Isolation and identification of a high molecular weight protein in sow milk.

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.

[1]  P. Theil,et al.  Colostrum and milk production of sows is affected by dietary conjugated linoleic acid. , 2012, Journal of animal science.

[2]  P. Theil,et al.  Predicting milk yield and composition in lactating sows: a Bayesian approach. , 2012, Journal of animal science.

[3]  C. Bagnell,et al.  Milk-borne lactocrine-acting factors affect gene expression patterns in the developing neonatal porcine uterus. , 2011, Reproduction.

[4]  F. DiMeco,et al.  Glycoproteomic analysis of glioblastoma stem cell differentiation. , 2011, Journal of proteome research.

[5]  H. Bovenhuis,et al.  Relationships between milk protein composition, milk protein variants, and cow fertility traits in Dutch Holstein-Friesian cattle. , 2010, Journal of dairy science.

[6]  F. Harte,et al.  Isolation of caseins from whey proteins by microfiltration modifying the mineral balance in skim milk. , 2009, Journal of dairy science.

[7]  K. Dewettinck,et al.  Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk. , 2009, Journal of dairy science.

[8]  Niclas G Karlsson,et al.  Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes. , 2008, Journal of proteome research.

[9]  A. I. Antonov,et al.  N-Glycosylation in Chrysosporium lucknowense enzymes. , 2008, Carbohydrate research.

[10]  Hsiu-Ling Chen,et al.  A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy. , 2006, Talanta.

[11]  J A Asenjo,et al.  Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge. , 2005, Biotechnology and bioengineering.

[12]  M. Erat,et al.  Purification of glutathione reductase from chicken liver and investigation of kinetic properties , 2005, Applied biochemistry and biotechnology.

[13]  Lindsay Sawyer,et al.  Invited Review: β-Lactoglobulin: Binding Properties, Structure, and Function , 2004 .

[14]  H. Swaisgood,et al.  Isolation and partial characterization of CD36 from skim milk. , 2002, Journal of dairy science.

[15]  L. Miclo,et al.  Separation and Characterization of Mares’ Milk αs1-, β-, κ-Caseins, γ-Casein-Like, and Proteose Peptone Component 5-Like Peptides , 2002 .

[16]  L. K. Rasmussen,et al.  Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone. , 2001, Journal of dairy science.

[17]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[18]  John R. Crowther,et al.  The ELISA Guidebook , 2000, Methods in Molecular Biology™.

[19]  Y. Huang,et al.  Glycoconjugates of the Rat Ciliary Body Epithelium: A Lectin Histochemical and Protein Blotting Study , 1999, The Histochemical Journal.

[20]  S. Gendler,et al.  The epithelial mucin, MUC1, of milk, mammary gland and other tissues. , 1995, Biochimica et biophysica acta.

[21]  W. Hurley,et al.  Electrophoretic comparisons of lactoferrin from bovine mammary secretions, milk neutrophils, and human milk. , 1993, Journal of dairy science.

[22]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[23]  E. Bergmann-Leitner,et al.  Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies , 2008, Malaria Journal.

[24]  Qin Yi-de.  The Evidence of the Origin of a Polymorphic High Molecular Weight Protein in Sow Milk , 2006 .

[25]  Zhang Sixiang.  The evidence of the origin of a polymorphic high molecular weight protein in sow milk , 2006 .

[26]  G. Kontopidis,et al.  Invited review: beta-lactoglobulin: binding properties, structure, and function. , 2004, Journal of dairy science.

[27]  Qin Yi.  Correlation between Polymorphism of High Molecular Weight Protein in Sow Milk and Milk Growth Factors, Litter Weight Gain , 2004 .

[28]  Qin Yi-de.  The Correlation between Gene Expressive Quantity of High Molecular Weight Protein in Milk and Sow Lactation Performance , 2004 .

[29]  Cheng Wei-hua.  Homogenous Identification of a Group of Polymorphic High Molecular Weight Protein in Sow Milk , 2004 .

[30]  Qin Yi.  The polymorphism of high molecular weight protein in sow milk and its relationship with lactation performance , 2003 .

[31]  Qin Yi-de.  Dynamic analyses of protein components in Erhualian sow milk , 2003 .

[32]  L. Miclo,et al.  Separation and characterization of mares' milk alpha(s1)-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides. , 2002, Journal of Dairy Science.

[33]  W. Hurley,et al.  Pig colostrum and milk composition:comparisons between Chinese Meishan and US breeds , 1992 .