Heterologous Expressıon Of An Isoenzyme Of Pycnoporus Sanguıneus Laccase In Yeast Saccharomyces Cerevısıae
Heterologous Expressıon Of An Isoenzyme Of Pycnoporus Sanguıneus Laccase In Yeast Saccharomyces Cerevısıae
Dosyalar
Tarih
2010-02-11
Yazarlar
Varışlı, Esra
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Fen Bilimleri Enstitüsü
Institute of Science and Technology
Institute of Science and Technology
Özet
Yapılan çalışmada Pycnoporus sanguineus lakkaz-2 cDNA ’sının Saccharomyces cerevisiae mayasında heterolog ekspresyonu gerçekleştirilmiştir. Bu amaçla, ~1600 amino asitlik ve ~60 kDa moleküler ağırlığında olan matür bir lakkaz izoenzimini kodlayan, tam boyda bir lakkaz-2 (Lcc2) cDNA ’sı, bakırla indüklenebilen CUP1 promotörü taşıyan pSAL4 ekspresyon vektörüne klonlanmıştır. Rekombinant plazmid, lityum asetat protokolü kullanılarak S. cerevisiae W303-1A suşuna aktarılmış ve transformant koloniler 0.2 mM ABTS ve 0.6 mM CuSO4 içeren seçici besiyerinde substratı okside edebilme özelliklerine göre seçilmiştir. Pozitif transformant koloniler arasında yapılan tarama deneyleri ile en yüksek aktivite gösteren koloni seçilmiş ve sonrasında optimum ekspresyon koşullarının eldesi için karbon kaynağına ve bakır konsantrasyonlarına göre farklı besiyerilerinin etkileri tespit edilmiştir.
In this study, heterologous expression of Pycnoporus sanguineus laccase-2 cDNA in yeast Saccharomyces cerevisiae was performed. For this aim, a full length laccase-2 (Lcc2) cDNA, encodes for a mature laccase isoenzyme of ~1600 amino acid residues with a predicted molecular weight of ~60 kDa, was cloned into the pSAL4 expression vector which bear copper-inducible CUP1 promoter. Recombinant plasmid was transferred into the S. cerevisiae W303-1A strain via using lithium acetate protocol and transformed colonies were selected in terms of the properties of oxidizing the substrate in selective medium supplemented with 0.2 mM ABTS and 0.6 mM CuSO4. The colony which shows the highest activity was selected with screening assays between the positive transformant colonies, and then the effects of different media in terms of the carbon source and copper concentrations were determined for obtaining the optimum expression conditions.
In this study, heterologous expression of Pycnoporus sanguineus laccase-2 cDNA in yeast Saccharomyces cerevisiae was performed. For this aim, a full length laccase-2 (Lcc2) cDNA, encodes for a mature laccase isoenzyme of ~1600 amino acid residues with a predicted molecular weight of ~60 kDa, was cloned into the pSAL4 expression vector which bear copper-inducible CUP1 promoter. Recombinant plasmid was transferred into the S. cerevisiae W303-1A strain via using lithium acetate protocol and transformed colonies were selected in terms of the properties of oxidizing the substrate in selective medium supplemented with 0.2 mM ABTS and 0.6 mM CuSO4. The colony which shows the highest activity was selected with screening assays between the positive transformant colonies, and then the effects of different media in terms of the carbon source and copper concentrations were determined for obtaining the optimum expression conditions.
Açıklama
Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2010
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2010
Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2010
Anahtar kelimeler
lakkaz,
heterolog ekspresyon,
Pycnoporus sanguineus,
laccase,
heterologous expression Pycnoporus sanguineus