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Phenylalanine-free protein for treatment of pku

一种苯丙氨酸、蛋白质的技术，应用在肽/蛋白质成分、含蛋白质食品配料、抵抗媒介传播的疾病等方向，能够解决蛋白质不再被表达、改变蛋白质结构等问题

Active Publication Date: 2019-04-19
METAX NUTRITION RES INST LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, substitution of Phe in native proteins is not always possible and can alter the protein structure such that the protein is no longer expressed

Method used

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  • Phenylalanine-free protein for treatment of pku
  • Phenylalanine-free protein for treatment of pku
  • Phenylalanine-free protein for treatment of pku

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 - Candidate Gene Discovery

[0064] To identify potential protein candidates meeting the required criteria for phenylalanine-free nutritional proteins, a self-designed search algorithm was used. Protein sequences from each genus or species were obtained from the UniProt database (http: / / www.uniprot.org) using the import feature of CLC MainWorkbench 6.6.1.

[0065] The proteins used are derived from common sources of food, such as vegetables (e.g. potatoes), but also from microorganisms (e.g. yeast) or animals (e.g. cattle). The Latin or common name of the species / genus (depending on which name resulted in a greater number of hits) was used as the search string. Downloading all hits except uncharacterized proteins yielded a total of 836,037 sequences from different species. The following search strings were used to identify the hits listed in Table 2.

[0066] Lists of amino acids present in each of the 836,037 genes were further generated using the "Creat...

Embodiment 2

[0079] Example 2 - Preparation of Bacillus subtilis

[0080] The synthetic gene GSP105 with the nucleotide sequence shown in SEQ ID NO 7 was inserted into the Bacillus subtilis expression vector pHT43 (MoBiTec, Germany), the recombinant protein was enabled to be secreted into the medium and introduced into protease-deficient Bacillus subtilis strain WB800N (MoBiTec), following the manufacturer's instructions.

[0081] After transformation, positive clones were selected on antibiotic selection plates; the presence of the expression vector was further confirmed by PCR, plasmid extraction and subsequent DNA sequencing. Glycerol stock cultures were generated and stored at -80°C.

[0082] A starter culture of recombinant Bacillus subtilis GSP105 was prepared as follows: 1 L of TB medium (Carl Roth, Karlsruhe, Germany) supplemented with chloramphenicol (Carl Roth) and neomycin (Carl Roth) was inoculated, and each final The concentration was 5 μg / ml and 1 ml of Bacillus subtilis...

Embodiment 3

[0087] Example 3 - Mouse Feeding Study (Pilot Study)

[0088] Research design:

[0089] Six adult, male, homozygous PKU mice (Pah enu2 / 2 ; Shedlovsky et al., Genetics 1993, 134:1205; http: / / www.pahdb.mcgill.ca / ? Topic=Information&Section=Mouse&Page=1) were divided into 3 groups with 2 mice in each group. Animals belonging to the same group share a single cage. These groups were fed as listed in Table 4. The main differences between the diets were protein composition and Phe content, as shown in Table 5. PKU mice were fed a standard mouse diet prior to the feeding study. The feeding study lasted 28 days, during which time the animals were given food and water ad libitum.

[0090] Table 4: Animal groups and animal diets (N=6)

[0091]

[0092] Table 5: Animal diets

[0093]

[0094]

[0095] "Phe-free GSP105 protein diet" or "Phe-free GSP105 diet" refers to a diet in which the only source of amino acids is recombinant dietary protein GSP105 supplemented with 0...

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Abstract

A recombinant dietary protein is provided that is free of phenylalanine and is a dietary protein with a high biological value. Further provided are a vector encoding said dietary protein, a microorganisms expressing said protein, a method for the production of said protein, and a dietary composition comprising said protein that in an embodiment is for use as a medicament and / or food for special medical purposes in patients with accumulations of phenylalanine in the body.

Description

technical field [0001] The present invention relates to a recombinant dietary protein or a dietary sufficient portion thereof, wherein said protein does not contain phenylalanine, for use in the diet of patients suffering from phenylketonuria. Background technique [0002] Phenylketonuria (PKU) (OMIM 261600, ORPHA716) is an inherited metabolic disorder with a prevalence of 1:10,000 in Europe. In most cases, this is a disorder of amino acid metabolism caused by loss of function or impairment of the liver enzyme phenylalanine hydroxylase (PAH). PAH deficiency in turn leads to excess phenylalanine (Phe) in the brain and plasma. The deficiency of PAH ultimately manifests itself as a lack of tyrosine, a precursor of neurotransmitters. Together with mutations in enzymes involved in pterin metabolism, PKU is associated with hyperphenylalaninemia (HPA). [0003] The disease is usually diagnosed during newborn screening after birth in most countries due to elevated blood Phe level...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195A61K38/00A23L33/195C07K14/32
CPCA61K38/00A23V2250/54C07K2319/21A23L33/195C07K14/32A61P3/00Y02A50/30C07K14/195A23V2002/00
Inventor B.霍夫曼Y.穆克S.拉奇N.贾布隆卡S.希尔伯格
Owner METAX NUTRITION RES INST LLC
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