Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum

Abstract

Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO(2)-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies.

Figure 1. In vivo localization studies on antibody heavy and light chain expression in P. tricornutum.

Localization studies demonstrate that heavy chain (HC) and light chain (LC) of the human IgG antibody CL4mAb fused to GFP accumulate in the endoplasmic reticulum of P. tricornutum. Hence, the endogenous signal peptide is sufficient to direct both antibody chains into the ER in the algal system. Scale bar represents 10 µm. PAF – plastid autofluorescence.

Figure 2. Analyses on antibody expression and assembly in P. tricornutum.

A. Twelve independent P. tricornutum transfectants were screened for expression of heavy and light chain of the antibody. Western Blot analyses demonstrate that expression levels are very similar. For further analyses clone number 12 was selected as expression seems to be slightly enhanced. B. Western Blot analyses demonstrate that both antibody chains are expressed in P. tricornutum cells and assemble into complete antibodies. Under reduced conditions both antibody chains are detected separately, whereas in non-reduced SDS-PAGE a high molecular weight signal is detected, which corresponds to the fully-assembled antibody. C. Monoclonal antibodies are expressed under a NO₃ ⁻-inducible promoter and different induction periods were tested to get highest expression levels. Best induction period turned out to be 1–2 days (lane 2+3), which was interestingly similar to expression levels with constant induction from the very beginning (lane 7). D. Antibodies expressed in P. tricornutum were purified using protein A-Sepharose beads. 5% of the eluate was loaded on a SDS-PAGE demonstrating that purification is very efficient with no contaminations detected by Coomassie or Silver Staining. Western Blot analyses with subsequent incubation with the lectin ConA revealed that both antibody chains get glycosylated in the ER of P. tricornutum. ConA – Concanavalin A, d – days.

Figure 3. ELISA with antibodies against the Hepatitis B surface protein expressed in P. tricornutum.

ELISA assays with purified antibody from P. tricornutum and whole cell extract of an antibody expressing clone demonstrate that algae-produced antibodies bind efficiently to commercial HBsAg reaching a binding maximum at 100 ng/ml. There was no antibody binding without HBsAg coated to the wells (negative control) or when using wild type protein extract (data not shown). Error bars indicate standard deviation (n = 3). The typical result of three experiments is shown.

Figure 4. In vivo localization studies on Hepatitis B surface protein (HBsAg) expressed in P. tricornutum.

HBsAg fused to GFP accumulates in punctual patterns, which partially co-localize with an ER-membrane protein (Der1-2) that was expressed as CFP fusion. The ER in P. tricornutum normally spans the whole cell, but is rather atypically aggregated in these clones. Punctual Fluorescence of HBsAg might be due to the formation of virus like particles (VLPs). Scale bar represents 10 µm. PAF – plastid autofluorescence.

Figure 5. Inhibitory ELISA using algae-produced HBsAg and the respective algae-produced antibodies.

Purified Anti-HBsAg antibody from P. tricornutum were mixed with serially diluted Hepatitis B surface protein (adr subtype) from P. tricornutum or S. cerevisiae (pos. control), respectively. HBsAg expressed in P. tricornutum inhibits antibody binding to solid-phase antigen slightly stronger than commercially available HBsAg from S. cerevisiae. Error bars indicate standard deviation (n = 3). The typical result of four experiments is shown.