Advances in molecular biology have revolutionized the field of biotechnology and allowed the development of recombinant protein production as an alternative to harvesting proteins from their natural sources. Production of target proteins in controllable host organisms offers scalable and economic approaches to meet market needs. Current host cell expression systems vary, and each has advantages and disadvantages. Photoautotrophic organisms, like microalgae, represent alternatives to fermentative microbes with the promise of recombinant protein production from sustainable inputs like carbon dioxide as a carbon source. In this thesis, a Chlamydomonas reinhardtii strain that was recently developed for phototrophic high- density cultivation and nuclear transgene expression was used to express target recombinant proteins from its plastid genome as a demonstration of possibilities for expansion of its potential value. Here, sequences of two anti-freeze proteins, the insect Choristoneura fumiferana (CfAFP) and grass Lolium perenne ice binding protein (LpIBP), and a yellow fluorescent protein (YFP, mVenus) were adapted to algal chloroplast genome expression plasmids, transformed, and protein titers characterized under various nutrient and growth regimes in alga. Rather than antibiotic selection, transformants were selected based on photosynthesis restoration in a knock-out recipient strain. LpIBP and mVenus expression were detected by Western blot and in gel fluorescence and estimated to be expressed up to ~7.65% and ~8.41% total soluble protein, respectively, whereas expression of CfAFP was not observed in any transformant. This work forms the basis of further investigation of recombinant protein expression in C. reinhardtii in high-density antibiotic-free culture and may influence feasibility assessments of scale up processes.
| Identifer | oai:union.ndltd.org:kaust.edu.sa/oai:repository.kaust.edu.sa:10754/691889 |
| Date | 30 April 2023 |
| Creators | Abdullah , Amna |
| Contributors | Lauersen, Kyle J., Biological and Environmental Science and Engineering (BESE) Division, Grunberg, Raik, Saikaly, Pascal |
| Source Sets | King Abdullah University of Science and Technology |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Relation | n/A |
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