HETEROLOGOUS EXPRESSION OF GENES ENCODING BACTERIAL LIGHT-HARVESTING COMPLEXES IN RHODOBACTER-SPHAEROIDES

One of the major problems in structural work on membrane-spanning prot eins is the identification of an expression system which will allow th e production of enough pure protein for structural studies; an inadequ ate expression system can lead, for example, to the formation of unwan ted protein inclusion bodies. In the present work we report the expres sion of genes encoding the light-harvesting 2 (LH2) membrane spanning proteins from a number of species of purple bacteria in mutants of Rho dobacter sphaeroides that lack the native LH2 antenna. The LH2 structu ral genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas ac idophila and Rubrivivax gelatinosus were amplified and tailed by polym erase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb, sphaeroides mutants; DBC Omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synt hesized LH2 complexes that were examined using absorption and fluoresc ence spectroscopy, and Western blotting. Thus, we have created a heter ologous expression system which supports the assembly of a functional ''foreign'' light-harvesting complex. This work opens up the possibili ty of creating site-directed LH2 mutants from bacteria for which no ge netic system is available; this is particularly significant in the cas e of Rps, acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic res olution.