Fare alfa klotho proteininin fotosentetik mikroalg Chlamydomonas reinhardtii'de heterolog ekspresyonu

Klotho (Kl) has been described as a powerful inhibitor of aging and a multifunctional regulatory protein. Klotho has been reported that homozygous mice with the mutant klotho allele show the typical aging phenotypes of atherosclerosis, ectopic calcification, pulmonary emphysema, skin atrophy and osteoporosis. It has been scientifically proven that mice that produce excess Klotho protein increase their lifespan by 30%. The antagonistic effect of Klotho protein on different types of cancer has been scientifically proven. It was observed that the amount of liberated KL (secreted KL: sKL) in blood system is significantly decreased in cancer patients and that external sKL supplementation has curative effects on most chronic diseases, including cancer types. In the next 5-10 years, many cancers, including diabetes, Alzheimer's, Parkinson's, arteriosclerosis, heart failure, high blood pressure, kidney failure, etc. It seems inevitable to supplement SKL for treatment and protection against diseases. In this study, the gene encoding mouse α-Klotho protein was cloned into the pCAMBIA1301 vector and then transferred to Agrobacterium tumefaciensis EHA105 cell lines. The mouse α-klotho gene was transferred to Chlamydomonas reinhardtii (CC-124) microalgae hosts by Agrobacterium-based transformation method. Positive transformants of CC-124 cells were selected on TAP agar media containing 30 µg/ml hygromycin antibiotics throughout three consecutive generations. Upon the isolation of genomic DNA from transformed microalgae, the molecular confirmation of transformed α-klotho gene in microalgae genome was performed by PCR amplification of the corresponding DNA region. Later, extracellular production of the mouse α-Klotho protein by transformed microalgae host cells was induced in liquid TAP medium. The successful production of α-Klotho protein in culture medium was then confirmed through the purification processes and following SDS-PAGE analysis. The functionality of the purified protein and its anti-carcinogenic effect were tested in two different cancer cell lines through MTT tests measuring the cell viability in mammalian cell lines. In conclusion, the recombinant production of the pharmaceutical α-Klotho protein was successfully carried out for the first time in model microalgae C. reinhardtii hosts extracellularly. In this way, the basis was established for the photosynthetically low-cost production of α-Klotho protein and this will definitely facilitate the accessibility of the Klotho protein for different medicinal applications for human health in near future.