The labile nature of membranes and organelles poses serious challenges to in situ biomolecule characterization in intact cells. Cell-free in vitro systems provide an alternative promising medium for the expression and characterization of protein conformation and function in a biochemical context that bears several similarities to the cellular environment. In addition, cell-free transcription-translation has recently emerged as a convenient method for protein selective isotope labeling, providing significant advantages for detailed NMR analysis. We report the cell-free expression of the model protein apomyoglobin (apoMb) in an Escherichia coli cell-free system and the effect of polyethylene glycol (PEG) on the expression yields. In contrast with in vivo protein production under control of the strong T7 promoter, apoMb is expressed in vitro in 100% soluble form. In-gel tryptic digestion followed by mass spectrometry were performed to confirm the protein identity. In order to probe the conformation of the newly expressed protein and investigate the feasibility of in situ structural analysis, high resolution protein characterization was carried out by 2D NMR spectroscopy. In vitro apoMb expression in a PEG-free environment is a convenient method for the production of soluble native-like protein under conditions amenable to selective isotopic labeling. Yields can be easily scaled-up by dialysis-assisted cell-free expression. (c) 2005 Elsevier Inc. All rights reserved.