A strategy for the production of soluble human senescence marker protein-30 in Escherichia coli.
Senescence marker protein-30 (SMP30) has been reported to hydrolyze diisopropyl fluorophosphate (DFP), a surrogate compound of chemical warfare nerve agents. Thus, SMP30 has the potential to be useful as a prophylactic against chemical warfare nerve agent toxicity. Our efforts to generate human SMP30 in bacteria using a variety of expression vectors invariably resulted in insoluble and inactive preparations. In this study, properly folded and active recombinant human SMP30 (rHuSMP30) was produced in Escherichia coli by coexpressing it with molecular chaperones in a combined strategy. The coexpression of rHuSMP30 with GroES/GroEL/Tf at 15 degrees C, combined with the addition of a membrane fluidizer, increased osmolytes, and a two-step expression resulted in the highest enhancement of solubility and DFPase activity. Our results pave the way for exploring the use of rHuSMP30 against organophosphate and nerve agent toxicity.
Choi
Moonsuk S
MS
Division of Bacterial and Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. moonsuk.choi@amedd.army.mil
Saxena
Ashima
A
Chilukuri
Nageswararao
N
D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012995:Solubility
D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; C513323:RGN protein, human; D011994:Recombinant Proteins