Design and construction of membrane-acting immunotoxins for intracellular and secreted protein expression in Pichia pastoris
Başlık çevirisi mevcut değil.
- Tez No: 402429
- Danışmanlar: DR. DAVID ELLAR
- Tez Türü: Yüksek Lisans
- Konular: Biyokimya, Biochemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 1999
- Dil: İngilizce
- Üniversite: University of Cambridge
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Biyokimya Ana Bilim Dalı
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 110
Özet
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Özet (Çeviri)
This work explored the design and construction of membrane-acting immunotoxins based on the active toxin core of Cyt2Aa1 and single-chain antibody fragments C6.5 and C6ML3-9. Intracellular and secreted expression of these recombinant proteins were investigated using the methylotrophic yeast Pichia pastoris. Cyt2Aa1 is a cytolytic, haemolytic and diptericidal -endotoxin from Bacillus thuringiensis subspecies kyushuensis. C6.5 and C6ML3-9 are of human origin and the target is ErbB- 2/HER-2, a surface glycoprotein which is overexpressed in approximately 30% of breast and ovarian cancers. C6ML3-9 is a mutant derivative of C6.5 with a six-fold increased affinity for the target antigen. An overlap extension PCR strategy was used successfully to create two immunolysin constructs based on the Cyt2Aa1 active toxin core and either C6.5 or C6ML3-9. The immunolysin design incorporated a flexible, soluble polypeptide linker between the toxin and the antibody domain, as well as a C-terminal hexahistidine tag for ease of purification. A novel P. pastoris secretory expression vector was also created by cloning the S. cerevisiae factor signal sequence into an existing intracellular expression vector. The two immunolysin constructs were subsequently cloned into both types of vectors, and used to engineer novel P. pastoris strains bearing stable chromosomal integrants of the expression cassettes. Initial attempts to express immunolysin from P. pastoris were not successful because previously undetected mutations in the immunolysin constructs resulted in truncated versions of the desired protein. However this was fortuitous because the truncated species corresponded to the scFv domain of the immunolysins and hence showed that the scFvs used in this study can be efficiently produced in P. pastoris. Subsequently P. pastoris strains bearing mutation-free immunolysin expression cassettes, as well as a novel Cyt2Aa1 toxin expression cassette, were constructed and screened for the production of the desired proteins. Initial experiments with the novel expression strains did not reveal detectable expression of either the Cyt2Aa1-based immunolysin or the individual Cyt2Aa1 toxin. Further detailed experiments analysing expression of the recombinant proteins could not be completed in the time available. However, preliminary experiments suggest that an inherent problem exists with the eukaryotic expression of Cyt2Aa1, either by itself or as a fusion protein. This problem may be attributed to the relatively AT-rich DNA sequence of Cyt2Aa1 which contains short stretches of nucleotides that may function as yeast polyadenylation sites causing premature transcript termination. It is suggested that further investigation of the expression of Cyt2Aa1-based immunolysins in P. pastoris should be carried out using a synthetic Cyt2Aa1 gene sequence with an increased GC-content and codon preferences for Pichia.
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