High level expression in E. coli of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat.
The high molecular weight mucin found in human milk fat globule and on the surface of mammary and other epithelial cells contains a 20 amino acid tandem repeat sequence that is highly immunogenic. We have immunoscreened lambda gt11 cDNA expression libraries from MCF7 cells and lactating breast tissue with 5 anti-mucin monoclonal antibodies. We isolated a group of cDNA clones that had the repeat sequence (HB11-2, HB11-6, HB11-10) and a group that had little or no homology with the repeat sequence (NP4, NP5, HB11-4). A fusion protein produced by NP4 bound preferentially BrE2, while HB11-4 bound only BrE2 and BrE3, NP5 produced a fusion protein that bound only Mc5 and not the other MAbs. Sequencing of the NP5 cDNA revealed it to be distinct from the mucin sequence and instead to have 97% identity with the estrogen induced transcript, pS2. An alternate reading frame was translated by the lambda gt11 fusion gene yielding a 44 amino acid protein having no homology with pS2 protein. Only a short region of homology (5 amino acids) with the breast mucin tandem repeat was found which was shown to be a mimotope for the Mc5 epitope on the breast mucin. High level expression of the NP5 cDNA was achieved by subcloning it into pEX2. The NP5 fusion protein has been useful for developing an assay for the presence of mucin derived antigen in patient serum.
Larocca
D
D
John Muir Cancer and Aging Research Institute, Walnut Creek, CA 94596.
Peterson
J A
JA
Ceriani
R L
RL
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001940:Breast / Q000276:immunology* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004848:Epithelium / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008969:Molecular Sequence Data; D009077:Mucins / Q000235:genetics* / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012091:Repetitive Sequences, Nucleic Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009077:Mucins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004247:DNA