Variant forms of urate oxidase and use thereof
View Patent ↗Genetically modified proteins with uricolytic activity are described. Proteins comprising truncated urate oxidases and methods for producing them, including PEGylated proteins comprising truncated urate oxidase are described.
1. A method for producing a uricase comprising the steps of:
(i) culturing a host cell comprising a vector comprising a nucleic acid sequence encoding a uricase
comprising a mammalian uricase amino acid sequence truncated at the amino terminus or the carboxy terminus or both the amino and carboxy termini by 1-6 amino acids and further comprising an amino acid substitution with threonine at position 7 (D7T), an amino acid substitution at position 46 (S46T), an amino acid substitution with lysine at position 291 (R291K) and an amino acid substitution with serine at position 301 (T301S), said truncation and amino acid substitution being relative to an amino acid sequence of SEQ ID NO:11;
wherein the nucleic acid sequence is operatively linked to a heterologous promoter under conditions such that the nucleic acid sequence is expressed by the host cell; and
(ii) isolating the expressed uricase.
2. The method of claim 1 , wherein the expressed uricase comprises an amino terminal amino acid selected from alanine, glycine, proline, serine, or threonine.
3. The method of claim 1 , wherein the expressed uricase comprises an amino terminal threonine.
4. The method of claim 1 , wherein the expressed uricase comprises an amino terminal methionine.
5. The method of claim 1 , wherein the uricase is conjugated with a polymer.
6. The method of claim 1 , wherein the heterologous promoter is an osmB promoter.
7. The method of claim 3 , wherein the expressed uricase consists of the amino acid sequence of SEQ ID NO: 8.
8. The method of claim 3 , wherein the expressed uricase comprises the amino acid sequence of SEQ ID NO: 8.
9. The method of claim 4 , wherein the expressed uricase consists of the amino acid sequence of SEQ ID NO: 7.
10. The method of claim 4 , wherein the expressed uricase comprises the amino acid sequence of SEQ ID NO: 7.
11. The method of claim 5 , wherein the polymer is selected from the group consisting of poly(ethylene glycol), dextran, polypropylene glycol, hydroxypropylmethyl cellulose, carboxymethylcellulose, polyvinyl pyrrolidone and polyvinyl alcohol.
12. The method of claim 5 , wherein the uricase is a polyethylene glycol—uricase conjugate.
13. The method of claim 12 , wherein the conjugate comprises 2 to 12 polyethylene glycol molecules on each uricase subunit.
14. The method of claim 12 , wherein each polyethylene glycol molecule has a molecular weight between about 1 kD and 100 kD.
15. The method of claim 13 , wherein each polyethylene glycol molecule has a molecular weight between 1 kD and 50 kD.
16. The method of claim 13 , wherein each polyethylene glycol molecule has a molecular weight between 5 kD and 20 kD.
17. The method of claim 13 , wherein each polyethylene glycol molecule has a molecular weight of 10 kD.