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Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

ABSTRACT: Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.

SUBMITTER: Hennicke J

PROVIDER: S-EPMC7067452 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

Hennicke Julia J Schwaigerlehner Linda L Grünwald-Gruber Clemens C Bally Isabelle I Ling Wai Li WL Thielens Nicole N Reiser Jean-Baptiste JB Kunert Renate R

PloS one 20200312 3

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, poly ...[more]

PMID: 32163462

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Project description:The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single [4Fe-4S]1+,2+ cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys. The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli. While the recombinant form of the wild-type and the D14S and D14C mutants contained a [4Fe-4S]1+,2+ cluster, the D14V, D14H, D14Y, and D14N proteins contained a [3Fe-4S]0,+ center, as determined by visible spectroscopy and electrochemistry. The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (Em -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, Em -203 mV). The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation. The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain. With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate. While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated DeltaS degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14. In contrast, there was a linear correlation between the DeltaH degrees' value and reduction potential. Kinetic analyses were carried out at 80 degrees C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P. furiosus). The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems. However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from POR and donate them to FNOR.

Project description:Current approaches to derive oligodendrocytes from human pluripotent stem cells (hPSCs) need extended exposure of hPSCs to growth factors and small molecules, which limits their clinical application because of the lengthy culture time required and low generation efficiency of myelinating oligodendrocytes. Compared to extrinsic growth factors and molecules, oligodendrocyte differentiation and maturation can be more effectively modulated by regulation of the cell transcription network. In the developing central nervous system (CNS), two basic helix-loop-helix transcription factors, Olig1 and Olig2, are decisive in oligodendrocyte differentiation and maturation. Olig2 plays a critical role in the specification of oligodendrocytes and Olig1 is crucial in promoting oligodendrocyte maturation. Recently viral vectors have been used to overexpress Olig2 and Olig1 in neural stem/progenitor cells (NSCs) to induce the maturation of oligodendrocytes and enhance the remyelination activity in vivo. Because of the safety issues with viral vectors, including the insertional mutagenesis and potential tumor formation, non-viral transfection methods are preferred for clinical translation. Here we report a poly(β-amino ester) (PBAE)-based nanoparticle transfection method to deliver Olig1 and Olig2 into human fetal tissue-derived NSCs and demonstrate efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. This approach is potentially translatable for engineering stem cells to treat injured or diseased CNS tissues.Statement of significanceCurrent protocols to derive oligodendrocytes from human pluripotent stem cells (hPSCs) require lengthy culture time with low generation efficiencies of mature oligodendrocytes. We described a new approach to enhance oligodendrocyte differentiation through nanoparticle-mediated transcription modulation. We tested an effective transfection method using cell-compatible poly (β-amino ester) (PBAE)/DNA nanoparticles as gene carrier to deliver transcription factor Olig1 and Olig2 into human fetal tissue-derived neural stem/progenitor cells, and showed efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. We believe that this translatable approach can be applied to many other cell-based regenerative therapies as well.

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Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

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Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

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