Phenylalanine-free protein and DNA coding therefor

Details Phenylalanine-free protein and DNA coding therefor Phenylalanine-free protein and DNA coding therefor

1996-01-16

2002-12-17

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S252300, C435S252330, C435S254210, C536S023100, C536S023500, C530S350000, C530S360000, C530S367000, C514S002600

Reexamination Certificate

active

06495344

ABSTRACT:

This application is a 371 of PCT/GB94/01046 filed on May 16, 1994, which claims benefit of GB9310472 filed on May 20, 1993.
This invention relates to an edible protein which has been modified so that it is phenylalanine free, to DNA coding for it, and to a method of producing it. Such a protein is a useful nutrient in the treatment of diseases which are associated with difficulty in metabolising phenylalanine. A particular example of such a disease is phenylketonuria (PKU).
PKU is a genetically acquired disease that occurs in a relatively fixed proportion of new births in a human population. A defect in the enzyme carrying out the pterin-dependent hydroxylation of phenylalanine to tyrosine prevents the body from metabolizing the amino acid phenylalanine. This amino acid occurs in varying proportions in all proteins in foodstuffs and is, in the correct amount, essential for human protein synthesis, and therefore for the growth and maintenance of the body. Patients with PKU cannot remove excess phenylalanine from the blood and tissues and the failure to achieve this control over phenylalanine levels leads to grave neurological damage, especially in the growing child.
PKU patients are at present fed with a synthetic diet which contains a metabolically-correct amount of phenylalanine along with a mixture of the other amino acids needed for growth. Such a diet is unpalatable and is presented in liquid form only and therefore has difficulty in achieving patient compliance.
An object of this invention is to provide an edible protein which when pure contains no phenylalanine and which can form the basis for a diet containing the optimal nutritional phenylalanine content for PKU patients. This object may be achieved by taking the gene from a known nutritional protein and modifying it so that the codons coding for phenylalanine are deleted or are replaced by codons coding for another amino acid.
An alternative approach is to synthesise by chemical means DNA coding for a phenylalanine-free polypeptide, starting either from fragments of genes coding for existing proteins, or from the nucleotides themselves.
According to one aspect of the invention we provide a DNA molecule coding for a food protein, modified in that the codons coding for phenylalanine have been deleted or replaced by codons coding for one or more other amino acids.
According to another aspect of the invention we provide an edible polypeptide which comprises a food protein modified in that the phenylalanine residues have been omitted or have been replaced by one or more other amino acids also occurring in protein.
We further provide a nutrient material comprising an edible polypeptide as defined above and other edible substances.
The food protein is preferably a common food protein such as ovalbumin or caesin.
We also provide a nutrient material comprising an edible protein or modified food protein as hereinbefore defined, and other edible substances.
The protein according to the invention is phenylalanine free when pure, but the diet of the patient must contain some phenylalanine, i.e. the amount required for metabolism, but with substantially no excess.
An obvious approach would be to add an appropriate proportion of normal food proteins, which contain phenylalanine, to a pure phenylalanine-free protein according to the invention.
On the other hand, proteins are notoriously difficult to purify to a high level. If only partially purified, the phenylalanine-free protein will be accompanied by other protein products of the host organism containing their normal amounts of phenylalanine. Thus, if the modified protein is only partly purified (which is much easier than complete purification), a protein mixture containing overall a reduced proportion of phenylalanine will be obtained. By controlling the degree of purification, a protein mixture containing a metabolically-appropriate proportion of phenylalanine can be produced. This invention also provides such a mixture.
Although codons for phenylalanine may simply be deleted from the gene for a food protein, in order to preserve as far as possible the tertiary structure of the protein the codons coding for phenylalanine are preferably replaced by codons coding for another amino acid, preferably those having the most similar properties, e.g. tyrosine.
We also provide an expression vector into which has been incorporated DNA for an edible protein or modified food protein as described herein. The expression vector is preferably a
Saccharomyces cerevisiae
expression vector because this yeast has a long history as a human foodstuff and is amenable to genetic manipulation. Other yeasts, e.g.
Pischia pastoris
, may also be used.
We further provide a host, for example a yeast such as
S. cerevisiae
or
Pichia pastoris
, transformed by such an expression vector.
Ovalbumin and caesin have been selected as preferred food proteins to be modified in accordance with this invention because they are naturally-occurring proteins which are commonly used as human foodstuffs, are widely acceptable, and also because the modified proteins are likely to behave in a similar manner to the native proteins when cooked or subjected to other food processing steps. A wide variety of other food proteins may, however, also be chosen.
Preferably, apart from omitting or substituting codons coding for phenylalanine, the DNA molecule coding for the edible protein is modified as necessary to ensure that the codon for each amino acid is the codon of preference for the selected host, e.g.
S.cerevisiae.


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