BIOC0003 Term 2 - Lecture notes All term 2 Lectures PDF

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Term 2 Lecture 1 Protein purification I Aim: to separate a particular protein from all other proteins and cell components Why purify proteins? - For functional and activity studies o Basis for characterization and for discovery of inhibitors and drugs - For structural studies o For example, by prote...

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Term 2 Lecture 1 Protein purification I Aim: to separate a particular protein from all other proteins and cell components Why purify proteins? - For functional and activity studies o Basis for characterization and for discovery of inhibitors and drugs - For structural studies o For example, by protein crystallography or NMR - To isolate pharmaceutically active proteins o For example insulin - For industrial processes o For example proteases for detergent industries - For biotechnology o Products include enzymes for diagnosis, restriction enzymes, other enzymes for molecular biology such as thermostable polymerases Two main frameworks in protein purification: 1-Purification from naturally available sources From organs, tissues (including blood) or cell culture The only option in “pre-genomic era” 2-Using recombinant technologies Following introduction of expression vectors encoding a protein of interest into host cells Expression vectors encoding for most human proteins are commercially or freely available Important considerations in planning a purification: Do we require a high yield of product? - Source/host o Animal cells: sometimes not many to work with o Bacteria: Often a lot of material from fermentation o Yeast: Often a good source of material Is activity essential? - Source/host o Has to allow correct folding and posttranslational modifications if these are required for activity-important when using recombinant technology - Take steps to protect by adding stabilisers, reducing the number of purification steps to minimise activity loss at each step and working quickly at low temperature General steps - Choice of framework (recombinant or not) and purification source/host - Extraction, solubilisation and choice of solvents - Protein separation procedures, many based on chromatography - Analysis of protein purity, properties and functionality Choice of framework (recombinant or not) and purification source/host: 1. Important factors in making choices about protein sources/hosts Yield, activity and more a. Characteristics of the desired protein properties of the protein can change with the source/host b. Source availability it should be readily available and easy to grow

c. Yield of product high levels of expression being desirable in most cases d. Safety considerations are of obvious importance since choice of vector and expression organism must be sensible with no dangerous by-products e. Regulatory constraints and patenting issues have a lot of bearing on the use of recombinant technology f. Consumer perception Comparison of expression systems:

Extraction, solubilisation and choice of solvents: Procedures include: - Cell breakage/homogenization - Centrifugation - Preservation of intact/functional components

Making a cell extract

A cell extract (homogenate) may contain: - Cell wall fragments - Membrane fragments (lipid) - Fibrous tissue Intracellular organelles - Soluble protein - Insoluble protein - Nucleic acids (can be removed by DNAse treatment, acidification etc) - Polymeric carbohydrate High speed centrifugation to prepare cytosol - Cytosol= portion of the cytoplasm that remains in the supernatant after centrifugation of a cell extract at 150,000 g for 1 hour o Contains concentrated solution of enzymes and other proteins - Particulate fraction = particles and organelles o Contains ribosomes, storage granules, mitochondria, chloroplasts, lysosomes, endoplasmic reticulum Differential sedimentation

Releasing membrane-associated and membrane-integral proteins - Ultrasonication - Detergent solubilisation - Organic solvent extraction - Digestion of phospholipid often followed by centrifugation

Preventing denaturation, inactivation and proteolysis: Denaturation: Best done by avoiding extremes of pH, temperature and organic solvents I. Use buffers around neutrality II. Carry out initial steps on ice III. Avoid organic solvents and chaotropic agents such as urea and guanidinium Inactivation: The active site of enzymes are extremely reactive and so we must I. Avoid oxidising conditions if the protein is SH requiring, can add reducing agents such as DTT, BME II. Avoid metal ions by adding chelators such as EDTA or EGTA III. Stabilise cofactor requiring enzymes by the addition of the appropriate cofactor to buffers Proteolysis: I. Speed and the use of low temperatures during the initial stages are important in avoiding proteolytic damage II. Can be avoided also by adding cocktails of protease inhibitors Buffers and buffering - A buffer consists of an acid and its conjugate base - Buffers absorb or release protons by a shift in equilibrium - The pH of a solution of a weak acid or base can be calculated using the Henderson Hasselbach equation - pKa (Ka=equilibrium constant) is the pH at which the concentration of acid and base are equal - The buffering capacity of most buffers drops off dramatically 1 pH unit either side of the pKa - As a consequence buffers should not be used outside the buffering capacity or range for a given buffer Buffer exchange: Desalting and buffer exchange are methods to separate soluble macromolecules from smaller molecules (desalting) or replace the buffer system used for another one suitable for a downstream application (buffer exchange) - Dialysis o Depending on size of the pores can be used to remove ions from proteins Protein separation procedures, many based on chromatography: Protein Properties - Basis for Separation - Size (3.3 to 1,600 kDa; most common 11-110 kDa) - Shape (globular, asymmetric) - Net charge, pI value (typical range of pI 4-9) - Charge distribution (uniform, clustered) - Hydrophobicity (extent and distribution) - Solubility (from 300 mg/ml) - Ligand (cofactors, substrates) and metal binding - Specific epitope (structural features for Ab recognition) Main variables that affect properties and interactions Interactions: - hydrogen bonds - hydrophobic interactions - ionic interactions Variables: - Temperature

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ionic strength ion type polarity of solvent (dielectric constant) pH

Protein separation procedures - “Crude” protein separation (e.g. salt precipitation) - Chromatography methods - Other methods Salt precipitation A protein is surrounded by solvent (water) A number of agents (e. g. salts) are capable of stripping away the hydration shell. Different proteins have a different ratio of hydrophilic/hydrophobic patches and precipitate at different salt concentrations.

General principle of chromatography: Stationary phase - solid porous matrix Mobile phase - solution from the reservoir and solution containing mixture of proteins (protein sample) Principle of separation - as proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. Individual types of proteins (such as A, B, and C) gradually separate from each other, forming bands and eluting separately

Lecture 2 Protein purification II Size-exclusion chromatography: - also called gel filtration, separates proteins according to size/shape - matrix is a cross-linked polymer with pores of selected size - Larger proteins migrate faster than smaller ones: o they are too large to enter the pores in the beads and hence take a more direct route through the column. o The smaller proteins enter the pores and are slowed by their more labyrinthine path through the matrix Ion-exchange chromatography -

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exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH matrix is a synthetic polymer containing bound charged groups: anionic groups are cation exchangers, cationic groups are anion exchangers the affinity of each protein for the charged groups on the column is affected by the pH and the concentration of competing free salt ions in the surrounding solution that can be gradually increased (salt Ionexchange gradient) to elute bound proteins A protein that has no net charge at a pH equivalent to its isoelectric point (pI) will not interact with a charged medium. At a pH below its pI, a protein has net positive charge and will bind to a negatively charged medium and can be eluted by + counter ions. At a pH above its pI, a protein has net negative charge and will bind to a positively charged medium and can be eluted by – counter ions.

Cation exchange:

Hydrophobic interaction chromatography: - Exploits differences in hydrophobicity of proteins - Matrix is a synthetic polymer containing bound hydrophobic groups - In contrast to ion-exchange, the binding is at a high salt, elution at low salt o Close to the surface of the hydrophobic ligand and solute, the water molecules are more highly ordered than in the bulk water and appear to ‘‘shield off’’ the hydrophobic ligand and the hydrophobic protein patch. o Added salt interacts strongly with the water molecules leaving less water available for the ‘‘shielding off’’ effect

Affinity chromatography: - separates proteins by their binding specificities - proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads - proteins that do not bind to the ligand are washed through and the bound protein of interest is eluted by a solution containing free ligand or other competing small molecule - Affinity-tags: inserted by recombinant technology Contemporary high-pressure, automated purification systems:

Stationary phase for column chromatography: The stationary phase comprises an inert material such as agarose, cellulose or other materials, some suitable for high-pressure (e. g. mono beads), further functionalized by: - level of polymerization that determines size of the pores for size exclusion - attaching chemical groups containing charge for ion exchange - attaching hydrophobic chemical groups for hydrophobic interaction chromatography - specific protein ligands for affinity chromatography - other modifications Analysis of protein purity, properties and functionality: Methods include - Measuring protein amounts/concentration - Assessing protein heterogeneity - Determining molecular mass - Enzyme assays and other functional assay Measurements of protein concentration

Bradford Assay: Coomassie brilliant blue binding - binds proteins primarily via aromatic residues and arginine - undergoes absorbance shift from 465 nm (brownish) to 595 nm (blue) Measurements based on light absorption: - Spectrophotometer - Purification systems have a flow cell instead of the sample cuvette- continuous detection of absorption SDS polyacrylamide gel electrophoresis - SDS PAGE - SDS= sodium dodecyl sulfate - In electrophoresis, the speed of protein movement depends on their size, their shape, and their electrical charge. - In SDS-PAGE, the protein mixture is treated in such a way that only the molecules’ mass affects their movement. - This is achieved by adding SDS. SDS molecules bind to the unfolded peptide chains (high temperature) and give them a strongly negative charge. - To achieve complete denaturation, thiols are also added in order to remove the disulfide bonds that contribute to correct folding. - Probe = sample containing a protein mixture Analysis of protein mixtures and mass determination of purified proteins by mass spectrometry: General principle: Molecules to be analyzed are first ionized in a vacuum. When the newly charged molecules are introduced into an electric and/or magnetic field, their paths through the field are a function of their mass-to-charge ratio, m/z. This measured property of the ionized species can be used to deduce the mass (M) of the molecule with very high precision. Two methods for analysis of proteins - ESI MS electrospray ionization mass spectrometry - MALDI MS matrix-assisted laser desorption/ionization mass spectrometry Enzyme assays: BASIC ASSAY COMPONENTS - Reaction mixture: Buffer, substrate, any cofactors or stabilisers. The reaction mixture is often brought to the temperature of the assay prior to initiating the reaction. - Initiating the incubation: Reactions are mainly initiated by addition of enzyme, or fractions from a purification protocol - Termination: some assays require termination by inactivating the enzyme, this can sometimes be combined with colour development. TYPES OF ASSAY - Continuous: This is the most common type of assay where the disappearance of the substrate or appearance of product is measured directly. - Discontinuous: In this type of assay the product has to be separated from the substrate to allow measurement.

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Coupled assays: This is an indirect measurement in which two reactions are involved. In these assays the substrate or products are not measurable and so we have to link or couple another enzyme into the assay that does produce a measurable product

Expressing enzyme activity Enzyme activity is usually expressed as: μmoles/min An international unit of enzyme activity is defined as: - The amount of enzyme that catalyses the conversion/transformation of 1 μmole of substrate per minute under defined conditions of temperature and pH. Activity = total units of enzyme in a solution Specific activity = number of enzyme units per milligram of total protein A purification table for a hypothetical enzyme:

Lecture 3 Protein purification II Recombinant technologies/genetic engineering and genetically modified (GM) organisms Recombinant proteins: Homologous protein production - The removal or isolation of a gene sequence/cDNA derived from a particular species and to subsequently produce its gene product by recombinant means in the same species. - In this case the host cell is producing a recombinant protein that it is capable of producing naturally. - In these cases the manipulation may allow the product to be produced in higher quantities and problems such as gene repression can be over come. Heterologous protein production - The expression of recombinant proteins in cells which they do not naturally occur is termed heterologous expression. - The vast majority of recombinant proteins are produced in this way Hosts and expression vectors Plasmids or viruses with a gene of interest can be introduced in: - Bacteria (E. coli) - Yeast (S. cerevisiae or P. pastoris) - Insect cells (Sf9) - Mammalian cells such as Chinese hamster ovary cells (CHO)

Expression vectors include a gene of interest that can be modified by adding “tags” to generate fusionrecombinant proteins: Tags for affinity purification, sometimes also solubility: - Poly-Histidine - Glutathione S-transferase (GST) - FLAG-peptide - Maltose-binding protein (MBP) One or more tags can be introduced at the N- or C-terminus or both

Tags in recombinant proteins:

Tags can allow simpler purification based on an affinity step:

Antibodies used to recognize a tag in a recombinant fusion protein can be used to detect the protein for example by Western blotting Some advantages and disadvantages of heterologous protein production in E. coli

*Causes for formation of insoluble formation or “inclusion bodies” include: Very high local concentrations of expressed protein in the cytoplasm lead to non-specific precipitation - Insufficient chaperones to aid in the correct folding with subsequent aggregation of partially folded and mis-folded intermediates -

Ways of facilitating the production of soluble heterologous proteins in bacteria: - Empirical determination of the optimal expression system in terms of host strain, plasmid, plasmid copy number and promoter sequence etc. - Expression of the desired product as a fusion protein, fused to a highly soluble and native host cytoplasmic protein. - Expression behind an export signal such as a signal sequence that directs the expressed protein to the periplasmic space or out of the cell completely. - Co-expression with chaperone proteins that improve the solubility of the expressed protein by aiding in the folding pathway of the protein. - Providing appropriate cofactors that may increase the soluble yield of a recombinant protein requiring that cofactor. - Growth of the recombinant cells at sub-optimal temperatures: growth of E. Coli cells at lower temperatures than optimal often discourages inclusion body formation. Overall impact of recombinant technologies: 1. Overcoming problems of source availability: Many naturally occurring proteins are expressed at low level (e.g. cytokines) or may only exist in difficult sources (e.g. human tissue). High level of expression achieved by recombinant technology results in high specific activity in the cell extract and consequently fewer purification steps and overall fold-purification is needed to achieve the required level of purity 2. Overcoming problems of source safety: some proteins are produced naturally by dangerous or pathogenic species (e.g. microbial pathogens, snakes etc.); there is a possibility of dangerous contaminants during purification or in the final product. Furthermore associated hazards may not be clearly defined (you may not know what problems are associated with a particular tissue). Recombinant technology procedures are developed within strict, standardized regulations.

3. Modifications are possible: target protein can be modified if desired by procedures (such as site-directed mutagenesis) which can lead to improved qualities of the desired product and/or facilitate purification (e.g. tags) The tag itself may enhance stability and solubility of the protein. Very large purification in a single step usually results from the use of affinity chromatography based on a specific tag. Some proteins of industrial and medical significance, traditionally obtained from animal sources

Factors that influence the acceptability of genetically engineered proteins are NOT just technical 1. Benefit to user (cures life threatening or not) 2. Intimacy with product (do you inject/consume/or just use ‘at a distance’) 3. May be the only source. Recombinant insulin: Insulin is used medically to treat patients with Type 1 diabetes because the hormone is no longer produced internally. Over 40% of patients with Type 2 diabetes also require insulin as part of their diabetes management plan. Recombinant Chymosin: Bovine chymosin is an acid protease used to produce milk solids for cheese manufacture

Recombinant proteases, lipases, amylases for ‘biological’ cleaning agents

Lecture 4 Protein Structure and Folding in Health and Disease The Building blocks - The peptide bond is planar - Has a certain degree of freedom, defined by the two angles which gives a secondary structure

Amino acid sequence, structure and function: - AA crucial for structure and function are conserved across species - Sequence alignment of homologues shows highly conserved regions - Clusters of conserved aa residues are called motifs The alpha helix - The carbonyl oxygen of residue ‘i’ forms a hydrogen bond with the amide residue of ‘i+4’ - Individual hydrogen bonds are relatively weak in isolation, the sum is stable - The propensity for a peptide to form a helix depends on its sequence The beta sheet - In a beta sheet, the carbonyl oxygens and amides form hydrogen bonds - Can be parallel or antiparallel - Sidechains are directed above and below the backbone Ramachandran Plot - Is a way to visualize dihedral angles ψ against φ of amino acid residues in protein structure. - Ramachandran recognized that many combinations of angles in a polypeptide chain are forbidden because of steric collisions between atoms. Tertiary structure: - The overall fold that a polypeptide chain adopts is call its topology

Protein folding

Intrinsically disordered proteins (IDPs): An intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure - Can gain structure on binding - Few hydrophobic residues and a high proportion of polar and charged residues Protein misfolding: Sometimes the lowest delta G is not the native state What happens if a protein cannot find the correct fold? - Genetic mutations can alter the process - Alternative conformations can be formed mediated by intermediates - Alternate forms c...