Production And Purification Of Mutl Gene And Uvrd Recombinant Protein For Helicase Dependent Dna Amplification
| dc.contributor.author | Buskaran, Kalaivani | |
| dc.date.accessioned | 2016-07-18T03:24:56Z | |
| dc.date.available | 2016-07-18T03:24:56Z | |
| dc.date.issued | 2015-08 | |
| dc.description.abstract | This research was conducted to optimize yield of recombinant UvrD helicase and to produce recombinant MutL protein for the helicase amplification dependent reaction. Recombinant UvrD helicase were cloned and expressed into Lemo21 expression host. The overall yield of the UvrD helicase protein was improved 10% using conventional shake flask cultivation method. The optimum recombinant UvrD helicase were successfully produced in terrific broth culture medium, at 0.2 mM IPTG inducer concentration, 9 hours of post-induction time and 37°C post-induction temperature. The cultivation of the recombinant UvrD/pET28a/Lemo21 in small scale fermentation using shake flask culture has optimized the yield to 1.35 ng/μg recombinant UvrD helicase proteins as compared to 0.143 ng/μg of recombinant UvrD helicase protein from previous studies. In order for, recombinant UvrD helicase function, recombinant MutL protein was produce from local isolate of Escherichia coli. The PCR amplified MutL protein had size of 1848 bp of sequence analysis. The MutL gene was then cloned into TOPO PCR 2.1 cloning vector. The successful clone was then analysed using Uniprot software produce 615 deduced amino acid 100% similarities to MutL protein from other interspecific species of E. coli. Later, the amplified MutL gene was cloned into pET/28a(+) expression vector and expressed in Lemo21 expression host. The recombinant UvrD and MutL was purified under non-denaturing conditions using immobilized metal affinity chromatography. SDS-PAGE was ran and confirmed the presence of the protein expression. Immunoreactivity of the recovered recombinant proteins was found to be sensitive and specific when tested with Western blot. MALDI TOF/TOF also conducted to confirm the recombinant protein at expected at 82 kDa and 68 kDa for UvrD helicase and MutL protein respectively. Subsequently, the activity of recombinant UvrD helicase was validated using unwinding helicase activity and helicase dependent amplification assay. The time course analysis of in vitro unwinding helicase activity at different concentration (250 ng and 500 ng) had shown a positive enzyme unwinding activity. Finally, the HDA reaction confirmed the ability of the in-house recombinant UvrD helicase and recombinant MutL gene to unwind a specific sequence from duplex DNA. In summary, the objectives of this research has been achieved successfully by optimizing and producing recombinant UvrD helicase and recombinant MutL protein which were validated by using in vitro unwinding helicase activity and helicase dependent amplification assay. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/2265 | |
| dc.subject | Other system of Medicine | en_US |
| dc.title | Production And Purification Of Mutl Gene And Uvrd Recombinant Protein For Helicase Dependent Dna Amplification | en_US |
| dc.type | Thesis | en_US |