Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli

Heather L. Cash, Cecilia V. Whitham, Lora V. Hooper

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIγ and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of β-strands which is typical of C-type lectins. Finally, both RegIIIγ and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.

Original languageEnglish (US)
Pages (from-to)151-159
Number of pages9
JournalProtein Expression and Purification
Volume48
Issue number1
DOIs
StatePublished - Jul 1 2006

Keywords

  • C-type lectins
  • Carbohydrate binding
  • Inflammatory bowel disease
  • Mucosal injury
  • Regenerating protein family

ASJC Scopus subject areas

  • Biotechnology

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