| 1. |
- Carlstedt, I, et al.
(författare)
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Characterization of two different glycosylated domains from the insoluble mucin complex of rat small intestine.
- 1993
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 268:25, s. 18771-81
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Tidskriftsartikel (refereegranskat)abstract
- The highly glycosylated domains of rat small intestinal mucins were isolated after reduction and trypsin digestion and separated into two populations (A and B) by gel chromatography. The molecular mass values were 650 and 335 kDa, respectively, and the relative yields suggest that the two glycopeptides occur in equimolar proportions. Electron microscopy revealed linear structures with weight average lengths of 230 nm (A) and 110 nm (B) corresponding to a mass/unit length of about 3 kDa/nm. The protein cores (17-19%) contain large amounts of threonine (over 40%), serine (17-24%), and proline (18-19%). Carbohydrate and sulfate account for approximately 80 and 0.5%, respectively, and gas chromatography-mass spectrometry showed that the patterns of neutral and sialic acid-containing glycans are very similar in the two glycopeptides. Both contain a significant amount (7-10 mol %) of single GalNAc residues, the average oligosaccharide is about 4 sugar residues long, and the largest species observed are heptasaccharides. The major neutral and sialic acid-containing oligosaccharides are Fuc1-2Gal1-3GalNAcol and GlcNAc1-6(NeuGc2-Gal1-3)GalNAcol, respectively. Sialic acid is present as both N-acetyl- and N-glycoloyl-neuraminic acid. Repeated extractions of the tissue with guanidinium chloride left approximately 80% of the mucus glycoproteins as an insoluble glycoprotein complex whereas exposure to dithiothreitol or high speed homogenization accomplished complete solubilization. The "subunits" obtained after reduction with dithiothreitol are larger than glycopeptides A and B, and fragments corresponding in size to the latter are obtained after cleavage with trypsin. Most of the mucins from rat small intestine thus occurs as an insoluble glycoprotein complex composed of subunits joined with disulfide bonds. The subunits contain two highly glycosylated regions with different lengths substituted with very similar oligosaccharides.
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| 2. |
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| 3. |
- Bergström, S, et al.
(författare)
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Cloning and sequencing of human kappa-casein cDNA.
- 1992
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Ingår i: DNA Sequence. - 1042-5179 .- 1029-2365. ; 3:4, s. 245-6
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Tidskriftsartikel (refereegranskat)abstract
- A cDNA encoding kappa-casein of human milk was cloned and sequenced. The kappa-casein cDNA was isolated from a lambda gt11 library generated from mRNA prepared from a mammary gland biopsy obtained from a lactating woman. The library was screened with polyclonal rabbit antibodies raised against purified native kappa-casein. The obtained nucleotide sequence contained an ORF sufficient to encode the entire amino acid sequence of a kappa-casein precursor protein consisting of 182 amino acids. This includes a tentative signal peptide of 20 amino acids and a processed protein of 162 amino acids.
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| 4. |
- Hansson, L, et al.
(författare)
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Expression of human milk beta-casein in Escherichia coli : comparison of recombinant protein with native isoforms.
- 1993
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 4:5, s. 373-81
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Tidskriftsartikel (refereegranskat)abstract
- Studies on physiological function and on structure-function relationships of human milk beta-casein have been limited. In this study, we have introduced the human beta-casein cDNA into vectors designed for expression in Escherichia coli. The inducible T7-based expression system resulted in high-level expression of recombinant beta-casein. The recombinant beta-casein, localized intracellularly in E. coli, was purified to homogeneity and compared with purified native beta-casein, in particular with respect to phosphorylation. The E. coli-produced beta-casein was found to comigrate with the full-length, nonphosphorylated native human beta-casein isoform on SDS-PAGE. An N-terminal peptide containing all tentative phosphorylation sites was isolated from the recombinant protein and analyzed by mass spectrometry. The molecular mass as well as the migration of this peptide on reversed-phase chromatography confirmed that it was unphosphorylated.
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| 5. |
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