Use of human growth hormone secretory signal sequence in recombinant protein production by Tetrahymena thermophila

Tetrahymena thermophila is a eukaryotic, ciliated, single-celled model organism with short division time and easy cultivation features. The recombinant capacity of the organism demonstrated by recombinant production studies of human DNaseI and human alkaline phosphatase proteins. The production of human growth hormone, which is one of the therapeutic proteins needed in the pharmaceutical sector and used to treat individuals with the growth disorder, is carried out in yeast, E. coli, and the human cell culture. It is hypothesized that T. thermophila, which is not used to produce this protein before, can recognize the E.R signal sequence of human growth hormone and secrete the recombinant protein outside of the cell. For this purpose, the codon sequence of the hGH gene with hGH E.R signal sequence was adapted to the organism and produced synthetically. The generated hGH gene cassettes were cloned into pNeo4 vectors providing genome integration over 45 copies and extrachromosomal pIGF circular vectors containing origin with 10.000 copy number, were transformed into conjugative/vegetative T. thermophila cells by biolistic particle gun. After inducing positive transformants by heat shock or CdCl2, two independent SDS-PAGE and Western Blot analyses of purified proteins from the cells showed recombinant protein has approximately 23 kDa molecular mass and the signal sequence was processed. As a result; it is understood that T. thermophila recognizes human hGH E.R signal sequence, uses the signal sequence, cuts it away and secretes the recombinant protein outside the cell.