Recombinant production and characterisation of human interferon beta (hIFNβ) therapeutic protein in Tetrahymena thermophila

Unicellular eukaryotic ciliate Tetrahymena thermophila with its cheap and simple culturing is used in health and biotechnological studies as alternative protein production host factory. Recently, anti-cancer monoclonal antibody (rituximab) drug has been produced as recombinant protein in this organism. T. thermophila is able to add correct disulfide bonds and form human-like small-size glycosylation modifications whereas E. coli could not. The capacitive analysis of T. thermophila to produce recombinant human interferon beta therapeutic protein which is used in treatment of Multiple Sclerosis (MS) auto-immune disease is the aim of this thesis. Synthesized according to the frequency of codon usage in Tetrahymena codon-optimised hIFNβ sequence, coding sequences of sfGFP and 12xHis affinity tag in its C-terminal position of hIFNβ-TtsfGFP cassette were placed and the fusion protein coding ORF was cloned into the pVGF vector under the control of cadmium inducible MTT1 promoter. Confirmation of the production of 48 kDa Tt-hIFNβ-TtsfGFP-12xHis fusion protein and 20 kDa broken Tt-hIFNβ by fluorescence microscopy and Western Blot analysis has showed that hIFNβ was successfully produced in T. thermophila as recombinant protein. Affinitic and GFP tag-free Tt-hIFNβ gene coding cassette with extracellular secrotary sequence was also placed under the heatshock promotor in the TtAC2 artificial chromosome vector. After the transformation of this vector into Tetrahymena cells, antibiotic selection and positive single cell clon isolation has been completed. Keywords: Tetrahymena thermophila, hIFNβ, recombinant therapeutic protein.