Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein.
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.
Yu
Liyan
L
Department of Biology, Georgia State University, Atlanta, 30303, USA.
Yang
Hsiuchin
H
Tai
Phang C
PC
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins