Purification of Hp Urease by Ion-Exchange Chromatography PDF

Purification of Hp Urease by Ion-Exchange Chromatography  ⚫LECTURE OBJECTIVES -Separate urease by ion-exchange (IEX) chromatography with Mono Q Sepharose anion exchanger -Analyze column fractions to qualitatively determine -protein concentration A280 -urease activ...

Purification of Hp Urease by Ion-Exchange Chromatography  ⚫LECTURE OBJECTIVES -Separate urease by ion-exchange (IEX) chromatography with Mono Q Sepharose anion exchanger -Analyze column fractions to qualitatively determine -protein concentration A280 -urease activity via PRUDA -Pool and concentrate the purified material to quantitatively determine -protein concentration A280 -Prepare an SDS-PAGE sample -indophenol activity test on pooled IEX fractions -Manuscript Writing SEMESTER-LONG PROJECT OUTLINE to Develop an Anti-ulcer personalized drug Wk1 - Good Laboratory Practice (GLP) Industry regulation Wk2 - Get a sample (biopsy or brush) of stomach tissue or fluid Microbiomics - Identify H. pylori: culture in lab in a special Brucella agar - Clone H. pylori urease into E. coli SE5000 Wk2 Genomics - Genotype E. coli SE5000 for H. pylori urease Wk3 Wk4 - Isolate urease from E. coli SE5000 using cell lysis & centrifugation - Purify urease protein (GFC & IEX) Wk5 Proteomics - Characterize urease protein (SDS-PAGE, immunoblotting) Wk6 Wk7 Wk8 - Perform enzyme kinetics Wk9 Enzyme Kinetics - Urease inhibition & statistical data analysis to find significance Drug Screening Wk10 - Computational modeling/docking/TBD Bioinformatics Wk11 - Good Manufacturing Practice (GMP) Industry regulation - Report findings (write assignment)Wk12 Dissemination  WHERE WE ARE NOW? IEX B Where are we now? Our protein is in retentate along with an unknown amount of other unwanted solute molecules. What do we need? (i) a way of quantifying the amount of desired protein (specific, sensitive, convenient). GFC Pool (ii) a way of systematically removing the remaining unwanted solutes. For part (i): There are several approaches: retentate filtrate (a) Spectroscopy: use electromagnetic radiations (b) enzyme assay: use substrate that will specifically bind to the enzyme to produce the product GF Pool (conc.) 5 ml  ⚫Chromatography: Ion Exchange - principle Separates ions and polar molecules based on their affinity to the ion exchanger Charged molecules bind to oppositely charged groups that are chemically attached to the matrix. Revision Anion exchange: binds to anions on the matrix Diethylaminoethyl (DEAE): Matrix—CH2–CH2–NH(CH2CH3)2+ Acidic gp on protein - ---O--- Cellulose or cation exchanger Agarose Diethylaminoethyl (DEAE) group (protonated form) Cation exchange: binds to cations on the matrix Carboxymethyl (CM): Basic gp Matrix—CH2–COO– - on protein Cellulose or Agarose Carboxymethyl (CM) group (ionized form)  Chromatography: Ion Exchange - procedure The binding affinity of a particular protein depends on: - the presence of the other ions competing with the protein for binding to the exchanger and - the pH of the solution, which influences the net charge of the protein. (i) Proteins to be separated are dissolved in a buffer of appropriate pH & salt concentration. Revision (ii) Any ions that bind tightly can be eluted using an eluant gradient buffer that has -a higher salt conc. (ionic strength) gradient or -a pH gradient at which the protein has reduced charge Surface net charge Protein titration curves, showing how net surface charge varies with pH Figure 5-6  Chromatography: Ion Exchange - Elution [NaCl] CV = column volume (ml) https://www.sigmaaldrich.com/technical-documents/  Chromatography: Ion Exchange - Elution Anion Exchanger + + 5-10 CV + - - + 5-10 - CV + [NaCl] 10-20 CV 5-10 CV https://www.sigmaaldrich.com/technical-documents/ Chromatography: Ion Exchange - Elution [NaCl] 10-20 CV 5-10 CV [NaCl] 5-10 CV [NaCl] https://www.sigmaaldrich.com/technical-documents/ ⚫ Purification of HP Urease by Ion Exchange Chromatography Class Experiments for Today Step 1: Preparative IEX separation of Hp urease via gravity filtration, collect fractions Step 2: PRUDA colorimetric urease assay to measure enzyme activity of each fraction Qualitative Step 3: Measuring protein concentration at A280 nm Step 4: Pool and concentrate active fractions as IEX pool Step 5: Measuring protein concentration of IEX pool at A280 nm Step 6: Preparing a sample for SDS-PAGE analysis Quantitative Step 7: Phenol-Hypochlorite urease assay of IEX pool to calculate enzyme specific activity Step 8: Graph protein conc (A280) and urease activity vs. IEX fraction number https://www.youtube.com/watch?v=pxVysix7wIc  Step 1: Preparative IEX separation of Hp urease via gravity filtration A-Preparation of gel and column packing amount estimated Column contains Mono Q Sepharose hydrate 4h, by hydrated bed RT in HCl -crosslinked 6% agarose beads w quaternary ammonium vol. (Table) or NaOH ours 30ml -45-165 µm (particle size), 90μm (average dimeter) -Preswollen in 20% ethanol decant half of supernatant remove fines by suction -allows constant fluid flow -even when the fluid level changes degas soln for 5-10 min affix a funnel on column pour even slurry, Acidic gp top, close column exit avoid trapping on urease air bubbles - 6% CH3 Agarose Quaternary ammonium gp (protonated form) Store at 4-30°C (20% ethanol) -glass fibers Pack a 2.5x5cm column @2mL/min Manufacture catalog: Bio-rad  Step 1: Preparative IEX separation of Hp urease via gravity filtration sample Equilibration injection gradient wash re-equiblibration 1-Equilibration volume elution regeneration -In IEX binding buffer (20mM NaPO4, pH 6.9, 1mM EDTA, 1mM ß-ME). GF Pool 5ml 2-Sample application -Load the 5ml of GFC pool sample by the drained-bed method -Use 2 column vol. of binding buffer to elute unbound proteins bed volume 3-Elution (Fraction Collection) 30ml (surface) -Employ a gravity gradient generator of 100 mL binding buffer + 100 mL elution buffer (20mM NaPO4, pH 6.9, 1mM EDTA, 1mM DTT, Unclamp Clamp Sample Sample Apply Unclamp; Add 1M NaCl) to develop the column at a flow rate of 1.5 mL/min drain buffer apply running drained to buffer drain buffer more mm to surface sample in surface; close To surface buffer clamp -Plug the eluent tubing into the automatic fraction collector -Collect at least 45, 4 mL fractions -Store all fractions on ice 45 tubes, 4ml each 4-Regeneration 4oC -Complicated -Usually, includes the use of strong acid & caustic agents https://www.youtube.com/watch?v=q3fMqgT1do8&feature=youtu.be  Step 1: Preparative IEX separation of Hp urease via gravity filtration GRAVITY (Conc.) GRADIENT GENERATOR Column chromatography Gel preparation Microfluidics RSC Adv., 2017, 7, 29966-29984 https://doi.org/10.1002/0471142727.mb1010s44  Step 2: Measuring protein concentration at A280 Fraction Number A280 1 2 3 -0.00184 0.00003 0.02052 SAMPLE DATA 1 4 5 0.02843 0.02549 0.1 6 0.02673 7 0.01819 8 0.00670 9 10 0.00686 0.00375 0.08 11 0.00111 12 0.00018 13 14 -0.00013 -0.00028 0.06 15 -0.00091 16 17 18 -0.00137 -0.00091 0.00298 A280 0.04 19 0.01291 20 0.02626 21 0.03697 22 23 0.04675 0.04039 0.02 24 0.03138 25 0.05451 26 0.06864 27 0.08680 0 28 0.07796 29 0.08991 30 0.08541 31 32 0.06787 0.05824 -0.02 33 34 0.05840 0.04737 0 10 20 30 40 50 35 36 0.03433 0.02331 Fraction Number 37 0.02005 38 39 0.01897 0.02269 -A280 values vs fraction number for the fractions yielded from the ion 40 0.03092 41 42 0.03759 0.04179 exchange chromatography column. 43 0.02362 44 45 0.01291 0.00810 Please practice plotting these values in Excel Biochem Mol Biol Educ. 2015 46 0.00437 Step 3: Phenol Red Urease Detection Assay (PRUDA): procedure 4oC 96-well plate map 2/11 1. Pipet 10 μL of +ve control (urease) in column 2/11 2. Pipet 10 μL of -ve control (gel mobile buffer) in column 2/11 3. Pipet 10 μL of -ve control (testing buffer; 3mM NaH2PO4, 10mM urea, 7 g/mL phenol red, pH 6.8) in column 12 4. Pipet 10 μL of protein fractions in columns 3 to 10 testing 5. Pipet 150 μL of testing buffer to all the wells with a p200 multichannel pipette. buffer 6. Cover the plate with film/lid and incubate at 37o C for 10 min 150 μL 7. Read results on the ePoch at 595 (595-630) nm. Note: -Pipet 2X up & down to mix, do not introduce ANY bubbles  Step 3: Phenol Red Urease Detection Assay (PRUDA) Fraction Number A595 21.00054041 21.99858631 0.819 1.307 SAMPLE DATA 2 96-well plate 23.00174707 1.155 1 2 3 4 5 6 7 8 9 10 11 12 23.99944592 1.348 25.00098079 1.807 2.0 13 14 15 16 17 18 19 20 21 22 23 24 25 1.5 A595 1.0 0.5 0.0 0 21 22 23 24 25 Fraction Number -A595 values vs fraction number for the fractions yielded from the IEX column Please practice plotting these values in Excel Biochem Mol Biol Educ. 2015  Graph A280 and urease activity vs. fraction number Superimpose SAMPLE DATA 1 & 2 filtration 0.1 Ultrafilteration were 0.08 ve various proteins ased upon their sizes. 0.06 e absorbance for A280 0.04 his conveys thatGradient Salinity red (0mM NaCl), orange (0.1M NaCl). black (2M NaCl) of the column 0.02 ame off in large ive fractions of ~5mL 0 wn in table 1, the 0 10 20 30 40 50 -0.02 ned many impurities Fraction Number eins that are relatively produced a tenfold-A280 values Figureversus fraction 2: A280 values for number for the the fractions fractions yielded yielded from the ion exchange in IEX chromatography. p to 0.077 U/mg) in chromatography column The box highlights fraction(s) 21-25 that tested positive for urease. The of urease within the dotted line indicated the salinity gradient; red indicates 0M NaCl, Biochem Mol Biol Educ. 2015  ⚫Step 4: Pool and concentrate active fractions 45 tubes, 4ml each Ultrafiltration (concentrating) 1. Do not pour >12 mL of pool into a labeled Amicon filter cup with mol. wt. cut-off (MWCO) of 100,000 Da. 4oC 2. Place the capped filter into the JA-18 rotor of the centrifuge; IEX counterbalance with another sample, Pool retentate & spin at 5000xg for 15 min. filtrate When using a fixed angle rotor, orient the urease +ve fractions device with the membrane facing up 5 (21-25) tubes, 4ml each “filtrate” flows through the pores, “retentate” is impeded 3. Discard the filtrate/retentate? Pooling 1. Pool all fractions 4. Repeat steps 1 to 3 until the vol. of 0.65ml with urease activity 4oC IEX pool is around 1mL into a 50 mL centrifuge tube. 20 mL IEX Pool IEX Pool (conc.)  Step 4: Pool and concentrate active fractions A B C D Manufacture catalog: Bio-rad  Step 5: Measuring protein concentration at A280 Total Protein Determination and Sample Yield 1. Determine absorbance at A280 of IEX retentate. 2. Use BSA protein std curve to calculate the conc. (mg/mL or µg/µl) & total protein (mg). Sample vol. to be measured (e.g. test sample + buffer = 100 μL) must be the same as the vol. of protein std. used (e.g., 100 μL) for accuracy. IEX Pool SAMPLE DATA Std. Curve for determining Relative3Mol. wt Protein conc. (µg/μl) Abs. 0.6 0.0 0.00 0.5 0.4 0.13 0.4 y = 0.3421x + 0.0029 A280 0.8 0.29 R² = 0.9989 0.3 1.2 0.42 0.2 1.6 0.55 0.1 2.0 0.68 0 IEX Pool 0 0.5 1 1.5 2 0.342 Protein (µg/µl) Please practice plotting https://www.youtube.com/watch?v=g0IB1Jd79CE these values in Excel  ⚫Step 6: Preparing a small sample for analysis by SDS-PAGE From now on, prepare and store a sample for later SDS-PAGE analysis. 1. In a 1.5 mL eppendorf tube, pipet 50 g of protein 2. Add the same volume of 2X SDS-PAGE sample buffer. 3. Boil the sample at 100oC for 5 min in the heat block. File:SDS-PAGE sample.png 4. Store the tube at -20oC Protein (50 g) + 2X SDS-PAGE sample buffer File:SDS-PAGE sample.png + 100oC for 5 min -20oC SDS-PAGE IEX Pool buffer  Step 7: Phenol-hypochlorite urease assay of IEX pool 1. Set up 6 polystyrene cuvettes in a rack, label as 15, 30, 45, 60, 90 and B (for 1.2 mL assay buffer + blank). 10 µl E + 2. Measure exactly 1.25 mL of reagent A into each cuvette. Revision 1.Add 1.2 mL of urease assay buffer to a 1.5 mL Eppi. 200 μL 2.Add 10 µl E (or your purification product) to the Eppi tube 3.Start reaction (& your timer), close cap & mix quickly by inverting 3X 15 30 45 60 90 B 4. At 15 sec add 200 μL from Eppi tube into the cuvette labeled 15 to denature E 5. Mix by pipetting up & down exactly 3 times (every time) 6. Immediately add 1.25 mL of reagent B to the cuvette. 7. Cover & mix by 3X inversion 1.25 mL Reagent A+ 0.2ml assay buffer (w E)+ 8. Add all reagents but E to the cuvette labeled “B” as your blank. 1.25 mL Reagent B 9. Transfer (in a rack) the reaction cuvettes to the 56oC waterbath for 6 min. 10. Read absorbance (O.D.637) after blanking the spec with the “B” cuvette. O.D.637 1. Repeat steps from 4 to 10 - until you have added reaction to the 90 sec cuvette. 56oC, 6 min 2. Record or print the data.  Step 8: Specific activity calculation of IEX pool SAMPLE DATA 4 Assay buffer=4.959ml (50 mM HEPES pH 7.5, 50 mM urea) Specific activity of GF purified, concentrated sample Enzyme=0.041ml Sample: 0.006 mL of 1.0 mg/mL protein (0.006mg/6µl) +0.74 ml (6µl E) Blank: 0.006 mL of buffer (no protein) 0 sec (∆Asample - ∆Ablank) x assay volume (in L) x 106 15 sec SAurease = Reag “A”=4.63ml ε x ∆time(min) x protein (mg) used in the assay Reag “B”=4.63ml Sample Blank Assay volume: 10mL 10mL 1 mL 1 mL 1 mL 1 mL ∆ Time (min): 0 15s 0 15s A637: 0.00 1.605 0.00 0.10 -wild-type H. pylori UMAB41 (100 U/mg) ∆ Abs: 1.605 0.10 Better to use cuvettes for Sp.A determination  Step 9: SEMESTER-LONG PURIFICATION DATA SAMPLE DATA - OVERALL Table 1: Purification table containing representative results, with activity of each fraction quantified using the phenol hypochlorite/indophenol assay. Cell lysate Specific activity = Total activity (µmol/min) Total protein (mg) Fold purification = Specific activity at particular step Specific activity of the initial step Yield (%) = Total activity at particular step x 100 Total activity of the initial step HOW TO REPORT RESEARCH FINDINGS? https://www.technologynetworks.com/ Different modes of disseminating information https://www.technologynetworks.com/ Typical Research Article Structure Literature review Problem (H.pylori infection) What has been done to solve the problem? (anti- ulcer, antibiotics) What is missing/gap? (no treatment for resistant specie) How can you fill the gap? (by offering personalized medicines, against resistant species) What is the impact of your proposed strategy? (Going to help people suffering from the resistant species) https://www.technologynetworks.com/ Typical Research Article Structure https://www.technologynetworks.com/ 6 Steps for tackling your first scientific paper Common misconceptions of first-time scientific authors Myth #1: A paper should be written intro-to-conclusion Myth #2: Results should be written in the order that they were obtained  Six Major Steps for Writing a Research Paper 1- Assemble your FIGURES, and write legends 2- Use your figures to write a concise RESULTS section 3- Start compiling your MATERIALS/METHODS with enough detail that someone could replicate your study 4- Interpret (but don’t repeat!) your results in the DISCUSSION, by addressing: a. How does my study support what others in the field have shown? b. How does my study challenge previous findings? c. What are the study’s main conclusions and how do they advance the field? d. What are the most important future directions? 5- Now that you’ve crafted your story and identified the major findings, set the stage for the reader in the INTRODUCTION 6- Finally, craft an ABSTRACT that briefly summarizes the paper’s highlights Hypothesis – objective – rationale --- 3 sample manuscripts  Bibliography Be sure to REFERENCE work done by others! Cite as much as you reasonably can Cite the original sources, rather than a review (or a source that cites the original source) Use a reference management program like Reference manager, Endnote or Mendeley -A free online reference manager software -Developed by Elsevier -create free account at www.mendeley.com -Add an extension or plug-in tool on google chrome toolbar How MENDELEY Works? amtul z How HowMENDELEY MENDELEYWorks? Works?  ⚫How MENDELEY Works? Bibliography:  ⚫How MENDELEY Works? Bibliography: ⚫ Windsor University: Writing Support Desk Video Tutorials https://www.uwindsor.ca/success/647/video-tutorials  Role of Co-Authors Co-author: -any person who has made a significant contribution to an article -share responsibility and accountability for the results -if more than one authors, choose one person to be the corresponding author https://www.nature.com/articles/ BIOC8790: PEER ASSESSMENT (5%) few of the reasons why disputes -Power dynamics occur b/w researchers -Competing priorities collaborating on -Personality differences projects/papers RUBRICS https://www.nature.com/articles/  MMB Lab Today: SDS-PAGE 1.Fill the inner buffer chamber with 1X electrophoresis running buffer (1X Tris-Glycine SDS (TGS), pH 8.8). 2.Carefully pull out the comb without tearing the edges of the wells. 3. Shoot buffer into the wells with a Pasteur pipette to clear any debris. 4. Fill both the upper & lower buffer chambers with 1X electrophoresis buffer. The lower buffer chamber takes about 200 mL. -Don’t overfill the lower chamber -makes electrophoresis -ve +ve precede more slowly. 1.Mix samples by pipetting (absolutely introducing no bubbles) 2.Spin down in the benchtop microfuge. 3.Load each sample using the special elongated gel loading tips 4.Place the cover on the chamber & insert the electrical leads into the power supply with the proper polarity black to black & red to red 1.Run the gel at 200 V (constant voltage). File:SDS-PAGE sample.png 2.When the yellow std. marker band (31,600 Da) is 1/2 way down the gel, stop the run by turning off the power supply. 3.The total run time should be 35-60 min. 31,600 Da http://en.wikipedia.org/wiki/File:SDS-PAGE_Electrophoresis.png  MMB Lab Today: SDS-PAGE 1. Remove the plates & dump the buffers contained in the gel box down the sink. 2. Disassemble the plates from the cell. 3. Pry the plates apart with the black/green wedge. 4. The gel sticks to the tall glass spacer plate. 5. Using the wedge (now a scraper) discard the stacking gel layer in regular trash. 6. Use the green wedge (now a knife) to cut the gel b/w lanes 5 & 6. 7. With wetted gloves, slowly peel the resolving gel off the plate into two separate Tupperware containers. Coomassie stained half Immunoblotting half http://en.wikipedia.org/wiki/File:SDS-PAGE_Electrophoresis.png  MMB Lab Today: SDS-PAGE 1. Follow the staining/destaining directions for Coomassie Bio- safe stain (Bio-rad). ddH2O, 3X, 5min =--SDS C. stain C. stain, 50ml, 1h 2. Take a photo of the Coomassie stained gel against a white light and save in your computer. Alternatively: -Microwave Coomassie stain with gel for 5 sec. -To ensure that the stain covers the gel evenly, repeat this procedure again -then incubate the gel with the stain for 10 minutes on the shaker. -Put the staining solution back in the tube at the conclusion of the 10 min -Incubate the gel in de-staining or stain-removing solution (~ 20 mL) for 10 min. -Repeat this three times June 17 - 21 reading week  Class & lab log (c&l-log) 1- Read the “6 steps for tackling your first scientific paper” pdf, and answer the below question: -At what step or in which section of a manuscript, as an author, you are supposed to set the stage for the readers? 2-Make a bibliography using Mendeley: Go on PubMed C/L part..n2 (https://pubmed.ncbi.nlm.nih.gov), type Amtul Z or any author(s) of your choice, copy and paste the title of my any five manuscripts in a Word file. Next, Jun 28th make a Mendeley library containing my those 5 manuscripts. Import and cite my manuscripts from Mendeley to the above Word file at their respective places. Insert the bibliography at the end of the Word file. Format the bibliography, according to the “Journal of Biological Chemistry” style. Upload the individual exercise on Bs with your participation. ASSESSMENTS DUE THIS WEEKend! - Lab report 4 – Jun 24, Mon 8 AM