Developing new tools for the expression of foreign proteins in tobacco chloroplasts

Abstract

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.