RECOMBINANT EXPRESSION OF THE PUFQ GENE OF RHODOBACTER-CAPSULATUS

Genetic studies have shown that the expression of the pufQ gene is req uired for normal levels of bacteriochlorophyll biosynthesis in Rhodoba cter capsulatus. Yet, the exact function of the pufQ gene is unknown, and a pufQ gene product has never been isolated. We describe the recom binant overexpression of pufQ in Escherichia coli, as well as the puri fication and characterization of its gene product, the 74-amino-acid P ufQ protein. Site-directed mutagenesis was used to facilitate the clon ing of the pufQ gene into various expression vector systems of E. coli , including pKK223-3, pLcII-FX, and pMal-c. Although high levels of pu fQ transcription were evident from constructs of all three vectors, hi gh levels of protein expression were apparent only in the pMal-c syste m. In vector pMal-c, the recombinant PufQ protein is expressed as a fu sion with an amino-terminal maltose-binding domain. After affinity pur ification on an amylose column, full-length PufQ protein was released from the fusion protein by limited proteolysis with the enzyme factor X(a). The PufQ protein demonstrated a strong tendency to associate wit h phospholipid vesicles, consistent with the view that it is an integr al membrane protein. The PufQ protein was subsequently purified by hig h-performance liquid chromatography and identified by amino-terminal s equence analysis. A possible role for the PufQ protein in the transpor t of bacteriochlorophyll biosynthetic intermediates is discussed.