Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

Hempel, Franziska; Lau, Julia; Klingl, Andreas; Maier, Uwe G.

doi:10.1371/journal.pone.0028424

Cited by 159 publications

(130 citation statements)

References 51 publications

Supporting

Mentioning

128

Contrasting

Unclassified

Order By: Relevance

“…Variants with and without an ER retention signal (DDEL) at the C-terminus were generated and introduced into P. tricornutum as described previously [19,42]. …”

Section: Methodsmentioning

confidence: 99%

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

2012

Self Cite

View full text Add to dashboard Cite

BackgroundAlthough 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.ResultsIn 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.ConclusionsMicroalgae 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.

show abstract

“…Variants with and without an ER retention signal (DDEL) at the C-terminus were generated and introduced into P. tricornutum as described previously [19,42]. …”

Section: Methodsmentioning

confidence: 99%

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

2012

Self Cite

View full text Add to dashboard Cite

show abstract

“…P. tricornutum was cultivated in f/2 medium under constant illumination (80 μmol photons·m −2 ·s −1 ) at 22°C as described previously (44). Liquid cultures were grown with agitation (150 rpm) in a volume of 300 mL to a density of approximately OD 600 = 0.8 with 1.5 mM NH 4 Cl as sole nitrogen source.…”

Section: Methodsmentioning

confidence: 99%

Evidence for glycoprotein transport into complex plastids

Peschke¹,

Moog

Klingl

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Diatoms are microalgae that possess so-called "complex plastids," which evolved by secondary endosymbiosis and are surrounded by four membranes. Thus, in contrast to primary plastids, which are surrounded by only two membranes, nucleus-encoded proteins of complex plastids face additional barriers, i.e., during evolution, mechanisms had to evolve to transport preproteins across all four membranes. This study reveals that there exist glycoproteins not only in primary but also in complex plastids, making transport issues even more complicated, as most translocation machineries are not believed to be able to transport bulky proteins. We show that plastidal reporter proteins with artificial N-glycosylation sites are indeed glycosylated during transport into the complex plastid of the diatom Phaeodactylum tricornutum. Additionally, we identified five endogenous glycoproteins, which are transported into different compartments of the complex plastid. These proteins get N-glycosylated during transport across the outermost plastid membrane and thereafter are transported across the second, third, and fourth plastid membranes in the case of stromal proteins. The results of this study provide insights into the evolutionary pressure on translocation mechanisms and pose unique questions on the operating mode of well-known transport machineries like the translocons of the outer/inner chloroplast membranes (Toc/Tic).

show abstract

“…Stable expression of the glycosidases might be considered to eliminate inter-batch yield variations. A priori the production in another “plant” host might also be considered, for example stable expression in lettuce ( Lactuca sativa ), algae or moss, in order to conceivably enhance yields and to avoid the high levels of phenolic and toxic alkaloids present in Nicotiana plants that might affect the purification procedures (Hempel et al, 2011; Chen et al, 2013; Xu and Zhang, 2014; Reski et al, 2015). Furthermore, recombinant human α-GAL has earlier been successfully produced in cultured Nicotiana tabacum cells and in an engineered moss cell line, eliminating possible transgene flow that might occur while using whole plants as production platforms (Kizhner et al, 2014; Shen et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Human Alpha Galactosidases Transiently Produced in Nicotiana benthamiana Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease

Kytidou¹,

Beenakker

Westerhof

et al. 2017

Front. Plant Sci.

View full text Add to dashboard Cite

Deficiency of α-galactosidase A (α-GAL) causes Fabry disease (FD), an X-linked storage disease of the glycosphingolipid globtriaosylcerammide (Gb3) in lysosomes of various cells and elevated plasma globotriaosylsphingosine (Lyso-Gb3) toxic for podocytes and nociceptive neurons. Enzyme replacement therapy is used to treat the disease, but clinical efficacy is limited in many male FD patients due to development of neutralizing antibodies (Ab). Therapeutic use of modified lysosomal α-N-acetyl-galactosaminidase (α-NAGAL) with increased α-galactosidase activity (α-NAGALEL) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional α-GAL, α-NAGAL, and α-NAGALEL enzymes for research purposes. All enzymes could be visualized with activity-based probes covalently binding in their catalytic pocket. Characterization of purified proteins indicated that α-NAGALEL is improved in activity toward artificial 4MU-α-galactopyranoside. Recombinant α-NAGALEL and α-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than α-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of α-NAGALEL, and to a lesser extent that of α-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified α-NAGALEL might reduce excessive Lyso-Gb3 in FD serum. This neo-enzyme can be produced in Nicotiana benthamiana and might be further developed for the treatment of FD aiming at reduction of circulating Lyso-Gb3.

show abstract

Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

Cited by 159 publications

References 51 publications

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

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

Evidence for glycoprotein transport into complex plastids

Human Alpha Galactosidases Transiently Produced in Nicotiana benthamiana Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease

Contact Info

Product

Resources

About