter) Larger molecules flow faster through the column because a smaller volume is accessible to them There will be a high concentration of proteins of high Mr emerging first from the column Proteins of medium and small Mr will emerge at intervals according to their delay in high concentration A graph of elution volume/column volume against the log of the M r produces a calibration line of know proteins – use this to work out Mr of an unknown protein

Ion Exchange Chromatography       

Depends on ionisable groups in the protein The column contains beads with e.g. carboxylate ions in order to negatively charge the bead If a protein has a net positive charge at pH 7 it will bind to a column of beads containing carboxylate groups Negatively charged proteins will not bind to the carboxylate ions and therefore will continue to flow through the column The bound protein can then be eluted (released) by increasing the [NaCl] or any salt in the eluting buffer Na+ ions will compete with positively charged groups on the protein for binding to the beads Proteins that have a low density of net positive charge will tend to emerge first

Protein Detection

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Most proteins are colourless Proteins absorb UV light at 280nm because of aromatic amino acid residues e.g. phenylalanine Concentration of assay for biological activity should coincide with a concentration of particular protein Biological activity

Affinity Chromatography 

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Most interesting proteins have a selective affinity for a particular structure: - Enzyme for substrate - Receptor for hormone - Antibody for antigen Specific affinity can be exploited, often with spectacular success, for purification This technique exploits the high affinity of many proteins for specific chemical groups Beads in the column contain chemical groups that the specific protein has a high affinity to The protein of interest will then attach, allowing all other proteins to continue flowing though the column The protein of interest is released from the beads by adding a concentrated solution of the same chemical that is contained within the beads The concentrated solution displaces the protein from the binding site of the bead The protein is then collected from the column

Proteins that Regulate Gene Expression by Binding to DNA Sequences    

A protein mixture is passed through a column containing specific DNA sequences attached to a matrix Proteins with a high affinity for the sequence will bind and be retained Final protein is formed however with an additional piece at one end The transcription factor can be released by adding a solution containing a high [salt]

Ni-Nitrilotriacetate Agarose        

Agarose with Ni2+ group attached Proteins without an affinity means other methods can’t be used to isolate the protein DNA is attached to the protein forming gene (fusuion protein) If attached DNA has an affinity it can be used to remove protein from the whole mixture Affinity chromatography can isolate proteins expressed from cloned genes Extra amino acids can be encoded for in the cloned gene – act as an affinity tag when expressed His tag – formed when repeats of the codon for histidine are added to the gene Tagged proteins are then passed through a column containing beads with covalently attached nickel II (or other metal ions)

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His tag binds tightly to the ions The protein can be eluted from the column by adding a chemical that binds to the metal ion and displaces the protein

How Do We Know the Protein is Pure? 

Need analytical methods to separate the proteins with high resolution

SDS-Page (Analytical not Purification Method)               

SDS = Sodium Dodecyl Sulphate (a detergent) – denatures the protein however still able to analyse Page = Polyacrylamide Gel Electrophoresis SDS unfolds protein by coating unfolded chain and give it charge Take protein and add SDS, reducing agents to break disulphide bonds and boil SDS binds to this molecule Each SDS is negatively charged because of the sulphate – removing natural charge of the protein by adding sulphate groups Charge: Mass ratio of all the proteins is now the same In gel, small molecules will move quite far and large molecules won’t move as far Staining the proteins will reveal its location Proteins of known size will be run alongside to for a ladder for comparison Use other methods alongside SDS-PAGE to identify unknown protein Purity is defined by clarity of band Asses quantity by the brightness of the band Size is assessed by the location of the markers Standard line helps to easily identify mass of protein

Isoelectric Focusing           

Technique depends on materials called amphoteric electrolytes – polymers with lots of ionisable groups (COO- and NH3+) Numbers of COO- and NH3+ groups on each molecules vary – molecules have different isoelectric points Isoelectric points (pI) is the pH at which the molecule has no net charge A pH gradient sis established by including the amphoteric electrolytes in a polyacrylamide gel The gel is then subjected to a high voltage The sample containing the mixture of proteins is amplified The proteins either move towards the positive or negative end depending on their net charge As they move along the pH gradient their net charges are reduced – subject to ionisable changes When they reach a pH where their net charge is 0 they stop This is the isoelectric point The gel is then stained with Coomassie Blue or Silver Nitrate

2 Dimensional Gel  

Isoelectric focusing first occurs Protein are embedded on SDS-PAGE gel to get a 2 nd separation on the basis of size

Determination of the Protein Structure

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Size of the protein can be found by size exclusion chromatography Estimating molecular mass by size exclusion chromatography Mass spectrometry can find the Mr of the protein Monomer size can be found by SDS-PAGE Combination of size exclusion chromatography and SDS-PAGE indicates quaternary structure Could be monomer, dimer, holodimer or hertrodimer

Determining the Sequence            

Edman degradation – uses Edmans reagent Sequential removal of N residues Put in Edmans reagent and make conditions alkaline Formation of an adduct – Edmans reagents adds to the N terminal of the amino goup Acidify with triflouroacetic acid – causes the peptide bond to break Compound can be purified from the mix to analyse Identify the protein by chromatography Most proteins are far too long for this process Long proteins will have to be broke up for this system to handle (chymotrypsin cuts after aromatic residues) Don’t know the sequence Cutting the protein with trypsin will give different fragments Sequence is most commonly found by sequencing DNA that encodes the protein

Mass Spectrometry     

Determines molecular mas with great precision Separation is based on mass/charge ratio Material being analysed must be in the gas phase Matric assisted laser desorption/ionisation MSMALDI-MS Electron ionisation ESI-MS...