Isolation of Recombinant Escherichia Coli Iptg Induced Taq Polymerase and Characterization Through Polymerase Chain Reactions%2c Western Blotting and Gel Electrophoresis

Topics: Polymerase chain reaction, Molecular biology, Thermus aquaticus Pages: 20 (7912 words) Published: February 26, 2013
Isolation of Recombinant Taq Polymerase for PCR Isolation of Recombinant Escherichia coli IPTG induced Taq polymerase and characterization through polymerase chain reactions, Western Blotting and gel electrophoresis * Braeden Cowbrough1, Michael Atkins2, Christopher Bonner3 From the Faculty of Biochemistry Lab 3006 B Carleton University, Ottawa, ON K1S 5B6 *Running title: Isolation of Recombinant Taq Polymerase for PCR To whom correspondence should be addressed: Braeden Cowbrough, Faculty of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, CAN, Tel: (613) 979-3534; Email: Keywords: PCR, Taq Polymerase, IPTG induction, salting out, thermostable polymerase Background: Taq polymerase is a thermostable enzyme essential in the PCR reaction that is isolated from recombinant E. coli. Results: Taq polymerase was isolated, the sample was not pure Taq, molecular weight found to be 114kDa. Conclusion: The methods isolated taq but an improvement of cation exchange and increase heat denaturation should be added. Significance: An improved method of Taq isolation offers a valuable resource for PCR. SUMMARY The aim of this experiment was to isolate recombinant Taq polymerase from E. coli bacteria induced by IPTG. This was done successfully through differential centrifugation, ammonium sulfate precipitation, and heat denaturation as confirmed by Western Blotting, rt-PCR and PCR reactions. The isolated protein sample was found to still contain remnants of host proteins as confirmed through SDS-PAGE with a Taq polymerase mass of 113.4 + 14.3 kDa. The activity of the isolated Taq was found to be 3922.3 + 192.9 bp per minute with a protein concentration determined through a Bio-Rad assay to be 1.88 + 0.11 mg/mL. The threshold of rt-PCR was found to be 20 cycles, with a melting temperature of 81°C confirming no primer dimer formation. A 1% agarose gel of PCR products revealed 5883.5 + 289.4 base pair lengths of double stranded DNA formed through the reaction. This study emphasized the need for 1 cation exchange columns for separation of high purity Taq polymerase as the methods were not superior to that of Engelke et al., 1990, as 300 + 17.7 mg of protein per litre of cell culture was isolated, but the protein still contained substantial impurities so the actual value would likely be reduced.

One technique important in both genetics and biochemistry is the Polymerase Chain Reaction (PCR), first developed in the 1960s, and then automated in 1983. Current PCR technology was not developed until the discovery of thermostable polymerases, specifically Thermus aquaticus (Taq) polymerase (1). The protein Taq polymerase was first isolated from the extreme thermophile T. aqauticus, where extreme thermopiles are bacteria that live in temperatures at or above 45°C. 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 74-75°C, allowing it the remain active in temperatures required for PCR double stranded DNA denaturation (3, 1) . The protein has an approximate molecular weight of 6263 kDa when isolated from T. aquaticus, and 94 kDa when isolated from recombinant Escherichia coli, and is still stable at temperatures of 93-95°C, hence the thermostability of the enzyme ). Taq specifically lacks any proofreading activity in the 3’ to 5’ direction, and therefore has a relatively high error rate of single base mispairings of 1 error per

Isolation of Recombinant Taq Polymerase for PCR 9000 nucleotides, as well 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 add up to 1000 base pairs in length on a template in under one minute under typical PCR conditions. The enzyme...

References: 10
Isolation of Recombinant Taq Polymerase for PCR TABLE 1 Taq Sample Dilution Factor 10 x Absorbance at 620 nm (+ 0.0005) 0.881 1.145 0.951 0.449 0.429 0.391 Protein Concentration (corrected) 4.10 + 0.47 mg/mL
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