Isolation and some effects of functional, low-phenylalanine κ-casein expressed in the milk of transgenic rabbits

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine κ-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit κ-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant κ-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine κ-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.