Cation-exchange purification of mutagenized bovine beta-casein expressed in transgenic mouse milk: its putative Asn68-linked glycan is heterogeneous.

Bovine beta-casein (A2 genetic variant) was mutagenized to (L70S/P71S) and expressed in transgenic mouse milk. This protein now carries the signal (N68S69S70S71) that mimics a consensus eukaryotic glycosylation signal (N-X-S/T) (3). Hypothetically this protein should be glycosylated at N68 by any eukaryotic organism producing it. This novel protein was purified from transgenic mouse milk by Mono-S cation-exchange fast protein liquid chromatography (FPLC). The novel beta-casein was separated without cross contamination from mouse caseins by using acetate buffer (pH 5.0) in the presence of 6 M urea, octyl-glucopyranoside and 2-beta-mercaptoethanol. The purified (L70S/P71S) beta-casein showed an N-linked oligosaccharide attached to Asn68 and different lectin binding profiles compared with the same protein expressed in yeast. The mouse-expressed beta-casein (L70S/P71S) was specific to Concanavalin A, wheat germ agglutinin, Erythrina cristagalli agglutinin, and Ulex europaeus, indicating its oligosaccharide structure is different in the mammary gland of mouse than the reported glycosylated beta-casein expressed in Pichia pastoris (4). In addition, the five serine residues located at amino terminus of wild type bovine beta-casein were shown to be normally phosphorylated as in native bovine beta-casein.

[1]  P. Fox,et al.  Advanced dairy chemistry-1: Proteins. , 2003 .

[2]  H. Swaisgood,et al.  Chemistry of the Caseins , 2003 .

[3]  A. Clark,et al.  Bovine beta-casein expressed in transgenic mouse milk is phosphorylated and incorporated into micelles. , 1996, Protein expression and purification.

[4]  M. Wheeler,et al.  Genetic Modification of Bovine β-Casein and Its Expression in the Milk of Transgenic Mice , 1996 .

[5]  R. Jiménez-Flores,et al.  Study of Putative Glycosylation Sites in Bovine β-Casein Introduced by PCR-Based Site-Directed Mutagenesis , 1996 .

[6]  D. James,et al.  N-Glycosylation of Recombinant Human Interferon-γ Produced in Different Animal Expression Systems , 1995, Bio/Technology.

[7]  J. Leaver,et al.  Chromatographic separation of the proteins of mouse milk , 1994 .

[8]  D. Dalgleish,et al.  Separation of beta-casein A1, A2, and B using cation-exchange fast protein liquid chromatography. , 1991, Journal of dairy science.

[9]  R. Jiménez-Flores,et al.  Expression of Bovine β-Casein in Saccharomyces Cerevisiae and Characterization of the Protein Produced in Vivo , 1990 .

[10]  J. Vilotte,et al.  Efficient tissue-specific expression of bovine α-lactalbumin in transgenic mice , 1989 .

[11]  J. Medrano,et al.  Milk protein typing of bovine mammary gland tissue used to generate a complementary deoxyribonucleic acid library. , 1989, Journal of dairy science.

[12]  J. Amiot,et al.  Application of reversed phase high performance liquid chromatography to the separation of peptides from phosphorylated and dephosphorylated casein hydrolysates , 1989 .

[13]  C. Polo,et al.  Gel filtration and high-performance liquid chromatographic analysis of phosphotungstic acid soluble peptides from blue cheeses , 1987 .

[14]  P. Højrup,et al.  Purification of goats' milk casein by reversed-phase high-performance liquid chromatography and identification of αs1-casein , 1987, Journal of Dairy Research.

[15]  G. Miranda,et al.  Analyse quantitative des caseines dans le lait de vache par chromatographie liquide rapide d'échange d'ions (FPLC) , 1987 .

[16]  L. Chaplin.  Hydrophobic interaction fast protein liquid chromatography of milk proteins. , 1986, Journal of chromatography.

[17]  D. Dalgleish.  Glycosylated κ-caseins and the sizes of bovine casein micelles. Analysis of the different forms of κ-casein , 1985 .

[18]  B. Gupta.  Determination of native and denatured milk proteins by high-performance size exclusion chromatography. , 1983, Journal of chromatography.

[19]  H. Segall,et al.  High-Performance Gel-Permeation Chromatography of Bovine Skim Milk Proteins , 1981 .

[20]  W. Donnelly.  Chromatography of milk proteins on hydroxyapatite , 1977, Journal of Dairy Research.

[21]  A. Law,et al.  An improved method for the quantitative fractionation of casein mixtures using ion-exchange chromatography , 1977, Journal of Dairy Research.

[22]  J. Chobert,et al.  Fractionation of whole casein on hydroxyapatite. Application to a study of buffalo κ-casein , 1977, Journal of Dairy Research.

[23]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.