Hp. Davey et al., IDENTIFICATION OF A ZINC-BINDING CYSTIC-FIBROSIS ANTIGEN IN HUMAN SALIVA BY ZN-65 PROBING AND N-TERMINAL SEQUENCING, Archives of oral biology, 42(12), 1997, pp. 861-867
Human whole saliva was collected at 4 degrees C into a protein inhibit
or cocktail to prevent proteolytic degradation. Saliva was clarified b
y centrifugation and fractionated by immobilized metal affinity chroma
tography (IMAC) after charging the column with zinc ions. Proteins wit
h an affinity for zinc were eluted from the IMAC column, characterized
by sodium dodecylsulphate-polyacrylamide gel electrophoresis, and pro
bed with (ZnCl2)-Zn-65 after electrotransfer on to polyvinylidenefluor
ide membranes. A zinc-binding protein of approx. 11 kDa was characteri
zed by N-terminal sequencing followed by a FASTA search of Genbank. Th
e first 40 residues were sequenced, of which the first 34 residues wer
e used to conduct a FASTA search and yielded an homology >97% coding f
or sequences of mRNA of two proteins, a cystic fibrosis antigen with a
n M-r of 10,938 and a calcium-binding inflammatory protein MRP8 with a
n M-r of 10,835 expressed in chronic inflammation. The identity of the
last 15 residues of the sequence and the likelihood that the protein
is secreted via saliva indicates that the 11-kDa protein is the cystic
fibrosis antigen, a protein not previously reported in saliva. (C) 19
97 Published by Elsevier Science Ltd. All rights reserved.