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oai:www.ideals.illinois.edu:2142/22183 2023-07-10 col_2142_5131 col_2142_14781 com_2142_5130 com_2142_687 com_2142_397

Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:55:53Z Item is restricted indefinitely. Jimenez-Flores, Rafael Choi, Byung-Kwon 2011-05-07T13:31:40Z 2011-05-07T13:31:40Z 10000-01-01 1996 The putative glycosylation site (Asn-X-Ser) was introduced to the bovine $\beta$-casein cDNA and genomic DNA (A$\sp2$ genetic variant) using PCR-based site-directed mutagenesis to increase its amphiphilicity. The two mutant bovine $\beta$-casein sequences containing the substitution of ${\bf Ser}\sb{70}$ for ${\bf Leu}\sb{70}$ (Asn$\sb{68}$-Ser$\sb{69}$-${\bf Ser}\sb{70}$-Pro$\sb{71})$ and the substitution of ${\bf Ser}\sb{71}$ for ${\bf Pro}\sb{71}$ (Asn$\sb68$-Ser$\sb{69}$-${\bf Ser}\sb{70}$-${\bf Ser}\sb{71})$ were constructed and expressed using Pichia pastoris and transgenic mice. The mutant constructs were placed under the control of the methanol-inducible alcohol oxidase (AOX1) promoter for P. pastoris and of the bovine $\alpha$-lactalbumin promoter for transgenic mice. The N-linked glycosylation of the mutant carrying Pro$\sb{71}$ was completely inhibited whereas the other mutant carrying Ser$\sb{71}$ was N-linked glycosylated in both expression systems. Thus, the C-terminal proline residue in the sequon was found to be a structural inhibitor for N-glycosylation. Glyco-$\beta$-caseins produced in both systems were phosphorylated to the same degree as authentic bovine $\beta$-casein. In addition, based on lectin blots, the oligosaccharides of glyco-$\beta$-casein from P. pastoris were high-mannose type whereas transgenic glyco-$\beta$-caseins contained various oligosaccharide chains. Mouse casein micelles containing glycosylated bovine $\beta$-casein showed the largest median diameter and rough outer surfaces, compared to normal mouse casein micelles and micelles from transgenic milk containing wild-type bovine $\beta$-casein. Made available in DSpace on 2011-05-07T13:31:40Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9625122.pdf: 5991212 bytes, checksum: c629897571643e48626e39e336256290 (MD5) Previous issue date: 1996 Restriction data tranferred 2014-07-01T11:26:05-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only AAI9625122 (UMI)AAI9625122 http://hdl.handle.net/2142/22183 eng Copyright 1996 Choi, Byung-Kwon Biology, Molecular Agriculture, Food Science and Technology Health Sciences, Nutrition Modification of bovine beta-casein using genetic engineering and its structure/function studies text Food Science and Human Nutrition Food Science and Human Nutrition University of Illinois at Urbana-Champaign Dissertation Ph.D.