Scott Franklin

Expression and assembly of a fully active antibody in algae

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5′ and 3′ RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5′ and 3′ elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes

Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5′- or 3′-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5′- and 3′-UTRs. In general, the 5′-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5′-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3′-UTR had little impact on mRNA and protein accumulation, as long as a 3′-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5′-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.

Recent developments in the production of human therapeutic proteins in eukaryotic algae

Antibody-based therapeutics have had great success over the last few years, and continue to be one of the fastest growing sectors of drug development. The efficacy and specificity of antibody-based drugs makes them ideal candidates for new drug development, but the specificity of these drugs comes from their complexity, and this complexity makes antibodies very expensive to produce. To address this problem, the authors have developed a system for the expression of recombinant proteins using the unicellular eukaryotic green algae, Chlamydomonas reinhardtii. As proof of concept, the authors have engineered microalgae to produce several forms of a human IgA antibody directed against herpes simplex virus. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian-based expression systems, as both the plastid and nuclear genomes are easily and quickly transformed, and the production of proteins in algae has an inherently low cost of capitalisation and production.

Safety evaluation of a high-lipid algal biomass from Chlorella protothecoides.

Chlorella are traditionally freshwater green algae that have been evaluated for dietary purposes because of their nutritional value. This study investigates the safety of Chlorella protothecoides in a 28-day study. Sprague-Dawley rats were administered 0 (control), 2.5, 5.0, or 10% of their diet for 28days using an FDA Redbook protocol. The average daily dietary intake of algal biomass was determined to be 0, 1794, 3667, and 7557 mg/kg body weight for males and 0, 1867, 3918, and 8068 mg/kg body weight for females. Hematological and biochemical analyses were conducted, and upon completion, gross and microscopic evaluations were performed. No signs of toxicity were observed. Although statistically significant alterations were noted in several parameters among males and females, these changes were deemed to be of no toxicological significance due to the lack of dose-response relationships, the fact that they occurred in only one sex, and the lack of any supporting gross or microscopic alterations. The no-observed-adverse-effect level for the algal biomass under the conditions of this study was considered to be 10% in the diet, the highest dose tested.

Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast.

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.

Safety evaluation of oleic-rich triglyceride oil produced by a heterotrophic microalgal fermentation process

Numbers of macro- and microalgae have been used as food sources in various cultures for centuries. Several microalgae are currently being developed as modern food ingredients. The dietary safety of oleic-rich microalgal oil produced using a heterotrophic fermentation process was assessed in a 13-week feeding trial in rats with genotoxic potential evaluated using in vitro and in vivo assays. In the genotoxicity assays, the test oil was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains (⩽5000μg/plate) with or without metabolic activation. Further, no clastogenic response occurred in chromosome aberration assays in the bone marrow of mice administered a single intraperitoneal dose (2000mg/kg). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000ppm oleic-rich oil for 90days. No treatment-related mortalities or adverse effects occurred in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights or histopathology. Although several endpoints exhibited statistically significant effects, none were dose-related or considered adverse. Taking all studies into consideration, the NOAEL for the oleic-rich oil was 100,000ppm, the highest concentration tested and equivalent to dietary NOAELs of 5200mg/kg bw/day and 6419mg/kg bw/day in male and female rats, respectively.

16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes.

Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships.

13-week dietary study and in vitro and in vivo genotoxicity studies of a structuring fat produced through a microalgal fermentation process

Highlights • The dietary safety of a unique microalgal structuring fat produced by a heterotrophic fermentation process was assessed.• The algal fat was non-mutagenic in Salmonella typhimurium and Escherichia coli tester strains at up to 5000 μg/plate.• The algal fat was non-clastogenic in mouse bone marrow after a single oral dose at 2000 mg/kg bw.• Dietary NOAEL of the algal structuring fat for rats in a 13-week study was 100 000 ppm, the highest concentration tested.• Based on consumption, the oral NOAEL for the algal structuring fat in rats was 5299 (male) and 6313.8 (female) mg/kg bw/day.