Study of Putative Glycosylation Sites in Bovine β-Casein Introduced by PCR-Based Site-Directed Mutagenesis

 University of Illinois.

 Author to whom correspondence should be addressed [telephone (805) 756-6103; fax (805) 756-2998; e-mail rjimenez@oboe.aix.calpoly.edu].

 California Polytechnic State University.

 Abstract published in Advance ACS Abstracts, December 1, 1995.

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.

Keywords: Bovine β-casein; mutagenesis; Pichia pastoris