Optimized expression and refolding of human keratoepithelin in BL21 (DE3).
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 1L of culture media. The recombinant KE was refolded in arginine-containing dialysis solutions and the recovery of bioactive KE typically was approximately 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.
Yuan
Ching
C
Department of Ophthalmology, University of Minnesota, Minneapolis, MN, USA. huang088@umn.edu
Reuland
Janice M
JM
Lee
Lyndon
L
Huang
Andrew J W
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002448:Cell Adhesion; D003001:Cloning, Molecular; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein