Obesity is characterized by an excessive accumulation of body fat, for which impaired adipogenesis is believed to play a crucial role. As a gatekeeper of early adipogenesis, protein delta homolog 1 (DLK1) has a pivotal role in deciding whether pre-adipocytes will differentiate, determining the balance between healthy and unhealthy fat tissue. Here, an expression system for the cysteine-rich soluble human DLK1 was established. DLK1 was overexpressed in Escherichia coli BL21(DE3)pLysRARE, purified by affinity chromatography and refolded by stepwise dialysis. Identity, purity, secondary structure and refolding efficiency were determined. Proteolytic digestion followed by mass spectrometry analysis proved correct disulfide bridge formation. The biological activity of DLK1 was examined by differentiation assays in murine pre-adipocyte-like 3T3-L1 cells. Thereby, recombinantly produced DLK1 was shown to inhibit adipogenesis in a concentration- and time-dependent manner. All in all, our approach gives access to large amounts of active DLK1 and can be transferred to related proteins. (C) 2015 Elsevier Inc. All rights reserved.