Protein Analysis Module PDF

PROTEIN ANALYSIS MODULE PA week 3 description: ★ MurA protein purification using AKTA FPLC ★ size exclusion chromatography ★ Bradford assay to determine a protein concentration PA week 4 description: ★ SDS-PAGE – Coomassie stained gel (analyzing MurA protein purification) ★ Bradford...

PROTEIN ANALYSIS MODULE PA week 3 description: ★ MurA protein purification using AKTA FPLC ★ size exclusion chromatography ★ Bradford assay to determine a protein concentration PA week 4 description: ★ SDS-PAGE – Coomassie stained gel (analyzing MurA protein purification) ★ Bradford assay MurA protein purification using AKTA FPLC objective: purify His(6x)-MurA protein using Ni-NTA affinity chromatography His(6x) tag has high affinity for Nickel, which will be immobilized on resin in the column after cell lysis, the lysate will pass through the Ni-NTA resin and allow the His(6x)-MurA protein to bind due to the interaction between the His tag and Ni the resulting flowthrough has unwanted proteins wash buffer applied, flowthrough contains unwanted proteins and contaminants buffer with imidazole is applied, which competes with the His tag for binding to Ni, allowing the protein to be eluted and collected WORKFLOW: Mura protein expression and purification 1. monitor O.D. (600 nm) of uninduced cells (E. coli cells transformed with pCA24N-His(6x)-MurA plasmid) – sample removed for uninduced cell sample a. to ensure cells are in optimal phase (log) for protein induction 2. once at desired O.D. (0.4-0.5), induce cells with IPTG a. IPTG triggers expression of His(6x)-MurA protein by activating plasmid 3. induced cells harvested and centrifuged – sample removed for induced cell sample a. separates pellet (cells) and supernatant (liquid) 4. cell pellet lysed with BugBuster reagent, centrifuged, then passed through 0.45 µm syringe filter a. BugBuster releases cell contents into solution, centrifuged to remove cell debris 5. Ni-NTA column connected to peristaltic pump, washed with equilibration buffer, lysate applied to column – sample removed for lysate sample a. lysate filtered to remove large debris b. equilibration buffer stabilizes Ni-NTA resin before adding lysate (corrects binding state) c. protein binds to resin due to His-tag and Ni ion affinity 6. direction of flow from buffer tube to top of column => elution  a. unwanted proteins washed out 7. connect Ni-NTA column to AKTA FPLC. two buffers are already attached: a. buffer A (50 mM Tris pH 7.5, 100 mM NaCl): equilibration buffer to stabilize column b. buffer B (50 mM Tris pH 7.5, 100 mM NaCl, 250 mM imidazole): elution buffer that competes with His tag, allowing MurA to be eluted 8. elute MurA protein size exclusion chromatography mobile phase: buffer that encompasses solvent and mixture of molecules requiring separation stationary phase: form of matrix (resin) through which mobile phase travels column/resin bed: mass/volume of resin/beads within a column void volume: volume of space between beads exclusion limit: upper limit of a bead/resin type; size above which proteins will elute in the void volume of the column types of chromatography size exclusion chromatography: separates mixture of molecules by size 1. the stationary phase contains microscopic beads in a column 2. a mixture of molecules in a mobile phase is applied to the top of the column, such that the larger molecules elute first 3. the small molecules enter the tiny holes in the beads and travel slowly down the column, then slowly out the beads affinity chromatography: isolates and purifies target protein by introducing a stationary phase with a resin coupled to a molecule (like antibody) that binds to the target protein ion exchange chromatography: separating proteins based on charge, utilizing a positively or negatively charged resin hemoglobin (MW: 65,000 Da): protein in RBC that is made up of 4 polypeptides vitamin B12 (MW 1350 Da): vitamin that breaks down fats WORKFLOW: separation of hemoglobin and vitamin B12 1. drain column till 2 mm buffer remains on top of column bed 2. protein mixture added (hemoglobin and vitamin B12) a. large hemoglobin molecules elute faster b. vitamin B12 enters the pores and travels through the column more slowly Bradford assay Beer-Lambert Law: linear relationship between absorbance and concentration breaks down when solution becomes non-ideal (high conc of chromophore) or when chemical processes occur Bovine Serum Albumin (BSA) is used to create a standard curve for protein quantification WORKFLOW: preparing a Bradford assay 1. prepare serial dilutions of BSA. 2. add dye reagent a. binding changes solution from brown to blue b. intensity of colour proportional to protein present 3. measure absorbance at 595 nm using a spectrophotometer. 4. plot absorbance against BSA concentration to create a standard curve. 5. measure the absorbance of test protein samples and use the standard curve to determine their protein concentration. SDS-PAGE 10 µL lysate + 10 µL 2X-SDS loading buffer ○ after cell lysis (BugBuster) ○ includes all proteins and contaminants ○ visualize wide-range of bands, MurA band seen ○ no pure MurA 10 µL flow through + 10 µL 2X-SDS loading buffer ○ passed through chromatography column ○ His(6x)-MurA protein has affinity for Nickel in resin, binds ○ proteins and contaminants in flow through ○ visualize wide-range of bands, no MurA band 10 µL wash + 10 µL 2X-SDS loading buffer ○ contaminants and other proteins still in column are washed ○ visualize wide-range of bands, likely less than flow through, still no MurA band 10 µL elution sample + 10 µL 2X-SDS loading buffer ○ elution sample contains higher concentration of imidazole that competes with His(6x) to bind with Nickel ○ His(6x)-MurA eluted from column ○ visualize MurA band (~45 kDa) CELL BASED ASSAY MODULE CBA week 1 description: ★ use growth curve to investigate effects of a gene knockout on E. coli bacterial cell growth at various pHs ★ use growth curve to investigate effects of increasing antibiotic concentrations on cell growth (endpoint measurement) CBA week 2 description: ★ investigate MurA protein overexpression to overcome fosfomycin inhibition bacterial growth curves E. coli grows and divides via binary fission ○ cell grows twice its size, then divides in half to produce 2 identical daughter cells ○ yields new generation every 20-30 minutes bacteria inoculated into liquid medium have 4 phases of growth: ○ lag phase: cells adjust to new environ, synthesize enzymes and materials ○ log phase: rapid chromosome replication, growth, reproduction; high metabolic rate and protein production ○ stationary phase: rate of reproduction decreases as nutrients are depleted and waste accumulates ○ death phase: cells die at faster rate than produced OD measured to determine light scattered by bacterial cells suspended in LB; turbidity reflects conc of cells antibiotic dose response assay cell-based assay output is endpoint measurement of cell growth in plate: ○ varying concentrations of same drug minimum inhibitory concentration: dose of drug the cell ceases to grow  specifically ampicillin ○ different drugs or library of compounds at same high conc determine new chemicals that have inhibitory potential strain 1 (WT – E. coli K12 cells): susceptible to ampicillin ○ determine what conc of Amp cell is inhibited ○ measure OD to determine turbidity strain 2 (WT-AmpR – E. coli K12 cells transformed with pUC19 containing amp resistance gene… beta-lactamase) ○ cells with resistance gene wil transcribe beta-lactamase ○ beta-lactamase inactivates ampicillin A ○ however, high conc of antibiotic, AmpR protein will not be enough to inhibit cell growth ○ therefore, takes a higher conc of amp to inhibit cell growth WORKFLOW: bacterial growth curve of WT and ∆mdtM E. coli cells at diff pHs 1. 2 LB-agar plates prepared with either WT cells or ∆mdtM cells overnight 2. dilute ∆mdtM cell culture in fresh LB at pH outlined… 2 tubes a. take OD and alternate between tubes b. leave lid loose so cells can grow 3. add antibiotic to well (ampicillin), along with cell culture (based on dilutions) MurA overexpression dose response assay determine the MIC of two strains of E. coli ○ strain 1: E. coli BL21 cells (WT) ○ strain 2: E. coli BL21 cells transformed w pCA24N-His(6x)-murA plasmid measure cell growth at increasing conc of fosfomycin and ampicillin (which does not inhibit MurA) controls in a high-throughput screening experiment high control: high signal due to growing cells ○ set up: cells, allow to grow without inhibitors, high OD low control: low signal due to lack of cell growth ○ set up: introduce inhibitor that can inhibit cell growth to result in low OD ○ added at a concentration that is two or three times the documented MIC MdtM paper  what is the purpose of kanamycin in our LB media for E. coli K12 ∆mdtM (containing genomic deletion of mdtM gene) cells? ○ mdtM protein is an efflux pump and antiporter that maintains the internal pH of the cell under alkaline conditions maintain acidic internal environment when external environment is basic (pH 9) ○ ∆mdtM (knockout strain): lacks mdtM gene add kanamycin to LB so that the cells can select for the plasmid that provides mdtM and kanamycin resistance basically adding back the mdtM gene to be replicated and expressed bacteria that did not take up the plasmid will be killed by kanamycin ○ WT: has mdtM gene do not need kanamycin bc they already have mdtM Fosfomycin Resistance in E. Coli paper objective: determine if overexpression of chromosomal genes in E. coli can confer resistance to antibiotic Fosfomycin findings: MurA overexpression resulted in clinical-level resistance ○ fitness cost lower Fosfomycin resistance ○ commonly acquired through reduced drug uptake, active efflux, target alterations, plasmid-encoded resistance chemical screening using ASKA library ○ only MurA was depicted as the chromosomal gene able to counter Fos resistance at a low fitness cost high throughput screening - assay type phenotypic-based drug discovery: grow cells in culture, measure growth ○ CBA target-based drug discovery: purify enzyme/develop assay to detect enzyme activity in presence/absence of potential inhibitor 1. You are purifying a His-tagged protein using a Ni-NTA column. After washing the column with a buffer containing 20 mM imidazole, you notice that the elution fraction has a faint band of  the target protein along with multiple contaminating bands. What could be the reason, and how would you optimize the washing step? to optimize the wash step, increase the concentration of imidazole in the wash buffer to ensure that it binds strongly and removes contaminants before eluting the target protein 2. During an AKTA FPLC run, you observe a sudden increase in absorbance at 280 nm after applying the sample to the Ni-NTA column. However, the peak diminishes before the elution step. What does this suggest about the interaction between your protein and the column, and what adjustments could you make to retain the protein on the column? an absorbance increase before elution could be because of weak protein binding to the column and slightly washing off during the loading or washing steps to retain the protein on the column, reduce the imidazole concentration in the binding buffer to strengthen the interaction between the His-tagged protein and the resin 3. You are using a Ni-NTA column to purify a protein with a His-tag. You notice that after the elution with a high imidazole concentration (300 mM), a significant amount of protein remains bound to the column. Suggest two potential reasons for this issue and how you would resolve it. 1. the protein remains bound to the column after elution with a high imidazole concentration because the protein’s His-tag might be buried, reducing its binding affinity 2. the protein remains bound to the column because the concentration of imidazole might not be sufficient to displace the strongly bound protein to resolve this issue and elute the rest of the protein, use a higher imidazole concentration or use EDTA to chelate to the Ni ions to release the protein 4. While performing a Ni-NTA affinity chromatography for MurA protein purification, you notice that the flow-through fraction contains a significant amount of your target protein. Describe two steps you could take to increase the binding efficiency of your His-tagged protein to the Ni-NTA resin. 1. to improve binding efficiency in the flow-through, lower the imidazole concentration in the binding buffer to reduce competition 2. increase the incubation time with the Ni-NTA resin to allow better binding of the protein 5. You have conducted a protein purification using the AKTA FPLC system with a Ni-NTA column. The chromatogram shows two distinct peaks during the elution step: one at 50 mM imidazole and another at 250 mM imidazole. What could be the reason for observing two peaks, and how would you confirm the identity of each peak? the first peak at 50 mM imidazole could be weakly bound contaminants  the second peak at 250 mM is the target His-tagged protein these samples from both peaks can be tested using SDS-PAGE to verify the identity of the proteins 6. During your AKTA FPLC run, you notice an increase in backpressure when applying the sample to the Ni-NTA column. What could be causing this increase in pressure, and how would you troubleshoot it? the pressure in the column could be because of high sample viscosity due to high protein concentration or DNA contamination to troubleshoot the high pressure, pre-filter the sample before loading or dilute the sample to reduce viscosity 7. You are eluting a His-tagged protein from a Ni-NTA column using an imidazole gradient (10 mM to 300 mM). If your target protein does not elute even at the highest imidazole concentration, what alternative elution strategy could you try, and why? if the target protein does not elute at the highest imidazole concentration, the pH of the elution buffer should lower to reduce the affinity of the His-tag for Ni ions 8. After purifying a protein using affinity chromatography, you perform SDS-PAGE and Western blot analysis. The Coomassie-stained gel shows a single band at 25 kDa, but the Western blot reveals an additional band at 50 kDa. Provide two possible explanations for this discrepancy. the additional 50 kDa band on the western blot could be because of non-specific antibody binding to a similarly sized contaminant protein 9. You are testing the efficacy of an enzyme inhibitor using a cell-based assay with *E. coli*. The assay involves measuring the growth rate of *E. coli* in the presence of different inhibitor concentrations. After adding the inhibitor, you observe an initial decrease in optical density (OD600) followed by a steady increase. What might be causing this pattern, and how would you interpret the results? the initial decrease in OD600 indicates inhibition of growth by the inhibitor the subsequent increase could be due to bacteria developing resistance or the inhibitor was reversible 10. You notice a faint band corresponding to your target protein in both the wash and elution fractions during Ni-NTA purification. How would you adjust the imidazole concentration in the wash buffer to improve the purity of the elution fraction?  to improve the purity of the elution fraction, the concentration of imidazole should be increased to better wash away weakly bound contaminants, ensuring a cleaner elution fraction 11. In a purification run using AKTA FPLC, you find that the protein elutes at a lower imidazole concentration than expected. What are two potential reasons for this, and how could you verify the cause experimentally? if the protein is eluting at a lower imidazole concentration, it may be because the His-tag is cleaved and has lower affinity for the Ni-NTA resin buffer conditions like low pH or the presence of chelating agents might reduce binding strength you could verify this using SDS-PAGE to determine any degradation or analyze the buffer for contaminants that could affect binding MdtM paper key findings MdtM Role in pH Homeostasis: MdtM contributes to E. coli's tolerance to alkaline pH, specifically between pH 9 and 10 It functions as a Na+(K+)/H+ antiporter that helps maintain a stable, acidic cytoplasmic pH relative to the external environment knockout strain generation: ΔmdtM knockout strain was created utilizing gene deletion methods from the Keio collection the ∆mdtM knockout strain was created by removing the mdtM gene and replacing it with a kanamycin resistance gene (if taken in = survives kanamycin) no mdtM gene, but has kanamycin resistance ∆mdtM knockout strain main finding: the ΔmdtM strain struggled to grow at high pH levels - cannot handle alkaline environments as well without the mdtM gene ∆mdtM strain generation: wanted to determine if adding mdtM back would help used a plasmid to test if adding it back helps the bacteria (plasmid contains mdtM gene AND carbenicillin resistance gene) carbenicillin resistance gene purpose was to see if the bacterium took in the plasmid (if taken in = survives in carbenicillin) growth in LB: [kanamycin] OR [kanamycin and carbenicillin] added to growth medium normal growth medium for WT [kanamycin] ensures knockout strain is growing (no mdtM)  [kanamycin and carbenicillin] ensures bacteria that picked up the plasmid are growing (new mdtM gene) phenotypes in liquid culture at different pHs: at pH 8.5, both wild-type and ΔmdtM strains had similar growth growth differences became significant at pH ≥ 9.0; wild-type strains grew better than the knockout strain no growth was observed at pH 10 in either strain. monovalent cation requirement: MdtM gene has ions that aids in keeping internal pH stable, allowing it to survive at high pHs without these cations, ΔmdtM cells could not grow effectively at high pH MurA Overexpression and Fosfomycin Resistance: overexpressing MurA, an enzyme involved in peptidoglycan biosynthesis, increased resistance to fosfomycin, an antibiotic targeting MurA