Recombinant expression of the pufQ gene of Rhodobacter capsulatus.
Genetic studies have shown that the expression of the pufQ gene is required for normal levels of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. Yet, the exact function of the pufQ gene is unknown, and a pufQ gene product has never been isolated. We describe the recombinant overexpression of pufQ in Escherichia coli, as well as the purification and characterization of its gene product, the 74-amino-acid PufQ protein. Site-directed mutagenesis was used to facilitate the cloning of the pufQ gene into various expression vector systems of E. coli, including pKK223-3, pLcII-FX, and pMal-c. Although high levels of pufQ transcription were evident from constructs of all three vectors, high levels of protein expression were apparent only in the pMal-c system. In vector pMal-c, the recombinant PufQ protein is expressed as a fusion with an amino-terminal maltose-binding domain. After affinity purification on an amylose column, full-length PufQ protein was released from the fusion protein by limited proteolysis with the enzyme factor Xa. The PufQ protein demonstrated a strong tendency to associate with phospholipid vesicles, consistent with the view that it is an integral membrane protein. The PufQ protein was subsequently purified by high-performance liquid chromatography and identified by amino-terminal sequence analysis. A possible role for the PufQ protein in the transport of bacteriochlorophyll biosynthetic intermediates is discussed.
Fidai
S
S
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Kalmar
G B
GB
Richards
W R
WR
Borgford
T J
TJ
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D005822:Genetic Vectors; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016354:Rhodobacter capsulatus / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D008081:Liposomes; D011993:Recombinant Fusion Proteins