Extracellular production of azurin by reusable magnetic Fe3O4 nanoparticle-immobilized Pseudomonas aeruginosa.

Azurin, found in the periplasm of Pseudomonas aeruginosa, has garnered significant attention as a potential anticancer agent in recent years. High-level secretion of proteins into the culture medium, offers a significant advantage over periplasmic or cytoplasmic expression. In this study, for the first time, P. aeruginosa cells were immobilized with magnetic nanoparticles (MNPs) to ensure effective, simple and quick separation of the cells and secretion of periplasmic azurin protein to the culture medium. For this purpose, polyethyleneimine-coated iron oxide (Fe3O4@PEI) MNPs were synthesized and MNPs containing Fe up to 600 ppm were found to be non-toxic to the bacteria. The highest extracellular azurin level was observed in LB medium compared to peptone water. The cells immobilized with 400 ppm Fe-containing MNPs secreted the highest protein. Lastly, the immobilized cells were found suitable for azurin secretion until the sixth use. Thus, the magnetic nanoparticle immobilization method facilitated the release of azurin as well as the simple and rapid separation of cells. This approach, by facilitating protein purification and enabling the reuse of immobilized cells, offers a cost-effective means of protein production, reducing waste cell formation, and thus presents an advantageous method.

Komagataella phaffii (Pichia pastoris) as a Powerful Yeast Expression System for Biologics Production.

Komagataella phaffii (K. phaffii) (Pichia pastoris), also called biotech yeast, is a yeast species with many applications in the biotechnology and pharmaceutical industries. This methylotrophic yeast has garnered significant interest as a platform for the production of recombinant proteins. Numerous benefits include effective secretory expression that facilitates the easy purification of heterologous proteins, high cell density with rapid growth, post-translational changes, and stable gene expression with integration into the genome. In the last thirty years, K. phaffii has also been refined as an adaptable cell factory that can produce hundreds of biomolecules in a laboratory setting and on an industrial scale. Indeed, over 5000 recombinant proteins have been generated so far using the K. phaffii expression method, which makes up 30% of the total cell protein or 80% of the total released protein. K. phaffii has been used to manufacture more than 70 commercial products in addition to over 300 industrial processes that have been granted licenses. Among these are useful enzymes for industrial biotechnology, including xylanase, mannanase, lipase, and phytase. The others are biopharmaceuticals, which include human serum albumin, insulin, hepatitis B surface antigen, and epidermal growth factor. Compared to other expression systems, this yeast is also considered a special host for synthesizing subunit vaccines, which have recently been supplanted by alternative vaccination types, such as inactivated/killed and live attenuated vaccines. Moreover, efficient production of recombinant proteins is achieved through multi-level optimization methods, such as codon bias, gene dosage, promoters, signal peptides, and environmental factors. Therefore, although K. phaffii expression systems are efficient and simple with clearly established process procedures, it is still necessary to determine the ideal conditions since these vary depending on the target protein to ensure the highest recombinant protein generation. This review addresses the K. phaffii expression system, its importance in industrial and biopharmaceutical protein production, and some bioprocessing and genetic modification strategies for efficient protein production. K. phaffii will eventually continue contributing as a potent expression system in research areas and industrial applications.