Recombinant production, purification and characterization of affinity-tagged human growth hormone with tetrahymena artificial chromosome vector system in tetrahymena thermophila

2023 National Vision plans to establish the pharmaceutical production substructure in the field of health through trained human resources and corporatization. In this thesis; human growth hormone (hGH), which is one of the human therapeutic proteins, is aimed to be produced in Tetrahymena thermophila as an alternative protein production host. The use of high copy number T. thermophila macronuclear artificial chromosome-2 (TtAC2) adapted for Good Manufacturing Practices (GMP) is planned as the vector infrastructure in hGH production. Codon-optimized hGH encoding DNA sequences were cloned into the TtAC2 vector, which is under the kontrol of the heat-inducible HSP70.2 heat shock promoter for protein expression. The vectors were transformed to the T. thermophila CU428 strain by the biolistic gun. The cytoplasmic production of the GFP-hGH fusion protein as 50 kDa monomer-101 kDA dimer, 6xHis-hGH and untagged-hGH cytoplasmic proteins as 17 kDa-22 kDa monomers and 34 kDa-44 kDa dimers were demonstrated by SDS-PAGE and Western Blot analysis. Secreted hGH was extracellularlly detected by ELISA analysis when 6XHis-hGH and untagged-hGH was produced by PLA22/23 signal peptide sequence directing the extracellular environment through endoplasmic reticulum. The literature based hypothesis about the degradation of secreted hGH by secretomic proteases has been confirmed by the results of in vitro proteolytic enzyme activity assay. In this thesis, the vector infrastructure developed for the production and purification of hGH could be used in the bioactivity characterization. As a result; the production amounts of hGH in T. thermophila can be increased by silencing protease genes of the organism and optimized bioreactor culturing.