Ever wanted to produce huge amount of your favorite recombinant protein? Here we will show you how! In this part of the project we aim to develop constitutive chimeric promoters that could allow for accumulation of different levels of recombinant proteins in Chlamydomonas reinhardtii chloroplast. The psbA promoter is one of the strongest promoters in green eukaryotic algae. This promoter has been used in the past (reference) to drive the expression of foreign proteins. Unfortunately, this promoter seems to render high level of protein accumulation only when the endogenous psbA product, the D1 protein, is absent. This has led some people to think that the D1 is somehow regulating the transcription of the ORF under the control of the psbA promoter.
We think that since the psbA mRNA levels are fairly constant throughout the growing cycle, the D1 protein is actually regulating translation of the mRNA by interacting with the 5´UTR. We reasonedº that if we can relieve the mRNA from this control element, but keeping its transcription at high level, a higher level of recombinant protein accumulation could be achieved. We think that replacing the psbA 5´UTR with other 5´UTR that have been shown to work well in prokaryotes can do this. Given the similarities of the chloroplast with prokaryotes, the effectiveness of these 5´UTRs should remain to a certain extent.
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