MAP kinase phosphatase 3 (MKP3, also known as DUSP6 and PYST1) is involved in extracellular signal receptor kinase (ERK) regulation and functions as a specific phosphatase to the activated (phosphorylated) forms of ERK1 and ERK2. MKP3 displays allosteric activation, which aids in tightly regulating its function to ERK substrates, but not other related MAPKs. Due to MKP3's specificity for the ERK signaling pathway, the development of specific activators or inhibitors to the enzyme have been suggested in order to expressly influence the ERK1 and ERK2 pathways. To produce the high yields of MKP3 protein necessary for physico-chemical characterization of MKP3 and for high throughput screening of its small-molecule activators and inhibitors, we have cloned, purified and, and identified refolding conditions suitable for producing full-length, human MKP3 from Escherichia coli inclusion bodies. Furthermore, we demonstrate the use of a 96-well plate format refolding assay in which the ERK-induced activity of MKP3 is simulated by 33% DMSO. The assay allowed for rapid detection of MKP3's function following a refolding screen in the absence of ERK and thus provides quick and inexpensive testing of MKP3 activity. Following screening, the refolded product was confirmed to be correctly folded by steady-state kinetic analysis and by the CD spectroscopy-determined secondary structure content. CD data were consistent with 36% helix and 14% sheet, which compared to an expected 32.9% helix and 12.4% sheet. These data indicated that MKP3 was properly folded, making it a suitable protein for use in functional studies. Crown copyright (C) 2007 Published by Elsevier Inc. All rights reserved.