Magnetic Immobilization of Pichia pastoris Cells for the Production of Recombinant Human Serum Albumin.

Seyedeh-Masoumeh Taghizadeh,Ali Dehshahri,Younes Ghasemi,Aydin Berenjian,Alireza Ebrahiminezhad,Mohammad Bagher Ghoshoon

doi:10.3390/nano10010111

Seyedeh-Masoumeh Taghizadeh, Ali Dehshahri + Show 4 more

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https://doi.org/10.3390/nano10010111

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Abstract

Magnetic immobilization as a novel technique was used to immobilize recombinant Pichia pastoris (GS115 Albumin) cells to produce human serum albumin (HSA). In this regard, magnetic nanoparticles (MNPs) coated with amino propyl triethoxy silane (APTES) were synthesized. P. pastoris cells were decorated with MNPs via nonspecific interactions. Decorated cells were magneto-responsible and easily harvested by applying an external magnetic field. The efficiency of magnetic immobilization (Ei) for cell removal was in direct relation with the MNP concentration and time of exposure to the magnetic field. By increasing the nanoparticles concentration, cells were harvested in a shorter period. Complete cell removal (Ei ≈ 100) was achieved in ≥0.5 mg/mL of MNPs in just 30 s. HSA is produced in an extremely high cell density (OD ~20) and it is the first time that magnetic immobilization was successfully employed for harvesting such a thick cell suspension. After 5 days of induction the cells, which were immobilized with 0.25 to 1 mg/mL of nanoparticles, showed an increased potency for recombinant HSA production. The largest increase in HSA production (38.1%) was achieved in the cells that were immobilized with 0.5 mg/mL of nanoparticles. These results can be considered as a novel approach for further developments in the P. pastoris-based system.

Highlights

Methylotrophic yeast, Pichia pastoris, is one of the most important and interesting eukaryotic microorganisms in the biotechnological process
Lactoferrin, human growth hormone, insulin, human serum albumin (HSA), hepatitis B vaccine, interferon alpha 2b, heparin-binding EGF-like growth factor (HB-EGF), and phospholipase A are just examples of several recombinant biopharmaceuticals that are produced by P. pastoris [4,5,6,7,8]
It should be clarified that the expression in the P. pastoris system is performed at extremely high cell densities (OD ~20) and this is the first time that magnetic immobilization was successfully used to harvest such a thick cell suspension

Summary

Introduction

Methylotrophic yeast, Pichia pastoris, is one of the most important and interesting eukaryotic microorganisms in the biotechnological process. The production of recombinant proteins in P. pastoris has gained increasing attention in pharmaceutical and biotechnological industries. In contrast to other prokaryotic and eukaryotic systems, this expression system provides outstanding advantages, such as post-translation modifications, proper protein folding, ease of manipulation, high expression level, and low processing cost [1,2,3]. Cell harvesting is the high-cost and critical point in downstream processing. There is a vast attempt to develop cell immobilization techniques as an efficient alternative for centrifugation. The P. pastoris system is not exceptional and several attempts were done to efficiently immobilize this cell for the production of valuable products, such as HSA, alcohol oxidase, l-alanyl-l-glutamine, and invertase [11,12,13,14]

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