Chromatographic Separation of the Milks of Native and Genetically Modified Mice

Stevenson, Elizabeth Mary (1993) Chromatographic Separation of the Milks of Native and Genetically Modified Mice. PhD thesis, University of Glasgow.

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Abstract

Selective breeding of dairy cattle has enabled milk composition to be modified by manipulation of the relatively small naturally occurring gene pool. Advances in molecular biology have widened our understanding of the control of gene structure and expression and may be utilised to construct genes encoding protein variants, whose physical properties have been altered. The mouse has proved to be an ideal choice of animal for the study of genetic manipulation of milk proteins because of its short generation time (21 days), large litter sizes and low cost in comparison to larger ruminants. The main drawback however is the relatively small volumes of milk, (about 1 ml) produced by the animals. The principal aim of this project was to develop methods for the quantitative separation of the main protein components of mouse milk and utilise these methods to analyse the expression of proteins in the milk of genetically modified animals. Separation of the major caseins and whey proteins was achieved by cation exchange FPLC. The conditions necessary for the complete dissociation of the murine casein micelles were more extreme than those used in the analysis of milk from other species, particularly the inclusion of the non-ionic detergent n-octyl beta-D-glucopyranoside together with 8M-urea. kappa-, beta- and alpha-caseins were identified by comparing the amino acid compositions of the purified proteins with their translated cDNA sequences. Another, unidentified acid precipitable casein was also detected. The concentrations of the major acid precipitable proteins in mouse milk were calculated to be WAP, 15.3; kappa-casein 4.7; beta-casein 25.2 and alpha-casein 51.7 mg ml-1 The separation was then scaled up using the Hi-Load system which gave sufficient amounts of the beta- and alpha-casein components to be used in the raising of antibodies to these individual proteins. Milk from two lines of transgenic mice which expressed a foreign protein in their milk was also analysed. The first line secreted normal ovine beta-lactoglobulin in their milk and the effect on the expression of individual mouse milk proteins was investigated. It had previously been shown that in this line, the amount of total protein secreted in the milk was unaltered even although it contained a relatively high proportion of ovine beta-lactoglobulin. The actual amounts of the individual murine milk proteins secreted were reduced, the most obvious of which was the alpha-casein. The concentrations of milk proteins were calculated as WAP, 9.6 mg ml-1; kappa-casein, 2.8 mg ml-1 beta-casein, 14.3 mg ml-1, alpha-casein, 20.4 mg ml-1 and ovine beta-lactoglobulin, 50.1 mg ml-1 The other line of transgenic mice expressed ovine beta-lactoglobulin which had been genetically modified to contain an oligomer containing a casein kinase recognition sequence on a readily accessible part of the protein molecule. It was hoped that the inclusion of this oligomer would result in the normally unphosphorylated protein being phosphorylated. As was demonstrated by its staining with the cationic dye Stains-all, the protein did not appear to be phosphorylated. To rule out the possibility of dephosphorylation due to the presence of endogenous phosphatases in the milk, the milk was collected and stored in a phosphatase inhibitor. Since this had no effect on protein staining with Stains-all, it can be concluded that protein phosphorylation requires more than just the appropriate kinase recognition sequence. The last line of mice to be studied were ones in which the murine beta-casein gene had been deleted. In this line the overall level of casein decreased by 11.5% and that of whey protein increased by about 16%. The increased level of whey protein could however be accounted for by changes in the partitioning of these proteins between the acid precipitated and supernatant fractions and suggests that beta-casein may be important in influencing the isoelectric precipitation of whey proteins in native mouse milks. Again, changes in the levels of expression of the remaining caseins were not uniform and may indicate that the proteins are secreted by different pathways.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J Leaver
Keywords: Animal sciences
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-74855
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:49
Last Modified: 27 Sep 2019 15:49
URI: https://theses.gla.ac.uk/id/eprint/74855

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