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Expression of membrane-acting immunotoxins based on the bacillus thuringiensis Cyt2Aa1 toxin in Pichia pastoris

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  1. Tez No: 402706
  2. Yazar: CEMAL GÜRKAN
  3. Danışmanlar: PROF. DR. DAVID J. ELLAR
  4. Tez Türü: Doktora
  5. Konular: Biyokimya, Biochemistry
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2002
  8. Dil: İngilizce
  9. Üniversite: University of Cambridge
  10. Enstitü: Yurtdışı Enstitü
  11. Ana Bilim Dalı: Belirtilmemiş.
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 254

Özet

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Özet (Çeviri)

This work explored the oncolytic potential of membrane-acting immunotoxins (ITs) based on the Bacillus thuringiensis (B. thuringiensis) Cyt2Aa1 toxin as novel anti-tumour agents using an in vitro model system for human cancer. Recombinant production of these ITs was assessed using the Pichia pastoris (P. pastoris) expression system. Cyt2Aa1 is a cytolytic, haemolytic and mosquito larvicidal toxin from B. thuringiensis subspecies kyushuensis. It was proposed that genetic fusion of the activated form of Cyt2Aa1 toxin with a tumour-specific cell-binding ligand might lead to the specific elimination of tumour cells bearing these markers. To this end, human single-chain antibody fragments C6.5 and C6ML3-9 were selected, which targeted p185HER-2, a cell-surface glycoprotein overexpressed in ~30% of human breast and ovarian cancers. Heterologous expression of C6.5 and C6ML3-9 was established in P. pastoris and the recombinant proteins were purified by a single-step immobilised metal affinity chromatography (IMAC). Final yields of C6.5 and C6ML3-9 were ~10 and ~70 mg.l-1 culture respectively. Next, de novo design and construction of a synthetic cyt2Aa1 gene was carried out since an earlier study had established that the heterologous expression of the native bacterial cyt2Aa1 gene was not feasible in P. pastoris. A recursive PCR strategy was used to construct the synthetic cyt2Aa1 gene, which was optimised for expression in P. pastoris by increasing the G+C-content to ~50% compared to ~30% for the native bacterial gene and using only the first and the second preference P. pastoris codons. Once the efficient P. pastoris expression of the synthetic cyt2Aa1 gene was demonstrated, DNA constructs were created where the synthetic gene was linked to either the scFv C6.5 or C6ML3-9 gene. Expression analyses of the C6.5-based construct in P. pastoris demonstrated that: (a) its intracellular expression was highly toxic for the host cells, (b) despite failure of proper secretion, secretory targeting of the recombinant protein led to its high-level accumulation in P. pastoris membranes, and(c) the intracellularly accumulated protein was partially glycosylated and still retained part of the secretion signal. A combination of an in vitro (on-the-column) refolding and IMAC purification strategies were then successfully used to purify the recombinant product at ~10 mg.l-1 culture. In vitro cytotoxicity and haemolysis assays showed that recombinant Cyt2Aa1-based IT-A was lytic to the target p185HER-2-overexpressing human cancer cell lines (SK-BR-3 and SK-OV-3). Significantly, the Cyt2Aa1-based IT-A was not cytotoxic to p185HER-2-negative control cell line (Swiss 3T3) and no longer exhibited the broad haemolytic activity associated with the activated Cyt2Aa1 toxin. A number of further attempts were also made to enhance the LC50 of Cyt2Aa1-based ITs against the target cells. However, these attempts were either unsuccessful or the requisite experiments could not be completed in the time available. Nevertheless, the main goal of this project was achieved since the specific activity of the P. pastoris produced Cyt2Aa1-based IT-A was demonstrated, confirming its potential for the targeted therapy of p185HER-2-overexpressing tumours.

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