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Experimental studies to determine the effect of protein characteristics on translocation of fusion proteins in Escherichia Coli

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dc.contributor Graduate Program in Chemical Engineering.
dc.contributor.advisor Hortaçsu, Amable.
dc.contributor.author Ütkür, Fatma Özde.
dc.date.accessioned 2023-03-16T11:08:16Z
dc.date.available 2023-03-16T11:08:16Z
dc.date.issued 2006.
dc.identifier.other CHE 2006 U85
dc.identifier.uri http://digitalarchive.boun.edu.tr/handle/123456789/14790
dc.description.abstract Previous experimental work to translocate maltose binding protein-glucose isomarasefusion protein from the cytoplasmic to the periplasmic space have shown that the fusionprotein mainly remained in the cytoplasm and only one per cent of the expressed fusion protein was transported to the periplasm.In the present study, genetic engineering techniques are exploited to investigate theeffect of protein size on translocation. For this purpose, two industrial enzymes of differentsizes were cloned using the pMAL-p2 vector. These two enzymes are: pullulanase and putative serine protease of Thermus Thermophilus HB8 which are 718 and 251 aminoacids long, respectively. Glucose isomease from the same organism is 395 amino acidslong, thus in between pullulanase and serine protease.The gene coding for serine protease and pullulanase from Thermus thermophilusHB8 (ATCC 27634) were amplified using PCR and inserted between XmnI and EcoRI site of the plasmid vector pMAL-p2. The fusion proteins MBP-serine protease and MBPpullulanasewere expressed in recombinant XL1 cells and the cellular distribution of thefusions were determined in cytoplasmic and periplasmic compartments by the appropriateenzyme assays and SDS-PAGE analysis. The experimental results showed that most of the MBP-serine protease fusion expressed was translocated to the periplasm with the highestvalue of 77 per cent and almost all of the MBP-pullulanase fusion expressed wastranslocated to the periplasm with the highest value of 99 per cent. On the other hand, the results for MBP-GI case was not satisfactory and not more than one per cent of the totalprotein produced was translocated to the periplasm. Therefore, it was obvious that thelength of the protein to be translocated didn̕t have any effect on translocation.
dc.format.extent 30cm.
dc.publisher Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006.
dc.relation Includes appendices.
dc.relation Includes appendices.
dc.subject.lcsh Membrane proteins -- Physiological transport.
dc.subject.lcsh Escherichia coli.
dc.title Experimental studies to determine the effect of protein characteristics on translocation of fusion proteins in Escherichia Coli
dc.format.pages xii, 81 leaves;


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