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Protein Purification
Biomolecules
University of Lincoln
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Protein Purification
● What is a protein? ○ Proteins are polymers ‘chains’ of amino acid molecules joined together by a common linkage ○ There are 20 different types of amino acid all with different shapes and charges ○ This chain folds up to give a 3D shape (conformation) and charge which is very important and gives the protein its function
● There are four levels of protein structure
● What characteristics do we use to purify proteins? ■ Affinity ● Charge
● Hydrophobicity ● Innate of engineered properties of the protein ■ Size
● Types of protein expression ○ Native vs recombinant ■ Recombinant gene expression under an artificial promoter generally gives much higher yields of proteins and allows the addition of affinity tags
● Why do we need pure proteins? ○ For research ■ Enzymes assays ■ Structural biology ■ Biophysics ■ Sequence determination ○ For therapeutic use - e. part of a vaccine ■ Insulin ■ Antibodies
● Sample preparation ○ Overall aim: ■ To isolate one protein from a mixture of thousands of other proteins
● Assessing the purity of proteins using SDS PAGE ○ Aim: ■ To separate out the mixture if different proteins according to size and view the purity of the target protein in relation to the contamination
● Protein purification ion exchange in a ‘nutshell’ ○ Aim: ■ To separate out the mixture if different proteins according charge ○ Use pH to induce a charge on the protein ○ This charge enables the target protein to bind to the ion exchange resin when loaded on the column ○ Some non-target protein flow through/or are washed off ○ A gradual increase in NaCl known as a gradient is applied to the column this out competes the proteins and caused them to ‘drop off’ the column ○ This is known as Elution
● Protein purification ion exchange ○ Protein contain many ionizable groups on the side chains of their amino acids including their amino - and carboxyl - termini ○ These include basic groups on the side chains of lysine, arginine and histidine ○ Acidic groups on the side chains of glutamine, aspartate, cysteine and tyrosine ○ The pH of the solution, the pK of the side chain and the side chain’s environment influence the charge on each side chain ○ As the pH of a solution increases, de-protonation of the acidic and basic groups on proteins occur, so that carboxyl groups are converted to carboxylate anions (R-COOH to R-COO-) and ammonium groups are converted to amino groups (R-NH3+ to R-NH2)
● Acid/base catalysis (side-chains) ion exchange
● Protein purification ○ Used a buffer to induce a suitable charge on the protein a target protein - heat is a good buffer ○ Q resin is positively charged ○ S resin is negatively charged ○ The isoelectric point (pl), is the pH at which a particular molecule or surface carries no net electrical charge ○ Avoid excess of pH and high buffer concentration (typically 10 mM hepes pH
8 for Q resin)
● Protein purification ion exchange
○ Proteins with weaker ion interactions will be released at lower salt concentrations ○ Proteins with stronger charges will have a higher affinity so will remain bound to the column longer
● The basic concept of ion exchange protein purification ○ Use pH to induce a charge on the protein ○ This charge enables the target protein to bind to the ion exchange resin when loaded on the column ○ Some non-target protein flow through/ or are washed off ○ A gradual increase in NaCl known as a gradient is applied to the column this out competes the proteins and caused them to ‘drop off’ the column ■ This is known as Elution
● Protein purification: hydrophobic interactions ○ Aim: ■ To separate out the mixture if different proteins via hydrophobic interactions ■ Elute by reducing the ammonium sulphate concentration on an gradient
● Protein purification: gel filtrations ○ Aim: ■ To separate out the mixture if different proteins according size
● The organisation of a size exclusion (gel filtration) system
● Immobilised metal ion affinity chromatography (IMIAC) ○ Aim: ■ To separate out the mixture if different proteins via a poly-His engineered tag
● Protein purification: buffer exchange/concentration ○ Dialysis and concentration ■ Dialysis: buffer and salt diffuse into a large volume of replacement buffer through a selectively permeable membrane ■ Leave overnight at 4C with stirring to exchange buffer ○ Ultrafiltration ■ Small molecules are forced through a membrane by centrifugation leaving the protein behind ■ A number of washes and needed to full replace the buffer this is also use to concentrate the protein
● Protein purification storage and preservation ○ Storage preservation ■ Proteins are normally stored at 4C ■ Azide is frequently added to prevent bacterial growth ■ Some can withstand freeze drying ■ Storage with 30% glycerol at -20 is also common ■ Columns a stored in 20% ethanol to prevent bacterial spoilage
● Purity vs expense/time/yield ○ What is your downstream application? ■ Therapeutic ■ Crystallography ■ Biochemical assays/characterisation ○ Are post translational modifications required, eukaroytic expression systems? ■ Cost depends on the labour reagents an facilities
Protein Purification
Module: Biomolecules
University: University of Lincoln
- Discover more from:BiomoleculesUniversity of Lincoln33 Documents
- More from:BiomoleculesUniversity of Lincoln33 Documents
