Institute of Functional Interfaces

Soluble full-length expression and characterization of snRNP protein U1-68/70K

  • chair:

    Richter, C. / Simon, T. / Asen, I. / Brenner-Weiss, G. / Hubbuch, J. (2014)

  • place:

    Protein Expression and Purification 104 (2014), 65-70

  • Date: 2014
  • Richter, C. / Simon, T. / Asen, I. / Brenner-Weiss, G. / Hubbuch, J. (2014): „Soluble full-length expression and characterization of snRNP protein U1-68/70K“. In: Protein Expression and Purification 104 (2014), 65-70

Abstract

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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, [16]). 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.