Cloning and expression of β-1,3-glucanase gene from Cellulosimicrobium cellulans in Escherichia coli DH5α
Bu çalışmada Cellulosimicrobium cellulans bakterisine ait β-1,3-glukanaz geni PCR ile amplifiye edilerek pUC18 klonlama vektörüne klonlanmış, böylece pTEG5 ve pTEG11 rekombinant plazmit DNAlar elde edilmiştir. Rekombinant plazmit DNAlar pTEG5 ve pTEG11 kompetent Escherichia coli hücrelerine transfer edilmişlerdir. SacI enzimi ile kesilmiş rekombinant plazmitlerin agaroz jelde elektroforezi sonucu 1.9 kbç büyüklüğündeki β-1,3-glukanaz gen bandının görünmesi, gen entegrasyonunun tamam olduğunu göstermiştir. β-1,3-glukanaz geninin rekombinant vektörlerden amplifikasyonu da 1.9 kbç büyüklüğündeki geni göstermiştir. Rekombinant enzim E. coli tarafından hücre içi olarak üretilmiştir. LB-laminarin-agar plağına damlatılan rekombinant E. coli suşlarının intraselüler içerikleri Congo-red boyaması ile pozitif zonlar üretmiş, böylece rekombinant bakterilerce üretilen proteinin aktif olduğu anlaşılmıştır. Zimogram analizinde, hücre içi üretilen rekombinant β-1,3-glukanaz enzimleri C. cellulans enzimi ile aynı moleküler ağırlığa ait aktivite bantları sergilemişlerdir.
Anahtar Kelimeler:
plazmitler, DNA klonlama, Escherichia coli, enzim aktivitesi, enzimler, gen ifadesi, genler
Cellulosimicrobium cellulansdan β-1,3-glukanaz geninin Escherichia coli DH5αda klonlanması ve ekspresyonu
In this study, β-1,3-glucanase gene of Cellulosimicrobium cellulans was amplified by PCR and cloned in pUC18 cloning vector to construct the recombinant plasmids pTEG5 and pTEG11. The recombinant plasmids pTEG5 and pTEG11 were transformed into competent Escherichia coli cells. Digestion of recombinant plasmids with SacI produced 1.9 kbp β-1,3-glucanase gene band on agarose gel which indicated the gene integration. β-1,3-Glucanase gene amplification on the recombinant vectors also indicated 1.9 kbp gene insert. Recombinant enzyme was produced by E. coli intracellularly. Intracellular components of recombinant E. coli strains with pTEG5 or pTEG11 dropped on LB-laminarin-agar plate, showed clear positive zones by Congo-red staining revealing the activity of secreted protein. Based on the zymogram analysis, the intracellular produced recombinant β-1,3-glucanase enzymes exhibited the same activity bands with C. cellulans enzyme with respect to molecular weight.
Keywords:
plasmids, DNA cloning, Escherichia coli, enzyme activity, enzymes, gene expression, genes,
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- ISSN: 1300-6045
- Yayın Aralığı: Yılda 6 Sayı
- Başlangıç: 1995
- Yayıncı: Kafkas Üniv. Veteriner Fak.
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