Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications. However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification. The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization processes are also discussed.
Albendazole (ABZ) is a therapeutic benzimidazole used to treat giardiasis that targets β-tubulin. However, the molecular bases of ABZ resistance in Giardia duodenalis are not understood because β-tubulin in ABZ-resistant clones lacks mutations explaining drug resistance. In previous work we compared ABZ-resistant (1.35, 8, and 250 μM) and ABZ-susceptible clones by proteomic analysis and eight proteins involved in energy metabolism, cytoskeleton dynamics, and antioxidant response were found as differentially expressed among the clones. Since ABZ is converted into sulphoxide (ABZ-SO) and sulphone (ABZ-SOO) metabolites we measured the levels of these metabolites, the antioxidant enzymes and free thiols in the susceptible and resistant clones. Production of reactive oxygen species (ROS) and levels of ABZ-SO/ABZ-SOO induced by ABZ were determined by fluorescein diacetate-based fluorescence and liquid chromatography respectively. The mRNA and protein levels of antioxidant enzymes (NADH oxidase, peroxiredoxin 1a, superoxide dismutase and flavodiiron protein) in these clones were determined by RT-PCR and proteomic analysis. The intracellular sulfhydryl (R-SH) pool was quantified using dinitrobenzoic acid. The results showed that ABZ induced ROS accumulation in the ABZ-susceptible Giardia cultures but not in the resistant ones whilst the accumulation of ABZ-SO and ABZ-SOO was lower in all ABZ-resistant cultures. Consistent with these findings, all the antioxidant enzymes detected and analyzed were upregulated in ABZ-resistant clones. Likewise the R-SH pool increased concomitantly to the degree of ABZ-resistance. These results indicate an association between accumulation of ABZ metabolites and a pro-oxidant effect of ABZ in Giardia-susceptible clones. Furthermore the antioxidant response involving ROS-metabolizing enzymes and intracellular free thiols in ABZ-resistant parasites suggest that this response may contribute to overcome the pro-oxidant cytotoxicity of ABZ.
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