Isolationand Purification of Alkaline Phosphatase
Purpose:Toisolate, quantitate and measure the enzyme parameters of E. coliAlkaline Phosphatase using common biochemistry laboratory techniques.
Proteinis not just a line of a nutritional information label. Most of theimportant components like medicines and other biotechnology products.Proteins contain a large number of distinct molecules. Some of theseproteins are enzymes that are useful in other metabolic processes inthe body (Devlin, 2011). Most proteins are naturally expresseddifferently and as such needs purification. There are five mainimportances of protein purification. Purification enables conductionof detailed studies on the function of these proteins anddetermination of their structures. Purification is the initial stepin the industrial production of pharmaceuticals and other products.Lastly, the process enables generation of antibodies anddetermination of amino acid sequence.
Thereare different methods of protein purification. Each of these steps isbased on the unique characteristics of these proteins (Scopes, 2013).The various properties for purification of proteins includesolubility, isoelectric point, binding to small molecules andsize/shape. While the techniques include salting out, ion exchangechromatography, affinity chromatography, and size-exclusionchromatography among others. The protein to be isolated and purifiedin this practical was Alkaline Phosphatase, which is a hydrolaseenzyme.
InE. coli, the enzyme is found in the periplasmic space and can bereleased from the location with a technique that weakens the cellwall (McComb, Bowers & Posen, 2013). The separation techniquethat was used is the SDS-PAGE. This technique. The techniqueseparates proteins based on their molecular weight. The proteins areseparated based on differential rates of migration through a gelunder the influence of an electric field (Scopes, 2013). Broadwayassay was used to quantitate the separated Alkaline Phosphatase. Thetechnique is dependent on the composition of amino acids on themeasured protein.
AlkalinePhosphatase Isolation Day 1
20mlof E.Coli-K12suspension was placed in a labelled 50ml polycarbonate centrifugetube and warmed with hands for five minutes. 0.2ml of lysozyme wasadden and gently swirled before incubation for 40 minutes. Next,0.2ml of DNAse was added and mixed followed by addition of 0.2ml 1MMgS04.The contents were then incubated for 20minutes followed by additionof 0.2ml of EDTA. The contents were again mixed through swirling.During the incubation, an appropriate length of dialysis tubing wascut and soaked in a beaker containing ddH2O.Next, the contents were centrifuged at 12000g for 20minutes. Thesupernatant was decanted to a fresh tube and the volume measured.1.0ml was removed for enzyme assay and protein estimation, andlabeled. All the excess Water was drained from the dialysis tubing.The remaining enzyme was transferred into the dialysis bag using aPasteur pipette. A beaker was then labelled appropriately after whichthe stage 1 enzyme in the bag was placed in the beaker and placedinside the refrigerator.
AlkalinePhosphatase Isolation Day 2
Thedialyzed Stage 1 enzyme was, first, retrieved and decanted into a50ml polycarbonate centrifuge tube. The volume was measured and tubeplaced into an 800Cwater bath for 15minutes. The tube was rapidly cooled by running coldtap water. The contents were centrifuged at 10000rpm for 15minutes.An appropriate length of dialysis tubing was cut and soaked in abeaker having excess ddH20.Stage 2 enzyme was then decanted into a 50ml polycarbonate centrifugetube, and the volume measured followed by removal of 1.0ml for enzymeassay and estimation. Next, 0.603g of ammonium sulfate ml-1was weighed and added while gently stirring until all the crystalsdissolved. This was followed by incubation for 15minutes. Thecontents were then centrifuged at 18000rpm for 30 minutes. Thesupernatant was the discarded, and the pellet re-suspended by adding10ml of Tris- HCl buffer. Excess water was drained from the dialysis,and a pair of knots tied in the dialysis tubing at one end. ThePasteur pipette was used to transfer the enzyme into the dialysis bagand a pair of knots tied to seal the other end of the dialysistubing. The bag was placed in the beaker of dialysis nuffer andplaced inside the refrigerator.
AlkalinePhosphatase Isolation Day 3
Thedialyzed stage 3 enzyme was, first, retrieved and decanted into 50mlpolycarbonate centrifuge tube, and measured before removing 0.6ml forenzyme assay and protein quantification. 20 3×100ml test tubes wereprepared and marked at 1.0ml level. Excess buffer above the packingmatrix was drained, and stage 3 enzyme added evenly to the column,and topped with 1ml buffer A. 5ml of buffer A was added and 1.0mlfraction was collected and 1ml of buffer B added. The process wasrepeated five times. The different fractions containing AlkalinePhosphatase were identified through spot test. The fractionscontaining high alkaline phosphatase activity were pooled togetherinto 1 tube, gently mixed, and the volume measured. The stage 4enzyme was covered and saved in the freezer for gel electrophoresisanalysis.
ProteinGel Electrophoresis of Alkaline Phosphatase
Assembling the Glass cassette and Casting Stand Assembly: The casting frame was placed upright with pressure cams in the open position and facing forward on a flat surface. A short plat was placed on top of a spacer plate, and the plate oriented so that the labeling is up. Two sandwiched glass plates were slide into the casting frame.
Casting the resolving gel: 5 µl of 25% Ammonium persulfate and TEMED were mixed well. 6.0µl of the mixture was then quickly poured between the glass plates up to the top part. A spacer comb was inserted between the spacers and allowed to polymerize for 10minutes.
The electrophoresis module was then assembled.
Next, the samples were loaded and the mini tank assembly.
Power was first applied to the mini tank to start the process at 200 volts. After electrophoresis was complete the mini tank lid was removed and carefully lift out the Inner Chambers Assembly. The gel was removed by gently separating the glass plates.
Staining the gel: The gel was placed in an old pipettor tip box and filled ½ full with Coomassie Blue Stain. The staining solution was then poured off and washed with water once before placing it in de-stain solution.
Theabsorbance of each of the samples were rea and the results tabledagainst concentrations. The average absorbance was calculated andcorrected as shown below.
Figure1. Tableshowing actual values of volume and concentration used of BSA toobtain corresponding absorbance at 595nm.
Thecorrected absorbance from previous table was plotted againstconcentration and gradient calculated as shown below.
Figure2. Protein quantification determined using BSA standard. Plot ofAbsorbance 595nm vs BSA concentration (ug/ml) to obtain trendline(y=mx, where y=Absorbance, m=slope (0.002), and x=concentration).
Figure3. Protein concentration determined using BSA standard. A) Tableshowing actual protein concentration for each stage, volume, andtotal protein concentration. C) Protein quantification of each Stageobtained by averaging corresponding absorbance, subtracting baselinevalues, and dividing it by slope (from trendlineconcentration=Absorbance/slope). B) Graph showing proteinconcentration of each stage. C) Graph showing total proteinconcentration in each stage of isolation.
Next,the isolated enzymes were then purified through SDS-PAGE, and theresults obtained are as shown below.
Figure4. Separation of AP through SDS-PAGE and calculation of MW via Rf. A)Picture of SDS-PAGE showing MW Ladder, proteins in each Stage ofIsolation, and Comm AP as control. B) Table showing values of MW, LogMW, distance traveled, and Rf. C) Plot of Log MW vs Rf to obtaintrendline. D) Calculation of MW of AP using trendline (y=mx +b).
Theisolation of alkaline phosphatase was possible through EDTA-lysozymetreatment. It was relatively easy to isolate substantial amount ofamount of crude alkaline phosphatase because of two main factors. Theenzyme biosynthesis is induced through in-situ phosphate starvation.Also, the enzyme is a periplasmic space enzyme that is readilyreleased from the cell due to cold-water shock, with minimalcontamination through cytoplasmic proteins. However, dilapidationthrough organic solvents reduced the protein concentration of crudeperiplasmic space proteins by almost 50%. Although the enzyme wasremoved from the aqueous phase, the enzyme was quantitativelyrecovered. It is possible that the organic solvents removed denaturedproteins and other fragments for the membrane of cells.
Themain limitation of the practical was the multiple operationsinvolved. The resuspension and re-incubation of the samples duringisolation was very involving and tiring. Some of the possible sourcesof mistakes include contamination and poor reading of measurements.The possible improvement from this is to conduct the procedure in acontrolled setting with minimal interference.
Thepurification of the isolated alkaline phosphate was done bysubjecting the samples to a separation technique that uses sodiumdodecyl sulfate, which is an anionic detergent that interferes withnearly all non-covalent interactions in the native protein (Burgess &Deutscher, 2009). The anions of SDS were bound to the main chains,and the complex formed a net negative charge. The negative chargebinds highly compared to the charge on the native protein. However,the charge is rendered insignificant. When the complex is subjectedto electrophoresis, the gel could be visualized by staining withCoomassie blue to identify the alkaline phosphatase (Janson, 2012).The mass could be obtained since the mobility of any protein in thegel is contrariwise comparative to the logarithm of their mass.
InSDS-PAGE, the proteins/enzymes are purified based on the density ofcharge while maintaining their biological activity. Differentproteins have different molecular weights which influence the rate atwhich they migrate through the sieving matrix facilitated by electriccurrent. The movement of the charged proteins through the sieve isaffected by the net charge, the magnitude of the current applied andthe molecular radius of the protein. However, for proteins that arenegatively folded, neither their molecular radius nor their netcharge is dependent on molecular weight. Their net charge is onlydetermined by the composition of amino acids. Therefore, in theirnative state, various proteins having similar molecular weight mayhave different speeds in the same electrical field. Therefore, thereis a need for destroying the tertiary structure of proteins so as toseparate them based on molecular weight alone. This is where SDScomes into play. The SDS was added in the experiment to destroy thetertiary structure and reduce the proteins into linear molecules andmask the intrinsic charge on the proteins.
TheSDS not only serves the purpose of destroying the tertiary structure,since it also covers the protein with an overall net (-) chargemasking the R-Groups charges. It also serves the purpose of ensuringthat the after linearizing the proteins and masking the net charges,they remain unchanged. The prominent feature in determining theSDS-coated protein is its molecular radius. The coated proteins areusually eighteen Angstroms wide and have lengths that areproportional to their molecular weights.
Proteinscoated with SDS moved to the positive anode in an electrical field atdifferent rates based on molecular weight. The rates of mobility wereexaggerated or amplified because of the high-friction environment inthe gel matrix. The polyacrylamide gel was selected because it ischemically inert. Additionally, the gel can easily be made indifferent compositions. This feature is helpful in producingdifferent sizes of pore for use in producing different conditions ofseparation. The TEMED was added in the experiment to help ininitiating the polymerization reaction of polyacrylamide by inducingthe formation of free radicals from ammonium persulphate. The freeradicals transferred electrons to the acrylamide, in turn,radicalizing the acrylamide and making it react with each other toform a chain.
Inthe experiment, the buffer was used to enable electrical conductionbetween the anode and the cathode. Discontinuous buffer system wasused in this case where the buffer that was suspended in the gel wasdifferent from the one in the holding tank. The continuous system wasmade up of a stacked gel at pH 6.8 and buffered with Tris-HCl, anelectrode buffer at pH 8.3 and a gel buffered at pH 8.8. When theelectric current was applied, the negatively charged glycine in theelectrode buffer (pH 8.3) were forced into the stacking gel (pH 6.8).The pH change made glycine switch to neutrally charged state. Theloss of charge reduces the rate of movement. The chloride ions,however, moved faster and formed an ion front. The separationdeveloped a narrow region having a steeper power gradient pulling theglycine along.
Theproteins contained in the gel have a moderate electrophoreticmobility between the mobility of glycine and chloride. Therefore, atthe time the fronts were sweeping through the sample, theconcentration of proteins in the narrow zone was increasing betweenchloride ion front and glycine front. The procession continued up tothe time it hit the running gel. In the running gel, the pH changedto 8.8. The molecules of glycine front accelerated past the proteinsresulting in proteins being dumped in a thin band at the loading andrunning gel interface. Because the running gel had a high acrylamideconcentration, it slowed down the movement of proteins based on size.It is here that the separation began.
TheSDS-Page that was used is sensitive, rapid and has a high degree ofresolutions (Scopes, 2013). However, there are still other techniquesthat can revolutionize the process. One such technique is isoelectricfocusing and 2D electrophoresis.
Thelab setting should have minimal activities to reduce incidences ofcontamination and interference. While conducting the practical, allthe aseptic techniques should be practiced to ensure that all sourcesof contamination of the samples are reduced. Lastly, further testingshould be done to confirm the purity of the final alkalinephosphatase.
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Harold,W. W. F., Handschumacher, M., Murthy, H. K., & Janusz, M. S.(2009). The three dimensional structure of alkaline phosphatase fromE. coli.Advancesin enzymology and related areas of molecular biology, 55,453.
Janson,J. C. (Ed.). (2012). Proteinpurification: principles, high resolution methods, andapplications (Vol.151). John Wiley & Sons.
McComb,R. B., Bowers Jr, G. N., & Posen, S. (2013). Alkalinephosphatase.Springer Science & Business Media.
Scopes,R. K. (2013). Proteinpurification: principles and practice.Springer Science & Business Media.