Cloning and expression of bovine corneal antigen cDNA.
OBJECTIVE
A cornea-associated antigen (CO-Ag) has been found to be the target for autoantibodies in patients with Mooren's ulcer. The study goals were to isolate a full-length clone encoding CO-Ag from a bovine corneal cDNA library and to express this clone in Escherichia coli (E. coli).


METHODS
A DNA fragment of CO-Ag was generated, using unique oligonucleotide primers and reverse transcription polymerase chain reaction. This fragment was used as a probe to obtain cDNA clones from a bovine corneal cDNA library. The clone with the longest cDNA insert was selected for sequence analysis. Expression of the CO-Ag protein in E. coli was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). The bacterially-produced CO-Ag was partially purified by calcium (Ca2+)-dependent hydrophobic interaction chromatography.


RESULTS
The cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail. The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal. This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons. The cDNA and deduced amino acid sequence of CO-Ag are completely identical to a S-100 protein, bovine calgranulin C. The cDNA was expressed in E. coli as a fusion protein consisting of 583 N-terminal amino acids of beta-galactosidase (beta-gal), 91 amino acids of CO-Ag, and possibly a number of additional N-terminal and C-terminal residues. The bacterially produced CO-Ag was fully functional with respect to hydrophobic interaction with phenyl-Sepharose matrix for its isolation. The fusion protein was recognized by antiserum raised against bovine CO-Ag protein on Western blots.


CONCLUSIONS
The isolation and analysis of a cDNA clone containing the complete coding sequence of the CO-Ag protein and the expression of the CO-Ag protein in E. coli is reported. The availability of a CO-Ag cDNA probe and larger quantities of the CO-Ag protein should aid in elucidating the possible pathogenic role of CO-Ag in Mooren's ulcer.
Gottsch
J D
JD
Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. JGottsch@Welchlink.welch.jhu.edu
Liu
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000235:genetics*; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D003001:Cloning, Molecular; D003315:Cornea / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005784:Gene Amplification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D000941:Antigens; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins