In this study, a strategy called gene splicing by overlap extension, &ldquo / Gene SOEing&rdquo / , was used for the construction of the fusion proteins with the purpose of increasing the thermostability of mesophilic enzymes by incorporation of stability domain from a thermostable enzyme.
Gene SOEing is a PCR-based approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. In fusion constructs, as the stability determinant Thermoplasma volcanium citrate synthase (CS) large domain has been used. This gene has recently been cloned in our laboratory. In two different fusions, as fusion partners, dehalogenase II (dehCII) gene of Pseudomonas sp. CBS3 and aminoglycoside-3' / -phosphotransferase-II (APH(3' / )-II) gene of E. coli were employed. Following the Gene SOEing, two fusion products, 1722 bp long CS Large Domain-dehCII and 1750 bp long CS Large Domain-APH(3' / )-II were constructed. Also a 1586 bp long dehCII-APH(3' / )-II fusion was prepared. Three fusion constructs were cloned in E. coli. Cloning was confirmed in each case, by restriction analysis of the isolated plasmids from recombinant colonies. APH(3' / )-II gene associated with CS Large Domain-APH(3' / )-II and dehCII-APH(3' / )-II fusion constructs were successfully expressed in E. coli as revealed by enzyme assay and antibiotic agar plate assay. CS Large Domain-APH(3' / )-II fusion protein retained 9.4% of the original APH(3' / )-II activity after 10 minutes at 60º / C. However, CS Large Domain-dehCII and dehCII-APH(3' / )-II fusions did not display any dehalogenase activity.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/2/12604778/index.pdf |
| Date | 01 June 2004 |
| Creators | Ozdogan, Seda |
| Contributors | Kocabiyik, Semra |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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