Modification of bovine beta-casein using genetic engineering and its structure/function studies
Choi, Byung-Kwon
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https://hdl.handle.net/2142/22183
Description
Title
Modification of bovine beta-casein using genetic engineering and its structure/function studies
Author(s)
Choi, Byung-Kwon
Issue Date
1996
Doctoral Committee Chair(s)
Jimenez-Flores, Rafael
Department of Study
Food Science and Human Nutrition
Discipline
Food Science and Human Nutrition
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Agriculture, Food Science and Technology
Health Sciences, Nutrition
Language
eng
Abstract
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.
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