title: Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii.
abstract: Microalgae are diverse photosynthetic microbes which offer the potential for production of a number of high value products (HVP) such as pigments, oils, and bio-active compounds. Fast growth rates, ease of photo-autotrophic cultivation, unique metabolic properties and continuing progress in algal transgenics have raised interest in the use of microalgae systems for recombinant protein (RP) production. This work demonstrates the development of an advanced RP production and secretion system for the green unicellular model alga Chlamydomonas reinhardtii. We generated a versatile expression vector that employs the secretion signal of the native extracellular C. reinhardtii carbonic anhydrase for efficient RP secretion into the culture medium. Unique restriction sites were placed between the regulatory elements to allow fast and easy sub-cloning of sequences of interest. Positive transformants can rapidly be identified by high-throughput plate-level screens via a coupled Gaussia luciferase marker. The vector was tested in Chlamydomonas wild type CC-1883 (WT) and in the transgene expression transformant UVM4. Compared to the native secretion signal of the Gaussia luciferase, up to 84% higher RP production could be achieved. With this new expression system we could generate transformants that express up to 10 mg RP per liter culture without further optimization. The target RP is found exclusively in culture medium and can therefore easily be isolated and purified. We conclude that this new expression system will be a valuable tool for many heterologous protein expression applications from C. reinhardtii in the future.
authors.affiliation: Center for Biotechnology-CeBiTec, Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, Germany.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Lauersen
authors.forename: Hanna
authors.initials: H
authors.lastname: Berger
authors.forename: Jan H
authors.initials: JH
authors.lastname: Mussgnug
authors.forename: Olaf
authors.initials: O
authors.lastname: Kruse
mesh_terms: D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D008156:Luciferases; D002256:Carbonic Anhydrases
keywords: ALG; CAH1; CHO; Chinese hamster ovary; Chlamydomonas reinhardtii; Chlamydomonas reinhardtii carbonic anhydrase 1; Enhanced nuclear gene expression; Gaussia princeps luciferase; High value recombinant protein production; Lollium perenne ice binding protein; LpIBP; Microalgae; PTM; RP; TAP; TSP; Tris acetate phosphate; asparagine linked glycosylation; cCA; gLuc; posttranslational modification; recombinant protein; secretion signal of CAH1; total soluble protein