Optimized expression and refolding of human keratoepithelin in BL21 (DE3)

Ching Yuan, Janice M. Reuland, Lyndon Lee, Andrew J.W. Huang

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Keratoepithelin (KE) is an extracellular protein participating in cell adhesion and differentiation. Mutations of the KE gene (on 5q31 in humans) cause deposition of abnormal proteins (amyloid and non-amyloid) in corneal stroma and lead to several corneal dystrophies in humans. However, further studies on the KE protein have been limited by the intrinsic difficulty of purifying this protein. A high-expression plasmid containing human KE gene was constructed to generate recombinant KE proteins in Escherichia coli. The plasmid was transformed into E. coli BL21 (DE3) and the recombinant protein was expressed as an insoluble His-tagged fusion protein and purified by nickel chelation affinity chromatography under denaturing conditions. On average, 12 mg of purified KE was routinely obtained from 1 L of culture media. The recombinant KE was refolded in arginine-containing dialysis solutions and the recovery of bioactive KE typically was ∼70%. The procedures developed in this report should enable reproducible production of KE and related mutant proteins in large quantities and facilitate future studies on biochemical and biophysical properties of KE and the pathogenesis of related corneal dystrophies.

Original languageEnglish
Pages (from-to)39-45
Number of pages7
JournalProtein Expression and Purification
Volume35
Issue number1
DOIs
StatePublished - May 2004
Externally publishedYes

Keywords

  • BIGH3
  • Keratoepithelin
  • Refolding

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