Isolation of Recombinant Escherichia Essay Example for Free

closing off of Recombinant Escherichia Essay one technique big in both genetics and biochemistry is the Polymerase Chain Reaction (PCR), freshman developed in the 1960s, and and so automated in 1983. Current PCR technology was not developed until the discovery of thermostable polymerases, specific in ally Thermus aquaticus (Taq) polymerase (1). The protein Taq polymerase was first free from the extremum thermophile T. aqauticus, where extreme thermopiles are bacteria that live in temperatures at or above 45C. The Taq enzyme is a member of the DNA polymerase I family (2, 3). The interesting property of Taq polymerase is that it has a temperature optimum at 74-75C, allowing it the remain active in temperatures required for PCR double stranded DNA denaturation (3, 1) . The protein has an approximate molecular(a) heaviness of 6263 kDa when isolated from T. aquaticus, and 94 kDa when isolated from recombinant Escherichia coli, and is still stable at temperatures of 93-95C, henc e the thermostability of the enzyme ). Taq specifically lacks any proofreading act in the 3 to 5 direction, and thitherfore has a relatively high hallucination rate of single base mispairings of 1 error perIsolation of Recombinant Taq Polymerase for PCR 9000 nucleotides, as s intimately up as a frame shift error rate of 1 per 41,000 basepairs (5, 6). Taq polymerase has an activity that is highly dependent on the environment of which it is in as it is thermostable, and has differing activities at nearly all temperatures up to the point of denaturation. Taq specifically can check up to ascorbic acid0 base pairs in length on a template in under one act under typical PCR conditions. The enzyme has a specific activity of cc,000 units mg-1, and can add around form 60 nucleotides per second at 70C (7). The isolation of Taq is essential for the PCR reaction. The most important reason for Taq being used in PCR is the thermostability at high temperatures (95C). This allowed for the pro cess of elongation, annealing, and denaturation to occur without the replacement of new enzyme, and thereby, was more cost-effective, faster, and cheaper because the reaction could be automated finished the use of a machine known as a thermocycler which basically is just a machine able to change temperatures of an isolated environment quick (7).Prior to the discovery of Taq, PCR was through with(p) development Klenow fragments of E. coli DNA polymerase I at 37C. The lack of thermostability required replenishment of enzyme after each PCR cycle (8). One of the initial difficulties of Taq polymerase was the organism in which it was expressed in, T. aquaticus, as it was difficult to culture and produce immense quantities of enzyme. E. coli bacteria were engineered to expressed the Taq polymerase gene to allow for retrieval of large quantities of enzyme ). The isolation of the Taq gene involved culturing T. aquaticus and whencece(prenominal) isolated the DNA of the cadres done lysing, proteinase K addition, extracting of aqueous and phenolic phases, dialyzing of extractions, addition of SDS, and then centrifugation of resolve to at last retreieve the DNA of the organism as outlined in Lawyer et al., 1989. With the isolation of the 2401+ BP gene of Taq, the gene was incorporated into a 6.58 kbp plasmid (pLSG1). The gene was inserted 171 bp distal to the lacZ friend/operator, and 109 bp distal to the BgII site, so the gene expression could be controlled by means of an inducible promoter.With the pLSG1 plasmid, the vector was introduced to E. coli bacteria to allow for plasmid uptake (4). Other experiments shoot been conducted towards the purification of Taq from recombinant E. coli. specifically En jellyke et al., 1990 developed a method for purfication of Taq. The E.coli strain 2 DH1 was used for the expression of the recombinant plasmid containing Taq polymerase. The bacteria were grown in 12 Litre batches of Luria Broth using 1 mL of saturated DH1 culture and 80g/mL of ampicillin. Isopropyl-1-thio--Dgalactopyranoside (IPTG) was added to 0.5mM and the cultures were grown for 16-20 hours. The cells were harvested in 2.4 L of buffer A (50 mM TrisHCL, pH 7.9, 50 mM dextrose, 1mM EDTA) and collected via centrifugation, resuspended in Buffer A with 4mg/mL lysozyme and incubated at room temperature for 15 minutes. Buffer B (10 mM TrisHCl, pH 7.9, 50mM KCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Tween 20, 0.5% NP-40) was added and incubated in 180 mL fractions, for 60 minutes at 75C in a water bath.The desegregatetures were centrifuged at 8000 rpm for 15 minutes at 4C. Taq then precipitated with polyethyleneimine (PEI) at room temperature, then isolated through centrifugation and suspended in buffer C (20mM HEPES, pH 7.9, 1 mM EDTA, 0.5mM PMSF, 0.5% Tween 20, 0.5% NP-40) containing 0.25 M KCL. PEI eluatents were diluted in 50mM KCL and buffer C and applied to a one hundred fiftymL BioRex 70 ion exchanger column, and then eluated using 200mM KCL. The protein was dialyzed for 12 hours against two changes of 1 L memory board buffer (20mM HEPES, pH 7.9, 100 mM KCL, 0.1 mM EDTA, 0.5 mM PMSF, 1mM dithiothreitol, 50% glycerol.The experiment resulted in 40-50 mg of protein per litre of cell culture (9). The methods used in this experiment differed in certain key aspects. First, Engelkes experiment made use of a higher concentration of ampicillin. The IPTG was added to the equal concentration, but was added after cell growth up to an optical density of 0.700. Instead of a water bath at 75C, this experiment made use of an air incubator for the temperature requirements. Engelkes experiment made use of PEI to precipitate Taq, while this experiment made use of 30g of (NH4)2SO4 per 100mL of supernatant. Buffer C was not used throughout this experiment, and no ion exchange columns were used. The dialysis procedure was done for twice as long with twice as many changes of resultant per 6 hours. The chan ges made from Engelkes experiment offers a different method for protein precipitation. The method used by Engelke made use of PEI which is an affinity precipitation method versus a salt precipitation method.The PEIIsolation of Recombinant Taq Polymerase for PCR method has the major drawback through the lack of selectivity, and can often precipitate nucleic acids as well (10). This is why the BioRex column needed to be used. Ammonium sulfate has the good that the precipitation can be controlled based on ionic strength of species involved, as well as has no negative effects on the activity of the tar possess enzyme. Salting out also has the advantage that only inbred state proteins are precipitated due to the hydrophobicity involved with native state proteins (10). Buffer C was not required for this experiment as no BioRex column was required. This experiment made use of various techniques and methods including SDS-PAGE, differential centrifugation, western sandwich Blotting, real t ime-PCR (rtPCR), PCR, agarose gel electrophoresis, and dialysis. 2 important techniques were PCR and rt-PCR. PCR does not allow for the quantification of DNA amplicons as it is an end-point PCR, but it does allow for confirmation of template duplication along with measurement of base pair length. gain of primer would confirm the presence of a thermostable DNA polymerase.The following agarose electrophoresis helps to adjust amplicon coat which can tell us the activity of Taq, as well as the specificity, as one template should only return one bar in PCR (7). rt-PCR allows for a quantitative assessment of PCR, and therefore the kinetics of the reaction, as it detects the amount of amplicons produced in the reaction. The point at which the banal curve reaches threshold in cycle number gives information on the activity of Taq, as a more active sample distribution of Taq reaches threshold earlier.Melt curve analysis also provides information regards DNA amplicons in solution (11). T he purpose of this experiment was the tribulation the methods for the isolation of PCR grade Taq polymerase from recombinant E. coli using differential centrifugation, salting out, and heat denaturation following lysation of cells to potentially improve isolation of Taq from past methods. The presence of Taq will be sustain through Western blotting, and rt-PCR and PCR reactions along with purity will be assessed through SDS-PAGE. The activity of Taq will be shew through rt-PCR and PCR. Finding the most efficient method for the isolation of Taq offers a valuable reagent source for any PCR reactions required. The isolation technique would also be applicable to any thermostable proteins. 3EXPERIMENTAL PROECDURES Isolation of Taq Polymerase Luria broth (500 mL + 100g/mL ampicillin) was inoculated with 50 L of frozen Taq polymerase expressing E. coli cell stock. Incubation was commenced for 12 hours at 37C until the opthalmic Density had reached 0.700. IPTG (0.5 mM or 0.112g/L culture ) was added and the culture was incubated for 12 to 14 hours at 37C. The 50mL of cells were then centrifuged (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810, and 5 mL of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1mM EDTA) was used to suspend the separated pellet. The solution was then centrifuged again (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810 and the pellet was once again suspended in Buffer A, with an additional 20 mg of lysozyme added. The reaction was incubated for 15 minutes at room temperature.Following incubation, 5mL of buffer B (10 mM Tris HCl, pH 7.9, 50mM KCl, 0.5% Tween 20, 0.5% NP-40, 1mM PMSF, 1mM EDTA) was added and incubated at 75C for 1 hour in a New Brunswick scientific-Innova 40 incubator shaker series, and shaken by hand just about every 5 minutes. The solution was then centrifuged (15000 RPM x 10 minutes at 4C) in a Thermoscientific Sorvall RC 6+ centrifuge and using a 603s Delta Range 30g of (NH4)2SO4 per 100mL of supernatant (8 mL of supernatant identical to 2.4g (NH4)2SO4 ) was added and incubated for 10 minutes at room temperature and shaken on the Innova 40 incubator.The lysate was then centrifuged again (15000 RPM x 10 minutes at 4C) in Thermoscientific Sorvall RC 6+ centrifuge and the resultant pellet was suspended in 2mL of buffer A. The solution was then dialyzed in a Spectra/Por membrane tubing institute at 6000-8000 Da molecular cargo selection in 1 L of depot Buffer (50 mM Tris HCl, pH 7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5 mM PMSF, 50% glycerol) for 24 hours at 4C changing the buffer every three hours. The dialysis solution was then diluted in a 11 ratio of storage buffer and stored at -70C until needed. Protein Concentration Determination A Bovine serum Albumin Bio-Rad stoppage standard curve was hustling (0 0.3 mg/mL) usingIsolation of Recombinant Taq Polymerase for PCR a 1mg/mL stock solution and an Asys Expert Plus spectrophotometer doctor at 62 0 nm. Bio-Rad assay was run in triplicate using 20L of protein dilution and 150 L of diluted Bio-Rad Dye Concentrate. 10x and 100x dilutions of the sample lively beforehandly were made and 20L were used with 150L of diluted Bio-Rad Dye concentrate. The solutions were incubated for 10 minutes and absorbances were tabulated. sandwich was then assembled with an additional ice blocking in the transfer apparatus. The apparatus was run at 180mA overnight in a refrigerator and the membrane was then stored in TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and refrigerated. 24 hours prior to the primary antibody (Anti-Taq monoclonal Antibody (8C1)) addition, the membrane was blocked in 1 gram of Carnation nonfat dry milk (5% w/v) and 20 mL of TBST Buffer.The primary antibody in TBST with SDS-PAGE 5% w/v nonfat dry milk at a 1800 dilution of A noncontinuous polyacrylamide gel was antibody was added to the membrane and shaken brisk using a Mini-PROTEAN Tetra Cell for 1 hour at room temperature. The membrane was module. The casting stand was assembled for 1mm then washed three successive times for 15 minutes gel and filled with National Diagnostics 12% with TBST buffer at room temperature. The settlement Gel (Protogel 2400 L, Resolving Gel secondary antibody (Peroxidase-conjugated Buffer pH 8.8 1560 L, dH2O 1974 L, 30% w/v AffiniPure Goat Anti-Mouse IgG (H+L)) was then APS 21L, TEMED 6L), casted to 1 cm below top applied in TBST with 5% w/v nonfat dry milk at a of glass plate, and then 4% Stacking Gel (Protogel 12000 dilution of antibody and shaken for one hour 390 L, Stacking Gel Buffer pH 6.8 720 L, dH2O at room temperature.The membrane was then 1830 L, 30% w/v APS 6L, TEMED 3L) was washed three successive times for 15 minutes with casted on top with a ten well gel comb. The TBST buffer at room temperature. The sharpnesss of the electrode set up was then submersed in 1 x Trismembrane were dried with a Kim and next 1mL of Glycine SDS rascal Run ning Buffer. To each 20 L Renaissance Western Blot Kit NEN Life Sciences of sample, 20 L of Laemmli buffer (0.5 M TrisProducts, Cat. No. NEL hundred and one luminol reagent with HCl, pH 6.8, 4.4% w/v SDS, 20% v/v glycerol, 2% 1mL of oxidizing reagent were uniteed together and v/v 2-mercaptoethanol, 10 mg/mL bromophneol then applied to the membrane. The membrane was blue) was added and boiled for 3 minutes and then then imaged with an exposure time of 40 seconds cooled for 5 minutes on ice. To one well 7L of using AlphaEaseFC software. New England BioLabs Inc. Prestained Protein Marker (7-175 kDa) was added. To the following PCR/agarose gel electrophoresis wells 15 L of standard Taq polymerase was added, A master mix for PCR was prepared (1x PCR along with 20L of six different samples, with the buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, fifth being prepared the anterior year with the same 0.5M forward primer, 0.5m Reverse Primer, 0.1 method of isolation as outlined previously. The gel ng Template DNA and Nuclease-free PCR water) was run at 200 Volts for 40 minutes, incubated in and 22.5 L of master mix and 2.5L of Taq fixing solution overnight and then stained with Bio- sample, the standard, or the Taq prepared a previous Safe Coomassie Blue for one hour at room year were added to PCR tubes and centrifuged temperature under agitation.The gel was then shortly on a Fisher Scientific Accuspin micro 17 analyzed used AlphaEaseFC software. just briefly using 1.5mL eppendorf tubes with no caps to contain the PCR tube. The PCR tube was Western Blotting then added to T3 Biometra Thermocycler and Using the method expound above for SDSdenatured at 94C for 3 minutes and then 35 cycles PAGE, a SDS-PAGE gel was taken prior to fixing. of PCR with the denature 94C for 45 seconds, The gel was then transferred to transfer buffer anneal 55C for 30 seconds, and extension at 72C (20mM Tris-HCL, pH 8.0, 150 mM Glycine, 20% for 1.5 minutes. The sample was then incubated at Methan ol). Immobilon-P transfer membrane with 72C for 10 minutes and then temperature was 0.45 m pore size and Whatman paper were cut to maintained at 4C. The samples were then stored at the size of the gel. The membrane was wet with -20C until agarose gel preparation. A 1% agarose 100% methanol, then transferred to MilliQwater gel was prepared through 1.5g of agarose (Sigma and soaked for several minutes. A standard blotting No. A-6877 Type II) to 150mL of Tris-Acetate4Isolation of Recombinant Taq Polymerase for PCR EDTA (TAE) buffer. The solution was microwave for 1 minute and mixed until in solution. Once cooled to 60C, 7.5 L of Biotium Gel Red Nucleic acid stain was added and mixed. The solution was then poured into the electrophoresis tray a comb was installed, and set at room temperature. One Litre of 1x Tae buffer was prepared through dilution of 50x TAE buffer and then the solution was poured onto the electrophoresis tray to cover the gel in 1mm of buffer. 20 L of PCR product prep ared previously and 4L of Gel Red tinge were mixed and 20L of each sample, the standard, and Taq prepared the previous year and Invitrogen life Technologies 1 Kb DNA ladder Cat. No. 15615016 was run at 150 Volts, 100 mA for one hour (or until dye reached the bottom of the gel). The chevrons were then visualized under 300 nm light and fluorescence was measured at 590 nm.The gel was analyzed using AlphaEaseFC software. concentration of the sample Taq was 1.88 + 0.11 mg/mL. The solution of proteins was not pure Taq as substantiate by the SDS-PAGE (Fig. 2) as various proteins created distinct bands (B to K excluding E). The standard Taq revealed only one band (A), indicating band E was most in all probability belong to Taq, as it was the darkest band in the gel. An analysis of the molecular weights of the bands through electrophoretic mobility (Tab. 3) showed the standard Taq having a molecular weight of 115.2 + 14.6 kDa, and the likely band (E) had a molecular weight of 113.4 + 14. 3 kDa. in that respect was a distinct distortion in the bands of the SDS page in all lanes with the exception of the standard Taq and the 2011 Taq (Fig. 3). The distortion is of a smile. The overall gel also has a large distortion, but of a frown. It would appear there was a similar protein to D E and F wassail in all samples, including the 2011 sample. The standard Taq did not contain the bands. Real Time PCR The Western Blot (Fig. 4) revealed distinct A master mix for PCR was prepared (1x PCR bands however, there were more than one band in buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, each lane with the exception of the standard Taq. 0.5M forward primer, 0.5m Reverse Primer, 0.1 Two distinct bands were present in 5, Taq, and 2 (b, ng Template DNA and Nuclease-free PCR water). c).The lanes of * and ? contained several bands To PCR tubes, 22.5 L of Master Mix and 2.5 L also. The overall gel also expressed a slight color of Taq sample or the standard Taq were combined, banding along t he solvent front edge which is mixed through vortexing and then centrifuged with shown in both Fig. 3 and 4. The 2011 lane did not a Fisher Scientific Accuspin micro 17 just briefly appear to have any Taq present, as no band was using 1.5mL eppendorf tubes with no caps to distinguished. The entire ladder expressed some contain the PCR tube. The Taq samples were antibody activity. prepared in triplicates. 20L of each sampled were The real time-PCR revealed a threshold reached then transferred to a 96-well PCR plate and then at 20 cycles, with the vast majority occurring at 24 sealed. The well was then placed in a BioRad CFX cycles. The melt curve showed an approximate connect Real Time System using the scheduling of melting temperature of 81C (Fig. 7). enzyme activation (95C, 30 seconds, 1 cycle), 40 The agarose gel electrophoresis revealed one cycles of Denaturation (95C, 1 second) and distinct band at approximately 5883.5 base pairs in annealing/extension (60C, 5 seconds), with a m elt length.The brightest bands, and therefore the curve of (60-95C in 0.5C intervals, 3 seconds per highest quantities of Taq enzyme were found in the step, 1 cycle). The samples were then analyzed std., 2 and 4. When the base pairs specific activity using AlphaEase FC software. of the enzyme was calculated it was found to be 834.5 + 63.9 bp/min/g of sample, or 3922.3 + 192.9 bp per minute. RESULTS The results of the Bio-Rad assay on the sample of Taq polymerases diluted to 10x and 100x revealed that the 10x dilution was far to concentrated and fell outside the linear curve of the Bio-Rad assay. The retrieval of protein from the Luria broth was found to be 300.8 + 17.7 mg protein per L of Luria broth. These results (Tab. 1) suggest the protein 5 DISCUSSION Through the analysis made through SDS-PAGE, the MW of the standard Taq was found to be 115.2 + 14.6 kDa and 113.4 + 14.3 kDa. This is different from the accepted literature value of 94 kDa (9). Even with error correction, the prot ein did not fallIsolation of Recombinant Taq Polymerase for PCR within the range of the accepted literature value. In total, the two proteins differ by 23% and 21% without error correction, or 21.2 kDa and 19.4 kDa respectively. In comparison to one another, the two bands have essentially the same molecular weight, indicating whatever error occured in the gel was equivalent on both the standard and the isolated Taq. One explanation for the difference in the molecular weights may be explained through the quantitiy of protein used. The darkest and thickest band ( E, fig. 4) likely belongs to the Taq protein. To get a more defined band, a dilution would be effective in making a higher resolution band (12). The amount of protein isolated per loudness of Luria broth was determined to be 300.8 + 17.7 mg per L of Luria Broth. Quite obviosuly, there are issues both with the heating of the gel, and distortion of the bands into smiles. The distoration of the gel likely was caused by unbalan ced heating of the gel causing the center of the gel to be hotter than the peripheries, as the walls of the apparatus act as heat sinks (13).The uneven heating can be removed by switching to a lower voltage for a longer period of time (12). The distortion of the protein bands within the individual lanes produced a smile structure. The distortion was likely caused by either an overloading of proteins, which can be solved by dilution of the protein sample, or was due to salt conditions of the loading sample. This step could be fix through extra steps of dialysis to decrease salt content of the loading sample. (14). One final issue with the SDS-PAGE gel was the distance between bands. The target molecular weight was near 100 kDa, so the concentration of the gel could be decreased to allow for a higher resolution of the higher molecular weight proteins, or allowed to run for a longer period of time (14). A purity assessment of the isolated Taq enzyme can be made through the SDS-PAGE ge l (fig. 2). Distinct banding occurs in ten different bands on the Taq lane, with 9 being distinct from Taq protein (E).This highlights that there were infact multiple proteins still present in the Taq solution. This would fence that the heat shock portion of the methods was insufficient in denaturing all of the proteins in the E. coli, allowing for precipitation upon salting out. This is based on the extra protein banding only occuring for the Taq polymerases prepared for this experiment. A factor that could have also played a role was the incubation at 75C was continually 6 interrupted through the need to shake the reaction vessel thereby lowering the temperature of the solution. This was due to mechanical difficulties of the equipment. It would be best to find a working New Brunswick Scientific-Innova 40 incubator shaker series to improve the protein isolation. To decrease the protein impurities, an increase heat cycle could be implemented, as Taq is thermostable at 75C, and coul d sustain structure at that temperature for long durations (7). The ammonium sulfate salting out would be more efficent after an increased heat cycle as even fewer native state proteins would remain (10). Another method to decrease impurities would be to add a purification step using another specific property of Taq polymerase. This could be the isoelectric point.This could be done through ion exchange columnsor isoelectric focusing (12). The extra isolation step would significantly decrease the impurities, and increase the specific activity per mg of protein of sample.The impurities were likely a result of other proteins present in E. coli bacteria lysate that were relatively thermostable, as those proteins would be most probable (9). The isolation of Taq can be confirmed through the Western Blotting and PCR reactions (Fig. 4-7), as a distinct band in the Western Blot, and measureable amplicon replication in the PCR and rt-PCR. In the standard of Taq of the Western blot (Fig. 4) th ere is a distinct band. The same band in the channel containing the isolated Taq can be seen. The band occurs in the same relative vicinity as the Taq molecular weight band in the SDS-PAGE (Fig. 2) so would fit best fit the Taq enzyme.The banding of the blot shows a common band across all lanes that line up with the standard Taq, emphasizing the isolation of Taq. There is a hesitation in confirmation of Taq due to the extra protein banding in the prepared fractions, as these bands were not seen in the standard Taq. The banding would suggest proteins transferred from the gel to the membrane and was still able to bind to the primary antibody or secondary antibody. There are various possible explanations for this. First and foremost, the banding occurred in areas wherever protein was present (ladder and lanes). This would indicate lack of specificity in the primary antibody which is intended to only find full sequence Taq and bind to it (15, 16). Another problem may be due to lack of b locking solution binding to the membrane, or Isolation of Recombinant Taq Polymerase for PCR inordinate washing removing blocking solution from the membrane. A final possible explanation may be binding of the secondary antibody to membrane bound proteins with the exception of casein (the blocking protein used) (15, 17).Antibody specificity can be corrected by finding a new antibody, lack of blocking simply requires longer blocking periods or increased blocking solution concentration, and washing can be minimized to see resultant effect on the membrane. Each of the possible problems with the Western Blot would have to be tested by altering the procedure used above by one method (washing, antibody, blocking solution). The PCR results show template replication through thermocycling, which indicates the presence of thermostable DNA polymerases in the PCR tube. From this, it can be conferred that Taq polymerase was indeed isolated. Further confirmation could be made through further pur ification of Taq. This could be done through 2-D SDS-PAGE vs Isoelectric point electrophoresis using the isoelectric point of Taq and using the bands emphasized as Taq, and a lower concentration gel (12). Another method would be to analyze the gel bands through other methods such as mass spectropscopy or NMR (18). There was distinct differences between three sets of Taq polymerases the standard, the sample prepared in the previous year, and the sample produced in this experiment. Most distinctly the proteins differ with respect to SDS-PAGE gels.Quite obviously, the purest of the enzymes was the standard Taq, followed by the 2011 sample, and the sample prepared in this experiment. The sample prepared through this experiment had a high amount of a salt concentration and resulted in distorted bands, along with numerous other proteins present in the sample. The enzymes also differed with respect to the Western Blot (Fig. 4). The 2011 sample failed to return 2 antibody response, indicati ng lack of Taq polymerase, or lack of primary antibody binding, while the standard and experimental sample both had representive banding. There may have been excessive blocking or drying of the lane containing the 2011 Taq, as the SDS-PAGE shows a representive band in the region of Taq, that is the darkest band in the lane (15). The protein concentrations as determined through the Bio-Rad assay (Tab. 1, Fig. 1) returned 7 drastically different results. The two protein concentrations differed by 2x concentration. The easiest explanation of this result is the 10x dilution was insufficient in reducing the absorbance to within the standard curve.Due to the absorbances being above the standard curve, the results are invalid, as the region in which the curve is linear is up to 0.5mg/mL (19). The 100 x dilution returned a result of 1.88 + 0.11 mg/mL. This coroborates the SDS-PAGE findings as the protein was not excessively overloading the lane. The SDS-PAGE could have been further diluted , but the concentration used was sufficient for the purposes of the experiment. In an analysis of the PCR results (fig. 7), the brightest fluorescence bands occurred in the std., 2 and (4/Taq) lanes. This would indicate the highest activities occuring in these lanes. When compared to the western blot, the darkest banding of regions of Taq (5,?,*) returned the bands with less fluorescence. This result shows that the amount of enzyme may inhibit the PCR reaction as the the bands with the highest recoveries returned the last fluorescence. With an assessment of the basepair length, reaction time, and amount of enzyme used, an approximately activity of 834.5 + 63.9 bp/min/g of protein, or 3922.3 + 192.9 bp per minute. In comparison to the literature values of the protein, this is slightly above the 60 base pairs per second value, however, that was at 70 C (7).The rt-PCR returned a consistent melting temperature of 81C (Fig. 6)for all amplicon samples indicating the lack of a primer-dime r formation. Threshold was ab initio reached at 20 cycles (Fig. 5), which an RFU value of approximately 9000. This indicated a high activity of the taq polymerase used, at least above 1.25 Units (20). Both PCR assays agree with one another. There was no primer dimer formation noted on the agarose gel, or the melt curve analysis. There was a high activity of the enzyme sample isolated as found through the bpmin-1 and cycle of reaching threshold, however, between the two assays, the rt-PCR has the significant advantage of time, and no electrophoresis required. Currently, Taq is widely available and would likely be cheaper to simply acquire commercially. This experiment does however outline a method for thermostable protein isolation which could be used for the more recent and more valuable thermostable enzymes (Pyrococcus furiosus Polymerase) which Isolation of Recombinant Taq Polymerase for PCR are passe-partout to Taq in both thermostability, and error rate due to proofreading a bility (21). Overall, the purpose of the experiment was met.Taq was indeed isolated from a culture of recombinant E. coli. This was confirmed through the Western Blotting, and thermostable DNA activity in the PCR and rt-PCR. The purity was assessed and found to be below that of the methods used by Engelke et al., 1990. The purity could be increased through use of a cation exchange column (9). The length of heat denaturation and an automatic heat controlled shaker would help to remove excess proteins and improve purity. The length of dialysis time would need to be increased for less band distortion in SDS-PAGE, and either more selective primary antibody, increased blocking or decreased washing would be required for improved Western Blotting. For further experiments, it is suggested testing the new method modifications, and or implementing recombinant Pyrococcus furiosus Polymerase.