Testing production of membrane proteins in cyanobacterial thylakoid by mean of the paradigmatic E. coli subunit b of F1Fo ATP synthase

Membrane proteins (MPs) play pivotal biological roles in living cells: through the transport of ions, solutes and water, they maintain cellular homeostasis; they sense and transduce changes in the cellular environment; they control the cell–to-cell contacts required to ensure coordinated cellular activity in all organisms. Knowing the structure of MPs is thus essential to decipher the molecular mechanism underlying their function. However, crystallization of MPs is by far lagging behind that of soluble proteins, essentially because of the difficulties in over-producing them as recombinant products in conventional biological systems. Dedicated surveys [1; 2] have shown that the T7 RNA polymerase-based expression system and the E.coli BL21(DE3) derivative strains C41(DE3) and C43(DE3), characterized by the proliferation of intracellular membranes upon overexpression of MPs [3; 4], accounts for more than 60 % of MP structures obtained after heterologous production. A large surface area of membranes (that is an unnatural feature in E.coli while is typical of photosynthetic systems) appears therefore as a prerequisite for hosting recombinant MPs. In the frame of a project aimed at enlarging the panel of tools and practicable strategies for the production of large quantities of MPs, we planned to test thylakoid membranes of the two unicellular organisms, the cyanobacterium Synechocystis PCC 6803 and the alga Chlamydomonas rehinardtii [5]. Here, we will report on the production of Synechocystis strains expressing the E. coli subunit b of F1F0 ATP synthase, used as model protein. Two strains, the first one based on the T7 RNA polymerase and a second one exploiting the endogenous constitutive Pcpc560 promoter, will be compared.