Flashcards
BIOC222: Mod4
Cards in this deck (100)
- 1Why may we purify a protein?So we can study its function & ascertain 3D structure, or the protein may have medical or industrial significance
- 2What is an example of a protein thats had its function identified by solving structure?hABCA1 transporter (cholesterol homeostasis). 3D structure shows a tunnel in the transmembrane region & a gate in the extracellular region
- 3How can understanding protein structure help us understand disease?Allows mechanistic determination for many disease-causing mutations
- 4What are the steps of protein purification? What temp is this best done at?1. Cell source
2. Cell disruption
3. Debris removal
4. Initial purification
5. Hi-res purification
Best done at 4C as this stabilises proteins & lowers protease activity - 5What are some potential sources for a protein to be purified?Cells, tissues
- 6How can you disrupt the cells to free up the protein?Chemically or enzymatically
- 7After cell disruption, how may the debris be removed?Centrifugation, filtration
- 8What are some ways of initially purifying the protein post-debris removal?Solvents, salts, heat denaturation
- 9What are some high resolution protein purification methods?Ion exchange chromatography, electrophoresis, affinity chromatography
- 10Why are assays excellent methods to track the protein prior to purification?Because we often don't know what the protein we want to purify is, rather just its activity
- 11What final purities correspond to what uses for the purified protein?Low purity: industrial use
<95%: antibody production & sequencing
95-99%: research (structure, function)
>99%: therapeutic use - 12What buffers are best for protein purification? Why?Ones that are >1pH unit away from the protein's PI, as proteins are least soluble at their PI.
- 13What other components may be added to a buffer to stabilise the protein in solution?Salts, cofactors, BSA or glycerol (stabiliser), reducing agents to reduce thiols, protease inhibitors
- 14Modern protein purification methods tend to use ______ as a source for the proteinRecombinant cells
- 15What eukaryotic & prokaryotic cells may be used as recombinant organisms for protein purification?Eukaryotic: Saccharomyces cerevisiae, mammalian, insect
Prokaryotic: E. coli, Bacillus subtilus - 16After disruption, it is ALWAYS necessary to what?Centrifuge at 20,000g for 20 minutes
- 17What are some common properties that are manipulated to purify the protein? What method corresponds with each?Solubility (salting out)
Charge (ion exch chrom., electrophoresis)
Polarity (hydrophobic interaction chrom.)
Size (SDS-Page, gel filtration chrom.)
Binding specificity (affinity chrom.) - 18What is the principle behind salting out a protein?When adding a salt to a protein soln, solubility increased to a supersaturated soln, where solubility peaks. From here, more salt = lower solubility, thus protein precipitates out of solution.
- 19Does the salt used when salting out affect the outcome?Yes, different salts carry different properties. These are outlined by in the Hofmeister series
- 20What salt properties make it a good salt for salting out? Thus, what salt is widely used?Strongly hydrated anions & weakly hydrated cations, kosmotropic, increases surface tension, non-polar substances dec. solubility. Ammonium sulphate
- 21Outline the molecular interactions that lead to salting outUpon addition of excess salt, water is removed from hydrophobic patches on protein (usually is forced to interact, becoming ordered). Patches can now interact with patches on other proteins = precipitation. Larger hydrophobic surface = better at salting out
- 22Understanding the solubility of your protein allows you to do what when salting out? How does this work?Salt out contaminant proteins before the protein of interest. If you know the salt conc. at which your protein will precipitate, any precipitation before this is contamination
- 23What is the basic principle behind all liquid chromatography methods?Column contains stationary phase of hydrophilic stable beads under high pressure. Liquid runs over them, interactions recorded
- 24What makes ion exchange chromatography a powerful separation technique?There are 2 resin types: positively & negatively charged
- 25What are the 5 phases of ion exchange chromatography?1. Initial stage
2. Adsorption of target
3. Starting of elution
4. End of elution
5. Regeneration - 26During ion exchange chromatography, we start with ____ salt and move to ____ saltLow, high
- 27Describe hydrophobic interaction chromatographySame principle as salting out. Beads have hydrophobic groups that interact with hydrophobic groups on proteins that are exposed under high salt concentrations
- 28What is the basis of affinity chromatography?Contains a stationary phase (beads) that have an affinity for the target protein
- 29For affinity chromatography, what 2 steps are essential for achieving purified protein?Washing - removes unbound protein from beads
Elution - removes target protein through the use of a competing substrate or disruption of interactions - 30Explain how His tags work in affinity chromatographyThe His residues interact with the free electrons in a metal ion (usually cobalt or nickel). From here, the protein can be purified by lowering the pH or adding imidazole
- 31Describe size exclusion chromatography & what it tells usA column containing beads with small holes in them. Small molecules can enter beads, large molecules can't. Thus, small molecules have a longer path to the bottom of the column than large molecules, creating a separation
- 32What is the basis behind Western Blots?Proteins are transferred from gel to membrane, which is treated with antibodies that bind the protein of interest, allowing them to become visible. A way of visualising only your protein of interest
- 33How are the antibodies used in a Western blot often labelled?Fluorescently
- 34What is the basis behind mass spectrometry?Ionises chemicals & sorts them based on mass:charge ratio, allowing determination of mass of components in a sample
- 35Describe in detail how proteins are analysed using a mass spectrometerProteins made into an aerosol by passing them through a capillary at high voltage or bombarding with a laser. A mass detector is used to ascertain mass through time of flight (smaller proteins fly longer than large proteins)
- 36How can a mass spectrometer be used to sequence a protein?Tandem mass spectrometry using electrospray ionisation (ESI) on protein digest fragments. Fragments become ionised (peptide ions), allowing m/z values to be ascertained, and thus the protein may be sequenced
- 37The two common methods used to validate the correct protein has been purified are:mass spectrometry & western blots
- 38Discuss the effectiveness of using natural sources for protein purificationLower in abundance, contamination likely, animals often need to be slaughtered.
- 39Discuss the effectiveness of using recombinant sources for protein purificationWidely used. No seasonal fluctuations, optimised yields, controlled properties
- 40What cells are used the most for recombinant protein production?Prokaryotic, notably E. coli
- 41Discuss how a gene of interest (insert) is prepared for insertion into a cloning vectorIt is amplified many times through PCR before ligation
- 42How is a cloning vector inserted into the host cell?Cells are treated in a way that makes them competent (calcium chloride in cold), and they become transformed with cloning vector
- 43What characteristics should a vector insert have?- Selectable marker
- Defined copy number
- Method of induction
- Tag removal
- May include promoter - 44Why is the pET recombinant system widely used?Uses the lacI repressor encoded by E. coli & has the promoter that lacI repressor acts on. Thus, the system is inducible through the addition of IPTG (mimicry of lactose)
- 45What is a widely used method for purifying expressed recombinant proteins?The use of tags for affinity chromatography purifications
- 46Once the protein is purified, how is the tag dealt with?Cleaved by proteases. It is important the protease site is specific enough that it isn't present in the protein of interest, & thus the protease won't cleave the protein of interest
- 47How may codons present an issue for recombinant expression? Offer the solutionSome cells use a codon to produce a particular a.a. more than others. e.g. Humans use one codon to prod. 40% of one a.a., but E. coli only uses it 5%. Solution is to use enhanced strains that utilise certain codons
- 48How can prokaryotic proteases be problematic in recombinant systems? What genes are strains used for this engineered to be without?Lon & OmpT (both genes for proteases that cleave eukaryotic proteins)
- 49How can protein aggregation present an issue for recombinant expression? Offer the solutionMay aggregate in an undesirable way upon expression, making puriifcation difficult. Solutions include re-engineering the protein, or altering expression conditions
- 50Why must eukaryotic cells be used to produce some recombinant proteins?To achieve the proper PTMs
- 51Discuss different eukaryotic cell types used in recombinant expression?Yeast: Secrete proteins. High yield (15g/L), proper PTMs, slow growing.
Insect: Close resemblance to mammalian cells
Human: Expensive, low yield (<1mg/L), generally cancer cell lines - 52What are the 3 most common human cell lines used for recombinant expression?HeLa cells
HEK293 cells
3T3 cells - 53Describe HeLa cellsCervical cancer cells from Henrietta Lacks who died in 1951. Rapidly growing, active telomerase, HPV in genome, 82 chromosomes.
- 54Describe HEK 293 cellsHuman Embryonic Kidney cells. Made through transformation of normal human embryonic kidney cells with adenovirus 5 DNA
- 55Describe 3T3 cellsMouse embryonic fibroblast cells. Don't grow @ low densities. Multiple chromosome alterations
- 56What are some examples of therapeutic proteins produced via recombinant methods?Insulin, human growth hormone
- 57Why did recombinant production of HGH initially prove a challenge?It contains 2x disulphide bond PTMs not synthesized in prokaryotic systems
- 58Before mammalian recombination was used to product HGH, how was it produced? What was this product called?Produced in prokaryotic system, refolded post-production. Has methionine on N-terminus (natural GH has this cleaved) & may refold incorrectly, thus may culminate in reaction. Known as somatrem
- 59Laundry detergents usually contain what?Enzymes
- 60How are the enzymes in laundry detergents altered?They are optimised in cold water & high pH environments
- 61What is the most common laundry detergent enzyme?Subtilisin (protease)
- 62Subtilisin is a ____ proteaseserine
- 63What is the name of the bond cleaved by serine proteases?Scissile bond
- 64Why is subtilisin an appropriate enzyme to have in industrial products like laundry detergents?It is non-specific, thus will cleave many types of proteins
- 65What are some common problems presented by enzymes for industrial use? What is the solution?Readily denatured, specific activites, low activities. Solution is site-directed mutagenesis
- 66What properties has site-directed mutagenesis introduced into laundry detergent enzymes?Increased efficiency, enhanced thermal & pH stability, cofactor requirement removed, decreased substrate specificity, improved protease resistance
- 67What is an example of a residue in original subtilisin that required replacement for it to be included in detergents? What needed to be understood before this could happen?Met222, as it is readily oxidised by oxidising agents (commonly found in detergents), rendering the enzyme useless. However, the structure-function relationship between Met222 & the whole protein needed to be understood before it could be replaced
- 68What fact about Met222 in subtilisin means that any alterations deem the enzyme inactive? What residue was used to replace Met222?It is directly adjacent to the catalytic serine. Ala222 used to replace Met222, as this provided greatest stability under oxidising conditions
- 69What is the function of lipases? What fold involved in catalysis is often conserved?Break TAGs into DAGs + FFAs, i.e. increasing solubility of lipids. Alpha-beta hydrolase fold often conserved
- 70What method of catalysis do many lipases use? Describe itInterfacial catalysis. Involves a buried pocket of hydrophilic residues & associated water molecules. Allows lipase to be in soln with substrate + solvent without clumping together due to hydrophobic interactions
- 71What proved to be the first challenge for mass production of lipases? Why?Mass production, as lipases are generally low copy-number enzymes
- 72What organism's lipase has been extensively engineered for mass production in detergents?H. lanuginosa lipase
- 73What residue substitution & where has allowed a decrease in the specificity of H. lanuginosa lipase?Trp in lid
- 74What part of H. lanuginosa lipase was targeted & modified in order to decrease susceptibility to proteases?Structural loops
- 75Why was subtilisin an ideal target for protein engineering?Standard Michaelis-Menten kinetics, highly expressed, easy to assay, known structure
- 76Describe the details of starchBranched (1,4 + 1,6) glucose polymer used to store glucose in plants & other crops. 15-30% amylose & 70-85% amylopectin. Bonds stable at high pH, but hydrolyse at low pH
- 77What are the 3 steps of starch degradation?1. Liquefaction
2. Saccharification
3. Isomerisation - 78Describe the liquefaction of starchAt pH 6 95-105C w/ steam, alpha amylase, & calcium starch is broken into linear oligosaccharides & dextrins to decrease viscosity
- 79Describe the saccharification of starchAfter liquefaction, the dextrins & linear oligosaccharides are degraded into a high conc glucose syrup by the hydrolysis of 1,4 & 1,6 bonds via exo-acting glucoamylase. Product is 95% glucose
- 80Describe the isomerisation of starchGlucose isomerised into fructose - isomer over twice as sweet. Done using immobilised glucose isomerase (GI).
- 81Describe the difficulties presented during the isomerisation of starchGI is expensive as it is an intracellular enzyme. It's difficult to obtain >40% fructose as it's unstable with a high Km for glucose
- 82What enzymatic characteristic allows certain bacteria to grow on PET plastics as their carbon source?Presence of aromatic polyesterases (PETase) with a conserved lipase/chitinase-like fold, conferring an ability to degrade PET plastics
- 83How can PETase have its efficiency improved?Simple structural modifications through protein engineering, such as the substitution of rate or efficiency-limiting residues
- 84Describe the difference between protein engineering & directed evolutionProtein Engineering: development of an ideal protein design followed by site-directed mutagenesis to achieve this structure
Directed Evolution: random mutagenesis followed by screening & selection of desired phenotypes - 85What are some difficulties when considering new drug development? What decisions must be made?Extremely high costs & very low success rates (<2% show suitable biological activity). Factors such as need for the drug, competition, & commercial opportunity must be considered prior to development
- 86Describe some potential drug targetsUsually proteins (enzyme inhibitors, blocking interactions or signals), but also often nucleic acids
- 87Why must drug targets be validated? What is a common validification method?To show they are critical in the disease process. Gene knockout to test targets is common
- 88Drugs must be extremely _____ to the target with no off-target _____Specific, interactions
- 89Regarding drug discovery, what is a lead compound?A compound from a series of related compounds that shows desirable biological activity. N.B. this is NOT a drug candidate
- 90Once you have a lead compound (drug discovery), what needs to be considered?- Potential toxicity
- How easy to synthesize?
- How soluble (application methods)
- Resemblance to competition - 91Define rational drug designThe design of drugs based off a structural & functional understanding of the target prior to design
- 92What disease resulted in one of the first rational drug/therapy designs?HIV/AIDS
- 93What 3 proteins does HIV encode?Reverse transcriptase, integrase, protease all synthesized as a single polypeptide. These are drug targets
- 94How was it validated that protease was essential for HIV proliferation?Pepstatin addition rendered HIV non-infective, as did the mutation of the catalytic Asp residue
- 95Pepsin & HIV protease are both ______Aspartic proteases
- 96What allows the array of protonation states seen in aspartic proteases such as pepsin & HIV protease?Aspartic acid residues with different pKa values
- 97Explain the importance of Asp residues to the mechanism of pepsinAsp32 and Asp215 bind a water molecule causing it to become deprotonated. Asp32 receives proton whilst Asp215 donates proton to substrate int. Protonation of Td intermediate causes it to break down into products
- 98How should the structure of an HIV protease inhibitor relate to the HIV protease substrate?It should be based off the peptide bond substrate, more specifically it should be a transition state analogue
- 99What is the catalytic active site sequence of Aspartic proteases?Asp-Thr-Gly
- 100Outline all the criteria for a peptidomimetic drug1. Structurally resembles peptide bond/transition state
2. Replaces amide bond
3. Minimises molecular weight
4. Enhances binding
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- Flashcards on BIOC222: Mod4, created by Guy Turner on 23/07/2018.
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