A Biochemical and Molecular Characterisation of Non-Casein Proteins of the Baboon (Papio hamadryas), Pig (Sus scrofa domestica) and Marsupial (Monodelphis domestica)

Andrew Hall (2010). A Biochemical and Molecular Characterisation of Non-Casein Proteins of the Baboon (Papio hamadryas), Pig (Sus scrofa domestica) and Marsupial (Monodelphis domestica) PhD Thesis, School Biomedical Sciences, The University of Queensland.

       
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Author Andrew Hall
Thesis Title A Biochemical and Molecular Characterisation of Non-Casein Proteins of the Baboon (Papio hamadryas), Pig (Sus scrofa domestica) and Marsupial (Monodelphis domestica)
School, Centre or Institute School Biomedical Sciences
Institution The University of Queensland
Publication date 2010-08
Thesis type PhD Thesis
Supervisor Dr Kevin Bell
Dr Peter Kaye
Total pages 146
Total colour pages 10
Total black and white pages 136
Subjects 06 Biological Sciences
Abstract/Summary As a result of studies undertaken for this thesis, a new protein expressed in the milk of pigs was identified. This new protein was identified as a highly polymorphic proline-rich protein (PRP) and observed to be present in high concentrations throughout lactation. N-terminal sequencing of an internal fragment identified the protein as having homology to the human salivary proline-rich proteins that have been attributed with the abilities to bind various fimbriated bacteria, precipitate tannins and inhibit calcium phosphate precipitation. This is the first time that PRP has been described in the milk of any species. Large numbers of porcine milk samples were examined by isoelectric focussing and resulted in the identification of five codominant alleles that were confirmed using dam/daughter data. As part of the study I isolated, sequenced and named the gene encoding porcine milk PRP, PREX1 (proline-rich protein EcoNI/XagI subfamily gene 1), and in the process demonstrated it to be composed of a large block of tandem repeat sequence which shows variable length polymorphism with five alleles being identified. Further, PREX1 was localised to SSC5q using a porcine somatic cell hybrid panel and confirmed by radiation hybrid mapping. The identification of PRP was serendipitous whilst further characterising the anomalous expression of porcine β-lactoglobulin described by Bell et al. (1981a). The cause of the anomalous β-lactoglobulin expression was not further investigated in this study following the identification of PRP in milk, which was considered to be of greater scientific and comparative interest. However, before investigation ceased, I was able to demonstrate that porcine β-lactoglobulin was present in both glycosylated and non-glycosylated forms in all porcine milk samples investigated. Following separation by isoelectric focussing in an immobilized pH gradient (pH 4.1-5.1) and using N-terminal amino acid sequencing, the threonine in position 4 was identified as being glycosylated. This is only the second β-lactoglobulin known to be glycosylated, the other is bovine β-lactoglobulin Dr (Bell et al 1970). Another significant finding was the identification of a colostral specific form of β-lactoglobulin which is present from day 1 through to day 5 of lactation. It too is glycosylated and has a characteristically lower than normal pI. In addition to the work investigating the whey proteins of pig milk, baboon and Monodelphis domestica milks were briefly characterised. This involved the identification and cDNA sequence determinations of β-lactoglobulin and α-lactalbumin in the baboon and Monodelphis milks respectively. Baboon β-lactoglobulin was identified and characterised by various electrophoretic techniques. Three variants were identified in a group consisting of both species and hybrid animals. The complete β-lactoglobulin cDNA sequence was determined using RT-PCR and routine cloning procedures with primers designed from N-terminal sequence data and from homologous regions of aligned sequences from a number of species. This was the first primate β-lactoglobulin to be sequenced and is the longest β-lactoglobulin (168 residues) yet identified. In order to compare the South American marsupials with the highly characterised Australian marsupials I undertook the qualitative characterisation of the non-casein milk proteins of Monodelphis domestica. Significant findings to arise from this work include the identification of a novel α-lactalbumin whose protein N-terminal sequence revealed 10 unique residues, not seen in any similar sequences, in the first 35 residues. The full-length cDNA sequence was determined and demonstrated that Monodelphis α-lactalbumin is unique compared with all known α-lactalbumins in having 21 amino acid residues, of a total of 125 residues, which are positionally unique. In addition Monodelphis α-lactalbumin has an insertion of two residues at positions 15 and 16 respectively which, to date, has only been documented in monotremes.
Keyword Milk proteins, proline rich proteins, baboon, pig, Gray short-tailed opossum
Additional Notes Colour pages:17, 20, 44, 55, 72, 80, 82, 83, 89, 144 Landscape: 72, 105

 
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Created: Mon, 22 Nov 2010, 12:40:56 EST by Mr Andrew Hall on behalf of Library - Information Access Service