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Characterization and high-yield production of non-N-glycosylated recombinant human BCMA-Fc in Pichia pastoris.

ABSTRACT: B-cell maturation antigen (BCMA) fused at the C-terminus to the Fc portion of human IgG1 (BCMA-Fc) blocks B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL)-mediated B-cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA-Fc (rhBCMA-Fc) in Pichia pastoris under the control of the AOX1 methanol-inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N-linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed-batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA-Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA-Fc possessed high-binding affinity to APRIL and dose-dependent inhibition of APRIL-induced proliferative activity in vitro through three-step purification. Thus, this yeast-derived expression method could be a low-cost and effective alternative to the production of rhBCMA-Fc in mammalian cell lines.

SUBMITTER: Hou W

PROVIDER: S-EPMC6999299 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Characterization and high-yield production of non-N-glycosylated recombinant human BCMA-Fc in Pichia pastoris.

Hou Weihua W Meng Xianchao X Wang Yuxiong Y Mo Wei W Wu Yi Y Yu Min M

Engineering in life sciences 20160706 2

B-cell maturation antigen (BCMA) fused at the C-terminus to the Fc portion of human IgG1 (BCMA-Fc) blocks B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL)-mediated B-cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA-Fc (rhBCMA-Fc) in Pichia pastoris under the control of the AOX1 methanol-inducib ...[more]

PMID: 32624756

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Project description:BackgroundPichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.ResultsAddition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD₅₉₅⁻¹) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (k(L)a) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.ConclusionsWe show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.

Dataset Information

Characterization and high-yield production of non-N-glycosylated recombinant human BCMA-Fc in Pichia pastoris.

Publications

Characterization and high-yield production of non-<i>N</i>-glycosylated recombinant human BCMA-Fc in <i>Pichia pastoris</i>.

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