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Fig. 1 | Microbial Cell Factories

Fig. 1

From: From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein

Fig. 1

Phaeodactylum tricornutum secretes fully assembled α MARV NP IgG antibodies into the medium. a In vivo localization studies on the expression of heavy and light α MARV NP antibody chains in P. tricornutum. Sequences for the light and heavy chain of the murine IgG2a antibody against the Marburg virus nucleoprotein were retrieved from a hybridoma cell line and expressed as GFP fusion proteins in the diatom P. tricornutum. Both fusion proteins are expressed in the heterologous system without codon optimization and accumulate within the ER of the cells demonstrating that the murine signal peptide is sufficient for ER targeting. Scale bar represents 10 µm. b Purity analysis of secreted α MARV NP IgG antibodies. Proteins of 30 ml culture medium were concentrated with centrifugal filter units (cut off of 10 kDa), separated by SDS-PAGE and stained with Coomassie. Besides the antibody heavy and light chain (marked with asterisks) two dominant signals were detected at ~130 kDa representing a known phosphatase (Phatr2 ID: 47612, verified by mass spectrometry). After protein A purification no background remained, but a weak signal for a HC variant with reduced molecular weight is detected (verified by mass spectrometry). c Biochemical analyses of secreted α MARV NP IgG antibodies. 100 ng of purified hybridoma produced antibody and antibody from the algal medium were prepared under reduced and non reduced conditions, separated by SDS-PAGE and analysed in western blot studies using an α mouse secondary antibody for detection. Under reduced conditions heavy and light chain of the antibody are clearly separated and appear as distinct signals of 55 and 30 kDa. In case of the algal produced antibody the light chain seems to accumulate to slightly higher amounts. Under non reduced conditions disulfide bridges stay intact and the assembled antibody consisting of two heavy and two light chains is detected with a size of about 170 kDa. Lower molecular weight signals are observed in case of both antibodies with no significant difference. Ab antibody, HC heavy chain, LC light chain, MARV Marburg virus, NP nucleoprotein, PAF plastid autofluorescence

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