Myelin protein zero (PO), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the PO gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter PO-PO interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human PO isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of PO extracellular domain (PO-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that PO-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human PO-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to similar to15 OD using orbital shakers and the use of diafiltration, which result in yields of similar to150 mg highly pure protein per liter of medium.