Study of Putative Glycosylation Sites in Bovine β-Casein Introduced by PCR-Based Site-Directed Mutagenesis
Published in Journal of Agricultural and Food Chemistry, Volume 44, Issue 1, January 1, 1996, pages 358-364.
The bovine β-casein gene, A2 genetic variant, has been mutated at positions 70 and 71 for the introduction of a glycosylation signal (Asn-X-Ser). These mutants have been constructed to study the functionality of β-casein glycosylated exclusively at Asn68. The mutation was generated using PCR-based site-directed mutagenesis, and it was derived from bovine β-casein cDNA. The mutant cDNAs including the wild-type β-casein gene have been subcloned into the yeast Pichia pastoris expression vector pHIL-D2, which contains the methanol-inducible alcohol oxidase (AOX1) promoter. Three expression vectors were constructed and designated pCJ-βWT (wild-type bovine β-casein gene), pCJ- β68 (substitution of Ser70 for Leu70), and pCJ-β6873 (Ser70-Ser71 for Leu70-Pro71). Bovine β-casein produced in yeast was found to contain a sugar moiety on Asn68 (N-linked glycosylated) when produced from a strain containing the pCJ- β6873 construct in its chromosome. N-Glycosylation of bovine β-casein at position 68 was completely inhibited in transformants carrying vector pCJ- β68 with the single substitution of Ser70 for Leu70. The concentration of bovine β-casein in this expression system was in the range of 0.7-1.0 g/L.