Functional Production, Extracellular Expression, and Antitumor Activity of Mouse Alpha-Klotho in Model Microalga Chlamydomonas reinhardtii

Abstract

Purpose Klotho is a human protein with versatile functions associated with longevity and well-being. Deficiency of α-Klotho (α-KL) in the circulatory system is associated with decreased life expectancy along with numerous disorders such as chronic kidney disease, atherosclerosis, infertility, skin atrophy, emphysema, defective cognitive development, various incurable cancers, osteoporosis, and so on. Here we aimed the recombinant, low-cost, photosynthetic production of α-KL in eukaryotic microalgal systems. Methods The gene corresponding to the mouse α-KL(mα-KL) protein was codon optimized and synthetically produced for Chlamydomonas reinhardtii (CCALA 928) hosts. Following Agrobacterium-based transformation, the recombinant protein was expressed under the Hsp70A/RbcS2 dual promoter system and produced extracellularly. The anticarcinogenic activity of recombinant mα-KL was examined on Rattus norvegicus AR42J pancreatic tumor cell lines. Results Heterologous, functional and extracellular production of mα-KL protein was achieved for the first time in the model microalga C. reinhardtii. Secretion of mα-KL into the extracellular environment facilitated downstream processes and enabled low-cost purification at high titers. Up to 4.37 µg/mL mα-KL protein was recovered from cell-free culture supernatants of transgenic strains. Furthermore, the anticarcinogenic activity of recombinant mα-KL was confirmed in AR42J pancreatic tumors. Conclusion With the establishment of cheap, sustainable and functional production of α-KL, an important obstacle to its widespread use in the treatment of various diseases has been overcome. This can also serve as an important cornerstone in the development of various cosmetics, pharmaceuticals and quality products containing α-KL.