Purification and Analysis of an 80-kDa Carcinoembryonic Antigen-Binding Protein from Kupffer Cells
| Autor: | Donald A. Lazure, Peter Thomas, Timothy M. Kelly, Aniruddha Gangopadhyay |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Male
Kupffer Cells Blotting Western Molecular Sequence Data Biophysics Biochemistry Rats Sprague-Dawley FLAG-tag Affinity chromatography Antibody Specificity Protein purification Protein A/G Animals Amino Acid Sequence Amino Acids Molecular Biology Polyacrylamide gel electrophoresis chemistry.chemical_classification biology Binding protein Molecular biology Carcinoembryonic Antigen Rats Amino acid Liver chemistry biology.protein Protein G Isoelectric Focusing Carrier Proteins Sequence Analysis Protein Binding |
| Zdroj: | Archives of Biochemistry and Biophysics. 328:151-157 |
| ISSN: | 0003-9861 |
| Popis: | The receptor-mediated interaction of Kupffer cells with carcinoembryonic antigen (CEA) has led to the identification of an 80-kDa CEA-binding Kupffer cell protein. This study is aimed at the isolation and analyses of this protein from rat Kupffer cells. The binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis (PPAGE), and affinity chromatography using a CEA–Sepharose column. Fractions obtained from the gel filtration produced two major and few minor peaks with CEA-binding activity. Maximum reactivity was detected in the first major peak. The first major peak protein was partially precipitated following fractionation with 30% loss of activity in the precipitate. Fractions with CEA-binding activity were pooled and separated on the basis of molecular weight (MW) in PPAGE. The fractions between MW 70 and 90 kDa were pooled and affinity purified using CEA–Sepharose affinity chromatography. The purity of the 80-kDa protein was demonstrated by a single protein band on SDS–polyacrylamide gel. The protein was further identified by an anti-80-kDa binding protein antibody in Western blot analysis. The p I of the 80-kDa protein is 4.95. Amino acid analysis demonstrated no histidine; higher percentages of glutamine (13.3%), leucine (11.2%), asparagine and alanine (10.4%), and lysine (9.2%) were observed. Protein microsequencing revealed two unique sequences, one with 16 amino acids and the other with 11 amino acids. The 16-amino-acid sequence has less than 50% homology with a large sample of unrelated proteins, whereas the sequence containing 11 amino acids has 60–70% homology with the α chain of collagen from a variety of species but no significant homology with other known proteins, suggesting the presence of collagen-like domains in the 80-kDa receptor. |
| Databáze: | OpenAIRE |
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