TB2

SP structure (3)

1) 1-5 +vely charged 2) 7-15 hydrophobic 3) 3-7 uncharged then cleavage site

MBP tag (3)

1) Maltose binding protein binds specifically to amylose columns 2) Then can be eluted using maltose to compete for MBP binding 3) Similar pros and cons to GST, but doesn't dimerise

Are there transmembrane regions or anchors? (2)

1) Shortest TM helices are ~18 residues long and hydrophobic. 2) Thus, a ~20 residue box is scanned along the sequence to assess its hydrophobicity. This enables TM helices to be predicted.

How do we initially check to see if our protein is correctly purified (2) + but then...

1) Single bad on SDS PHAGE gel 2) Symmetrical peak on a gel filtration column - but then we need to double check that we have purified the correct protein, and that it is homogeneous in other ways: - Charge, PTMs, effect of proteolysis, mass

Why purify proteins? (4)

1) Structure determination 2) Determination of binding proteins 3) Analysis of enzyme function 4) Reconstitution of functional systems from components

Is it my protein? (3)

Western blotting 1) Proteins transferred from gel to membrane 2) Surface of membrane blocked with milk proteins 3) POI detected using an antibody (then a secondary antibody) coupled to an enzyme - e.g. HRP (horseradish peroxidase -> substrate -> light)

Harsher disruption methods (2)

- For E. coli and yeast (also malaria parasite) 1) Cell disrupter forces cells through small hole at high pressure 2) Sonicator used high pressure sound waves to cause cavitation - However, can cause suspension to heat up (keep sample on ice before)

GST tag + pros and cons (5)

- Larger, more specific tag - Glutathione-S-transferase protein interacts specifically with glutathione Advantages: 1) Very specific - fewer contaminants 2) Reagents are all commercially available 3) Quick and easy Disadvantages: 1) GST is large so needs to be removed before many applications (inc. crystallography) 2) GST dimerises which will lead to false results when looking at multimeric state of fusion proteins

Proteins from native sources (3)

- Some proteins are abundant and highly enriched in specific natural sources: 1) Haemoglobin 2) Lysozyme (chicken egg white) 3) Acetylcholine receptors

Ion exchange column rules (4)

1) A protein with a PI of 6 would be -ve at a pH of 8 2) Therefore we would use an anion exchange column (+ve column) 3) We need to keep the pH as close to physiological pH as possible (~6-8). Otherwise IEC is not possible (denaturation). 4) pH should be 2 units from pI

How much pure protein? (2)

1) Abs at 280nm - due to presence of aromatic Trp, Tyr, Phe 2) Extinction coefficient can be estimated from sequence. A= εcl - l is path length.

Extracellular proteins: controlling glycosylation (3)

1) Adding inhibitor which blocks processing (e.g. kifunensine) 2) Using mutant cell-like which lacks processing enzyme (GnTI-) 3) Cleaving glycans away using an enzyme (e.g. endo H which cleaves between GlcNAc residues)

Selecting better domain boundaries (4)

1) After purification, we can use limited proteolysis to trim the protein or to cut away stable fragments 2) The POI is treated with small quantities of proteases, and samples are taken at different. time points. 3) SDS phage is then run to study the results, showing us protease resistant fragments - can be identified using N-terminal sequencing. 4) If stable fragments are identified, we may choose to redesign the protein construct with new domain boundaries - NOTE: proteases will only cut their target in disordered regions

Immunoaffinity resins (2)

1) Antibody c an be raised against the POI, by injecting POI into mouse or rabbit. 2) These resins are highly specific and bind tightly to the protein, but it can be hard to elute it. - For this, a competitive peptide may be used, or sometimes by lowering pH

Re-suspension solution components (7)

1) BUFFER MOLECULES - Used to control pH of a solution to prevent denaturation - Choose pH which protein is found in (N.B. can vary with temp + ionic strength) - e.g. Tris-HCl 2) SALTS - Maintains proteins. NaCl can be used to increase solubility. 3) REDUCING AGENTS - Inside of cell is a reducing environment - e.g. DTT 4) LIGANDS - Some proteins only stable in presence of ligands (NADH, ATP, heme etc.) 5) PROTEASE INHIBITORS 6) DETERGENTS - Detergents have hydrophobic and hydrophilic parts. - They are able to surround hydrophobic parts of the protein, helping them remain in solution. (prevents aggregation) 7) SUCROSE or GLYCEROL - Increase density of solution, maintaining protein folded shape.

Making the resins (2)

1) Can chemically link the molecule to the resin 2) Can use protein A or G resins which are bacterial defence proteins that have high affinity for different antibodies such as IgGs

What is the domain architecture? (2)

1) Can use Phyre of Fugue that use known structures in PDB 2) AlphaFold - de novo structure prediction

Types of protein homogeneity (4)

1) Charge - Proteolysis may have removed charge residues - Differences in phosphorylation 2) PTMs - Glycans can be removed and identified using mass spec 3) Degree of proteolysis (at ends or nicking in the middle) 4) Degree of aggregation or oligomerisation

Ion exchange chromatography (5)

1) Charged resins will bind to proteins at different strengths based on their surface charge 2) These interactions can be broken using salts such as NaCl - Na+ and Cl- act as counter-ions, blocking teh interaction. 3) Different proteins will bind with different strengths to the resin and elute at different NaCl concentrations 4) Ion exchange resins can be positive or negative and weak and strong - e.g. Q sepharose - strong anion exchanger (+-ve charge) 5) Need to estimate pI (to compare with pH) based on aa sequence

Running an ion exchange column (4)

1) Choose appropriate combination of resin and buffer 2) Transfer protein into binding buffer (should have correct pH and little NaCL 3) Allow protein to interact with resin material and wash with low salt buffer 4) Elute, either with pH change - or more commonly a gradient of NaCL

Early stages of purification outline (3)

1) Choose solution in which the protein is likely to be stable and folded 2) Break the cells in this solution to extract the protein 3) Centrifuge to remove debris of broken cells

Hydrophobic interaction chromatography (HIC) (4)

1) Differnet proteins have different amounts of hydrophobic surface. 2) We can use hydrophobic material (containing phenyl, butyl, or octyl groups) to bind to protein of interest. 3) Hydrophobic interactions are strongest in high ionic strength buffers, so bind protein to the resin in high salt condiftions, and elute with a decreasing salt gradient. 4) Sample loaded onto a column in 1-3M salt, and then eluted with decreasing NaCl conc. gradient. - Therefore ideal for ammonium sulphate precipitated samples.

The degree of disorder (4)

1) Disordered loops or termini in an ordered protein 2) Disordered linkers between domains 3) Larger disordered regions as part of an otherwise ordered protein 4) Largely natively unfolded proteins - Disordered regions generally have a higher content of hydrophilic residues, and can be low in sequence complexity, enabling prediction.

Heterologous expression systems (4)

1) E. coli -> cheap and cheerful and often does the job 2) Yeast -> can make more complex proteins 3) Insect cells/baculovirus -> often works better for mammalian proteins (glycosylated proteins etc.) 4) Mammalian cells.

Making a clarified lysate (2)

1) First supernatant is growth media (5,000g) 2) Cell pellet centrifuged again (100,000g) - Second supernatant contains soluble protein mixture - Cell debris discarded 3) Differential centrifugation can be used to separate out a series of different cell components - Nuclear - Mitochondrial - Lysosomal - Microsomal

Fragmentation and mass spectrometry (2)

1) First we fragment the protein in the band by different methods: - Chemical reaction : cyanogen bromide cuts proteins to the C-terminal side of Met residues, dilute acid cleaves Asp-Pro - Protease digest : most commonly trypsin 2) Then mass spec is performed and spectra is compared to a theoretical spectra or a broader database.

Why are you making the protein? (4)

1) For crystallisation - Minimise disorder 2) To reconstitute a biological system - As close to native as possible 3) To assess pathogen binding - Properly glycosylated and processed 4) To dissect function of different domains - Need to define these domains

Removal of the tag (3)

1) Fusion proteins that need to be cleaved will contain a specific protease site. 2) Protease cleavage is followed by passing the protein through a resin which will remove uncleaved protein and the tag. 3) Can tag protease and MBP/GST with His for this 'reverse chromatography'

Protein purification considerations (3)

1) If possible, start with affinity method - quick, and lead to large percentage purification - Can also remove proteases 2) Finish with gel filtration - Can remove aggregated/misfolded proteins 3) Possibly use something else in between, tailored to the specific contaminants you are trying to remove.

Fancy expression strains: disulphide bond formation (2)

1) Inside of E. coli is a reducing environment, so disulphide bonds do not form. 2) Strains like Origami have mutations which change the intracellular environment to make it more oxidising. - Mutations in genes such as thioredoxin reductase

Purification of membrane proteins (6)

1) Membrane proteins are evolved to be stable in membrane conditions, in which they experience lateral pressure from the bilayer. 2) To extract them from the membrane environment into a solution, detergents are used. 3) These amphiphatic (containing both hydro-phobic and -philic portions) molecules screen the hydrophobic parts of the protein from the aqueous environment. 4) It is important that the detergents don't form micelles, so they are kept at a concentration just below the critical micellar concentration (cmc). 5) In purification, fractionation is used to acquire a pellet of membrane fragments. 6) The membranes are then solubilised by incubating with detergent for an hour, and then centrifugation is used to remove unsolubilised material. - Usual techniques then used (with detergent present at concentration about cmc)

Detergent-based cell lysis (2)

1) Non-ionic detergent mixtures supplemented with lysozyme and DNase to break cell wall and degrade DNA 2) Can selectively lyse cell. surface membrane, but not nuclear membrane - e.g. B-PER

Purifying extracellular proteins (3)

1) Often contain disulphide bonds, glycosylation and multiple domains 2) Best approaches: - Origami E. coli (enable disulphide bond formation but good to avoid glycosylation) - Secretion from insect cells (simple glycosylation) - Secretion from mammalian cells (e.g. HEK293) for human-like glycosylation 3) To purify protein from culture media, we can use tangential flow systems to concentrate sample from large volume, before standard chromatography methods.

Approaches to produce macromolecular complexes (3)

1) Produce components separately and then mix - Often a final SEC column will be used to separate complex from free components - Frequently used for 1:1 complexes, particularly when proteins are from different species (host-pathogen e.g.) 2) Express the components in the form of a complex and purify the assembled complex - May not express in folded form alone - Can be co-expressed in the same cells with a purification tag - e.g. anaphase promoting complex (APC/C) (14 different proteins) expressed in baculovirus-infected insect cells 3) Endogeneously tag one of the native genes and pull out the native complex from cells - When it cannot be expressed or no full understanding of composition - CRISPR/Cas9-based methods to add a tag (HA and Flag tags) - Lysed cells passed over resin - e.g. Human BAF complex (nucleosome-remodelling complex) - In order to capture in most native state

Mammalian cells expression system (4)

1) Properly processed human proteins 2) Most commonly used for protein overexpression: - HEK 293 - Human embryonic kidney - COS - African green monkey kidney - CHO - Chinese hamster ovary 3) HEK cells can be transfected with plasmid using chemical methods such as liposomes - Then grown in roller bottles if adherent or suspension culture 4) CHO cells cannot grow in glutamine free media -> glutamine synthetase (GS) gene is introduced.

Peptide expression (3)

1) Proteins susceptible to proteolysis during expression can be deliverately produced in the form of inclusion bodies - This is by fusion to a ketosteroid isomerase tag - Then purified under denaturing conditions 2) KSI is then cleaved by cyanogen bromide to release peptide. 3) This is then purified using reverse phase chromatography/

Affinity chromatography (2)

1) Purification often starts with this. 2) POI binds to ligand attached to a column material. - ligand can bind to tag - can bind specifically to POI - can be antibodies

Which proteins are present? (4)

1) SDS phage (sodium dodecylsulphate polyacrylmalide gel electrophoresis) 2) Protein heated in presence of detergent SDS 3) This binds to hydrophobic regions of the protein chain, unfolds it and coats with a negative charge. 4) Smaller proteins more rapidly down through the gel.

Considerations for each chromatography column (4)

1) Selectivity - the ability of the column to bind only the POI 2) Capacity - the amount of protein which can bind to the column - best to work at close to max column capacity - reduces free surface for contaminants 3) Resolution - how much will peaks from the protein and impurities overlap? 4) Compatibility - will the eluted sample be suitable for the next step?

Reverse phase chromatography (4)

1) Separates molecules based on hydrophobicity 2) Proteins loaded into aqueous buffer (sometimes trifluoroacetic acid (TFA) to control pH) 3) Proteins then eluted with an increasing concentration gradient of an organic solvent (e.g. acetonitrile) 4) However, organic solvents can denature proteins - Thus, usually used for peptides.

Histidine tag (4)

1) Six or more (usually up to 10) histidine residues. 2) Small, simple so rarely interferes with function of protein - Thus, often does not need to be removed before protein function is studied. 3) It interacts with metal containing resin during immobilised metal affinity chromatography (IMAC) - Most commonly used resins contain Ni2+ or Co2+ ions. - These are chelated by an NTA group attached to the resin 4) Each metal ion has two coordination sites free to interact with two histidines

Salting in and salting out of proteins (2)

1) Solubility increases as more salt is added (salting in) - salt molecules stabilise protein by replacing electrostatic interactions between proteins 2) When the ionic strength of a protein solution increases, solubility decreases leading to protein precipitation (salting out) - Salt molecules compete with the protein molecules in binding with water.

Purification of His-tagged protein (2) + Buffers (3)

1) Soluble cell extract is loaded onto a Ni2+-NTA column. 2) Column is washed and then protein is eluted using imidazole (competes with tag for binding to Ni2+) For all: - 20mM Tris pH 8.0 1) Lysis buffer - 300mM NaCl - 0.5% Triton X-100 - 15mM imidazole 2) Wash buffer - 500mM NaCl - 15mM imidazole 3) Elution buffer - 300mM NaCl - 200mM imidazole

Refolding purified proteins (4)

1) Some E. coli expressed proteins will be in the form of inclusion bodies - aggregates of misfolded, insoluble protein. However, they can sometimes be refolded 2) First, they must be denatured using denaturing agents such as Urea or guanidine HCl, as well as a reducing agent to break disulphide bonds sometimes. 3) Need to gradually decrease concentration of Urea. 4) Often, the protein is eluted while denatured and then diluted into buffer which lacks denaturing agents.

SEC column selection (3)

1) Superdex 10 is for <10 kDa 2) Superdex 75 is for 3-70 kDa 3) Superdex 200 is for 1-600 kD

Designing a purification strategy (3)

1) The ideal purification step leads to a large increase in percentage purity of the POI, while losing little of it 2) Different chromatography steps can be linked together in sequence to remove different impurities: - Capture - Intermediate purification - Polishing 3) Should use minimum number of steps to give 'clean' protein to reduce sample losses

Membrane protein tricks: screening for solubility (3)

1) To work out conditions in which the protein is stable, we can use fluorescent SEC (FSEC) 2) Here we use the gel filtration column mobility of a protein to assess whether it is correctly folded. 3) The protein is expressed with GFP fused to C-terminus (with thrombin cleavage site)

Is the protein folded? (3)

1) Use circular dichroism 2) Secondary structure of protein determines protein's optical activity 3) Compare with standard curve.

Enzyme assay (3)

1) Used to test function of an enzyme at various stages during purification. 2) Assays will look at the loss of substrate or gain of product - e.g. cleavage of ONPG by β-galactosidase to ONP (yellow) 3) Many other assays have been developed to screen kinase, phosphatase, or protease activity.

T7 expression system (3)

1) Uses IPTG with lac repressor upstream of GOI and separate (already present) plasmid with T7 pol CDS. 2) Pre-existing plasmid has gene for T7 pol under control by bacterial promoter. 3) Addition of IPTG relieves repression, enabling expression of T7 pol, enabling expression of GOI. N.B. codon optimisation - Or use Rosetta cells N.B codon harmonisation relies on finding that non-optimal codons are used to slow the ribosome, enabling correct folding

Thermal shift analysis (4)

1) Uses fluorescent dye such as SYPRO orange -> only fluoresce in hydrophobic environment (core of protein) 2) Then, protein sample is heated to expose hydrophobic surfaces, increasing intensity. 3) Changing conditions can be shown graphically to show which conditions stabilise the protein. 4) TSA can also be used to locate ligands - All carried out in multi-well plates

E. coli expression system limitations (4)

1) Very very rarely glycosylate proteins 2) Don't add membrane anchors 3) Proteins tend to be simpler and single domain, so often fail to make more complex proteins (but sometimes they can) 4) Membrane composition is different (no cholesterol)

Secreted or intracellular (3)

1) When using insect or mammalian expression systems, the proteins can be expressed inside the cytoplasm or cause it to be secreted 2) For an extracellular, or secreted, protein, it is better to design it to be secreted - Then it will experience the right chaperones in the ER/Golgi and form disulphide bonds 3) To cause a protein to be secreted, an N-terminal secretion signal must be added before the start of the gene - gp67 works for insect cells.

N-linked glycosylation (3)

1) attachment of carbohydrate to nitrogen atom of asparagine side chain 2) N-x-S/T 3) Context matters so prediction programs take sequence context into account. NetNGlyc To remove modification sites -> N in N-x-S/T can be changed to Q-x-S/T

Pros and cons of a His-tag (4)

Advantages: 1) Short tag that generally doesn't affect function 2) Quick, easy, cheap and applicable to many proteins 3) It works in denaturing conditions (e.g. in presence of urea) Disadvantages: 1) Proteins with surface Histidine clusters can interact non-specifically - so high level of contaminants - But can add additional purification steps.

How much total protein is present? (2)

Bradford assay 1) Green/brown -> blue 2) Protein residues reduce the Cu2+ to Cu+

Ligand binding assay example (3)

CTX (Charybdotoxin) binds to many K channels 1) Radiolabelled CTX is incubated with protein at different concentrations of unlabelled CTX 2) Samples are washed on filter papers using a vacuum before radioactivity is counted. 3) This gives binding affinities

Assessing charge (4)

IEF (isoelectric focusing) gel system 1) IEF gels have a pH gradient across the gel 2) The pH gradient is formed by placing the anode in an acidic condition and the cathode in a basic buffer 3) The gel contains ampholytes which move within the gel until they reach the pH of their pI, reinforcing the pH gradient 4) When the pH > pI of protein, they move towards the cathode.

Gentle methods of cell disruption (4)

If too harsh a method is used, proteins can degrade or aggregate - thus, gentlest method is ideal. 1) Osmotic lysis - add cells to buffer with higher WP - they will expand and burst 2) Dounce homogenisation - Move dounce up and down in order to homogenise cells 3) Blade homogeniser - Blender (e.g. pig brain). 4) Squeeze through bent needle

MALS (2)

Multi-angle laser light scattering 1) Another method (other than SEC) to assess mass (intensity of scattered light) and size (angular distribution of scattered light) 2) Scattering results from absorption and re-emission of photons

Ammonium sulphate precipitation/fractionation (3)

Often precipitation causes aggregation 1) However, ASP leads to salting out of protein, reducing solvent molecules available to interact with protein 2) ASP doesn't seem to disrupt proteins or cause irreversible protein unfolding. 3) To determine the conditions in which the protein will precipitate, we can run a small scale trial - Choose concentration below which POI will precipitate - Then choose concentration just above where POI will precipitate

Example of ligand resin (3)

Purification of β-adrenergic receptor using alprenolol 1) Alprenolol is a potent antagonist of the receptor. 2) The receptor can be purified by passing through column with resin with attached alprenolol 3) Has major advantage that only correctly folded protein will be purified

Chromatofocusing (3) + disadvantage

Rarely used but very powerful at separation based on pI 1) Relies on pH gradient being created on the column as weak ion exchange resin interacts with a strong buffer 2) Proteins will move down with pH gradient, binding and unbinding to the resin - move at different rates based on pI 3) Good separating power but low capacity - Disadvantage of this AND ion exchange is that it has low selectivity for average protein as many proteins have similar pI values so will elute in overlapping peaks

Yeast expression (2)

S. cerevisiae, P. pastoris 1) Can carry out PTMs: - N-linked glycosylation - Palmitoylation 2) Can be constitutive expression vectors or inducible - AOX1 promoter can be used in methanol inducible system in P. pastoris

Insect expression systems (3)

Sf9 or S2 cells 1) More complex eukaryotic systems involve these - complex folding - many PTMs 2) S2 cells are derived from D. melanogaster - can grow as culture in suspension, or monolayer 3) Or can use baculovirus to transfect Sf9 cells - Replace polyhedrin gene with GOI - Co-transfection of plasmid with GOI and linearised baculovirus DNA - or large plasmids containing both components

How to separate by size & shape? (3) + Considerations (3)

Size exclusion chromatography (AKA gel filtration) 1) Uses porous resin 2) Molecules smaller than the pores will enter them and be slowed down 3) Molecules bigger than the pores will pass around the beads and move more quickly Considerations: 1) Sample should not interact with resin 2) The sample is diluted during SEC (so should be concentrated before) 3) Larger columns are used for purification, and smaller for analytical work