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.
The Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037, USA.
Anna T
AT
Coragliotti
Maria Verónica
MV
Beligni
Scott E
SE
Franklin
Stephen P
SP
Mayfield
D001709:Biotechnology / Q000379:methods; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases