An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency

Abstract

Background: Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins.

Results: In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient.

Conclusions: Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins.

Figure 1

Analyses on antibody secretion byP. tricornutum. Light and heavy chain of the human IgG antibody CL4mAb against the Hepatitis B Virus surface protein were expressed in P. tricornutum for two days either with (+DDEL) or without an ER-retention signal (−DDEL). Proteins of the cellular fraction (c) and the cell-free medium (m) were subsequently separated by gel electrophoresis and analyzed by Western Blot and Coomassie/Silver Staining. In cells expressing an ER-retention signal hardly any antibody is detected in the medium, whereas the deletion of the retention signal leads to efficient antibody secretion. Under non-reduced conditions a high molecular weight signal corresponding to the fully assembled antibody (consisting of two heavy and two light chains) is detected. For Western Blot analyses/Coomassie and Silver Staining proteins of 15 ml/30 ml cell-free medium were concentrated and precipitated. 10 μg protein of the cellular fractions was loaded.

Figure 2

Comparison of antibody secretion efficiency of differentP. tricornutumtransfectants. In a small scale screening twelve independent transfectants were tested for antibody expression and secretion efficiency after two days of protein expression. Proteins of the cellular fraction (c) and the cell-free medium (m) were precipitated and separated by SDS-PAGE. Western Blot analyses demonstrate that except for cell line #4 all transfectants express the antibody and secrete it efficiently into the medium. Cell lines #3, #8, #11 and #12 were selected four broader analyses on antibody functionality and quantity. As a loading control for cellular proteins and to check that the medium is not contaminated with intracellular proteins of destroyed cells an antibody against α-tubulin was used. 10 μg protein of the cellular fraction and protein of 15 ml cell-free medium were loaded.

Figure 3

Analyses on functionality and quantity of secreted antibodies in four independent cell lines. Antibody expression in cell lines #3, #8, #11 and #12 was induced at time point T0 with a culture density of 0.65. Subsequently, samples of the medium were taken over three days (T1, T2, T3) followed by a medium exchange (T0*) and two additional days of sampling (T1*, T2*). Functionality of the antibody, i.e. the efficiency of the antibody to bind to its antigen, was measured by ELISA assays. The secreted antibody binds to the antigen with an efficiency that basically correlates with the antibody amounts measured in the samples. During the first two days antibody quantities within the medium increased to concentrations of 450–850 ng/ml. After day two the amount of antibody in the medium slightly decreased, which is consequently accompanied by a loss in binding efficiency. The removal of the antibody containing medium and addition of fresh nitrate-containing medium restored antibody expression/secretion and resulted in antibody concentrations of 1550–2550 ng/ml at day T2* depending on the cell line tested. Error bars indicate standard deviation (n = 3).