Abstract
Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.
Highlights
Membrane proteins are involved in an array of biological processes including photosynthesis, respiration, signal transduction, molecular transport and catalysis [1] and constitute around 30% of the proteome [2]
We show here that Cr-PTOX1 can be expressed in a functional form in tobacco chloroplasts, and that chloroplasts might be a suitable host for expressing certain types of foreign membrane proteins
Not yet demonstrated experimentally, PTOX1 is likely to function as a plastoquinol oxidase: it shows 47% sequence identity with Arabidopsis thaliana PTOX (IMMUTANS) and has the typical features of a PTOX including the six putative iron-binding sites (E-183, E-222, H-225, E-273, E-324, H-327; numbering according to [17]) [18] and conserved Exon 8 [19], which is required for both the structural as well as functional stability of PTOX [18] (Figure 1B)
Summary
Membrane proteins are involved in an array of biological processes including photosynthesis, respiration, signal transduction, molecular transport and catalysis [1] and constitute around 30% of the proteome [2]. Due to their involvement in cellular communication, they are the targets of more than 50% of current drugs [3] and the focus of drug-discovery programs. Obtaining sufficient amounts of membrane protein, usually through heterologous expression, is often a major barrier for further detailed structural studies. A large number of recombinant proteins have been expressed in the chloroplast, the potential of the chloroplast to express and process heterologous membrane proteins remains largely unexplored, despite the advantage of having an extensive thylakoid membrane system for targeting proteins [7]
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