Renaturation of the catalytic domain of PDE4A expressed in Escherichia coli as inclusion bodies.
Owing to simplicity, speed, cost advantage, and a generally high product yield, expression in Escherichia coli is the method of choice for the production of large amounts of protein. However, because of the high expression level, proteins often accumulate within the cells as insoluble aggregates called inclusion bodies. The inclusion body protein is misfolded and biologically inactive and, thus, needs to be refolded into its native conformation. There is no universal method for refolding inclusion bodies and optimal conditions have to be determined empirically for any given protein. Here, we describe a simple and efficient refolding protocol for the catalytic domain of type 4 cyclic nucleotide phosphodiesterases (PDE4s). This method has the potential for adaptation to other PDE subtypes.
Department of Obstetrics and Gynecology, Stanford University School of Medicine, California, USA.
Wito
W
Richter
Thomas
T
Hermsdorf
Dietrich
D
Dettmer
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004925:Escherichia / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human