title: Soluble full-length expression and characterization of snRNP protein U1-68/70K.
abstract: The autoantigen U1-68/70K 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, [16]). Using cell-free expression screening, we successfully produced the snRNP protein U1-68/70K 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.
authors.affiliation: DIARECT AG, Freiburg, Germany; Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Electronic address: Carolin.Richter@kit.edu.
authors.forename: Carolin
authors.initials: C
authors.lastname: Richter
authors.affiliation: DIARECT AG, Freiburg, Germany. Electronic address: Thomas.Simon@diarect.com.
authors.forename: Thomas
authors.initials: T
authors.lastname: Simon
authors.affiliation: DIARECT AG, Freiburg, Germany. Electronic address: Iris.Asen@diarect.com.
authors.forename: Iris
authors.initials: I
authors.lastname: Asen
authors.affiliation: Karlsruhe Institute of Technology, Campus Nord, Institute of Functional Interfaces, Analytical Biochemistry, Eggenstein-Leopoldshafen, Germany. Electronic address: Gerald.Brenner-Weiss@kit.edu.
authors.forename: Gerald
authors.initials: G
authors.lastname: Brenner-Weiss
authors.affiliation: Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Electronic address: Juergen.Hubbuch@kit.edu.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D001324:Autoantigens / Q000096:biosynthesis / Q000302:isolation & purification*; D004926:Escherichia coli; D006801:Humans; D011487:Protein Conformation; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000096:biosynthesis / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry
chemical_list: D001324:Autoantigens; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human
keywords: Autoimmunity; Cell-free expression; Chaperones; snRNP complex