Expression, purification, and characterization of Pseudomonas aeruginosa SecA.
A secA gene from Pseudomonas aeruginosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13) under conditions where E. coli SecA was depleted. The binding of P. aeruginosa SecA (PaSecA) to the SP-Sepharose column was facilitated by ammonium sulfate fractionation but was not necessary for E. coli SecA (EcSecA) as the later bound more efficiently. PaSecA and EcSecA were purified by the single chromatographic step to greater than 98% purity and had a recovery of more than 20 and 40%, respectively, from the soluble fraction. This simple step purification obtained a higher homogeneity than previously reported. Cross-reactivity by immunoblotting showed that the purified PaSecA contained little EcSecA if any. The purified PaSecA is a dimer in solution, as judged by size exclusion chromatography, and is slightly larger than its counterpart EcSecA with an estimated molecular weight of 240 kDa. Further studies by the sedimentation velocity method indicate that PaSecA tends to remain as a monomer in solution. The purified PaSecA possessed ATPase activity; the intrinsic and liposome-stimulated ATPase specific activities of PaSecA were approximately 50% of EcSecA.
Yu
Liyan
L
Department of Biology, Georgia State University, 24 Peachtree Center Avenue, 402 Kell Hall, Atlanta, GA 30303, USA.
Yang
Hsiuchin
H
Ho
Quynh
Q
Tai
Phang C
PC
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D003429:Cross Reactions; D019281:Dimerization; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D008081:Liposomes / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D000906:Antibodies; D001426:Bacterial Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins