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Expression of cysteine‐rich C‐terminal domains of Pig Gastric Mucin in Pichia pastoris
Author(s) -
Turner Bradley,
Bansil Rama,
Afdhal Nezam H
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1318
Subject(s) - pichia pastoris , mucin , glycoprotein , microbiology and biotechnology , complementary dna , glycosylation , biochemistry , chemistry , amino acid , signal peptide , western blot , biology , peptide sequence , gene , recombinant dna
Pig gastric mucin (PGM), a large secreted glycoprotein (10MDa) produced by the epithelial lining of the stomach, is responsible for the viscous properties of the mucus layer that protects and lubricates the gastric epithelium. The PGM cDNA is homologous to human MUC5AC, contains a large tandemly repeated Ser‐Thr‐Pro domain that is O‐glycosylated and Cys‐domains at the C‐terminus that are globular, less glycosylated and contain von Willebrand factor subdomains (vWFd, vWFc, cystine‐knot [CK]). The Cys‐domains are important for the pH dependent gelation in PGM. To investigate the biophysical properties of these PGM domains, we have expressed them in the Pichia pastoris system. The C‐terminus of PGM and its subdomains were amplified by domain specific PCR primers, cloned into pDEST911 and transfected into Pichia strain SMD1163. Resulting clones were screened for protein production by Western blot analysis using antibodies specific for PGM peptide core and human MUC5AC. His6‐tagged peptides corresponding to the PGM C‐terminus, vWFd, vWFc and CK were secreted in the medium with molecular weights 200, 160, 51, 30 kDa respectively, which reacted with the antibodies. All peptides exhibited evidence of disulfide linked multimer formation and glycosylation. We have successfully expressed PGM C‐terminal peptides which will be used for biophysical investigations of PGM gelation. Study funding: NIH and NSF