Chloroplast transformation technology is an exciting platform for the safe, inexpensive and
large-scale production of recombinant proteins in plants, enabling them to be used as cellular
factories. However more work is required to test this technology for the expression of
membrane proteins, purification of recombinant proteins from the chloroplasts and high
throughput cloning strategies. This work was undertaken with a view to expand the utility of
chloroplast transformation technology.
In the first part of the thesis, the potential of chloroplasts to express various membrane
proteins was explored. The test proteins included a plastid terminal oxidase from
Chlamydomonas reinhardtii (Cr-PTOX1) and a human G-protein coupled-receptor,
adenosine receptor A (A2AR). Both proteins were successfully expressed in tobacco
chloroplasts and in the case of Cr-PTOX1 in an active conformation. However, its
transplastomic expression rendered the plants sensitive to high light. Interestingly protection
was observed at the germination stage against salt stress in Cr-PTOX1 seedlings. The A2A
receptor was also expressed; however it was not targeted to thylakoids.
In the second part of the project, the expression in tobacco of two widely used affinity tags,
glutathione S-transferase (GST) and His-tagged maltose-binding proteins (His6-MBP) was
investigated as a mean of improving the purification of chloroplast expressed proteins. Their
expression in transplastomic plant leaves reached approx. ≥7% and 37% of total soluble
proteins, respectively. GST could be purified by one-step-affinity purification using a
glutathione column. Much better recoveries were obtained for His6-MBP by using a twin
affinity purification procedure. Interestingly, the high level expression of GST led to
cytoplasmic male sterility. Similarly, the feasibility of adapting the Gateway® cloning system
for chloroplast transformation was also tested. The results suggested that Gateway® cloning
does not give a high expression of recombinant proteins in tobacco chloroplasts.
Overall this work opens up the opportunities for the expression of foreign membrane proteins
in chloroplasts and expands the tools available for purifying recombinant proteins from the
chloroplasts.