Gjs. Fowler et al., HETEROLOGOUS EXPRESSION OF GENES ENCODING BACTERIAL LIGHT-HARVESTING COMPLEXES IN RHODOBACTER-SPHAEROIDES, The Journal of biological chemistry, 270(40), 1995, pp. 23875-23882
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.