title: Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter.
abstract: Microalgae have potential as platforms for the synthesis of high-value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co-expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl-1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl-1. This study suggests that recombinant protein expression is product-specific and needs to be optimized individually.
authors.affiliation: Department of Biochemical Engineering, University College London, United Kingdom. stephanie.braun.10@ucl.ac.uk.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Braun-Galleani
authors.affiliation: Department of Biochemical Engineering, University College London, United Kingdom.
authors.forename: Frank
authors.initials: F
authors.lastname: Baganz
authors.affiliation: Institute of Structural and Molecular Biology, University College London, United Kingdom.
authors.forename: Saul
authors.initials: S
authors.lastname: Purton
mesh_terms: D001709:Biotechnology; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts; D017930:Genes, Reporter / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins
keywords: Chlamydomonas reinhardtii; Chloroplast; Fluorescence; Protein expression; Reporter protein