The autoantigen U1-68/70 K is the dominant diagnostic marker in Mixed Connective Tissue Disease (MCTD) that until recently could not be expressed in its full-length form (Northemann et al., 1995, (161). Using cell-free expression screening, we successfully produced the snRNP protein U1-68/70 K in a soluble full-length form in Escherichia coli cells. The protein length and identity was determined by Western Blot and MS/MS analysis. Additionally, its reactivity in the autoimmune diagnostic was confirmed. Establishment of a cell-free expression system for this protein was important for further elucidation of protein expression properties such as the cDNA construct, expression temperature and folding properties; these parameters can now be determined in a fast and resource-conserving manner. (C) 2014 Elsevier Inc. All rights reserved.