Method

Abstract

A method is described for releasing a soluble or membrane associated intracellular protein of interest (POI) comprising the steps of: providing a cell comprising a soluble or membrane associated intracellular POI; contacting the cell with a membrane extracting composition; and causing the POI to be released from the cell under conditions sufficient for the specific release of the POI and in a soluble form.

1 . A method for extracting a soluble or membrane associated intracellular recombinant protein of interest (POI) from a bacterial, yeast or fungal cell comprising the steps of:
 (a) providing a bacterial, yeast or fungal cell comprising a soluble or membrane associated intracellular recombinant POI;   (b) releasing the recombinant POI from the cell by contacting the cell with a membrane extracting composition comprising a quarternary ammonium compound at a concentration of between 0.05% to 0.6% by weight, under conditions sufficient for the release of the recombinant POI and in a soluble form   (c) recovering the recombinant POI from the membrane extracting composition.   
     
     
         2 . A method according to  claim 1  in which the bacterial, yeast or fungal cell is a transformed cell. 
     
     
         3 . A method according to  claim 1  in which the intracellular recombinant POI is produced by recombinant DNA techniques. 
     
     
         4 . A method according to  claim 1 , in which the cell is a yeast cell. 
     
     
         5 . A method according to  claim 1 , in which the quarternary ammonium compound is selected from the group consisting of Lauroyl Trimethyl Ammonium Bromide (LTAB), Myristyl Trimethyl Ammonium Chloride (MTAC), CetylTrimethyl Ammonium Chloride (CTAC), Cetrimide, Cetyl Trimethyl Ammonium Bromide (CTAB), Stearoyl Trimethyl Ammonium Chloride (STAC), Stearoyl Trimethyl Ammonium Bromide (STAB), Benzalkonium Chloride (alkyldimethylbenzylammonium chloride), N-Cetylpyridinium Bromide (N-Hexadecylpyridinium bromide), N-Cetylpyridinium Chloride (N-Hexadecylpyridinium chloride), Benzyl Dimethyl Tetradecyl Ammonium Chloride, Benzyl Dimethyl Hexadecyl Ammonium Chloride and a combination of any two or more thereof. 
     
     
         6 . A method according to  claim 1  in which the bacterial yeast or fungal cell is contacted with the membrane extracting composition at temperatures from 4° C. to 40° C. 
     
     
         7 . A method according to  claim 1  in which the bacterial, yeast or fungal cell is contacted with the membrane extracting composition at a pH of from 2.0 to 11.0. 
     
     
         8 . A method for screening for mutated cells or transformed cells producing elevated levels of a soluble or membrane associated intracellular recombinant POI comprising the steps of:
 (a) growing the mutated cells at 30° C.;   (b) incubating the mutated cells or transformed cells with the membrane extracting composition in a method as defined in  claim 1 ,   (c) recovering the cell free medium;   (d) screening the cell free medium for elevated levels of the intracellular recombinant POI;   
       such that the presence of the intracellular recombinant POI in the cell free medium is indicative that the intracellular recombinant POI has been released, in which the mutated or transformed cell is a bacterial, yeast or fungal cell. 
     
     
         9 . A method of using a membrane extracting composition comprising a quaternary ammonium compound to extract a soluble or membrane associated intracellular recombinant POI from a bacterial, yeast or fungal cell, in which the membrane extracting composition comprises a quaternary ammonium compound at a concentration of between 0.05% to 0.6% by weight and is contacted with the bacterial, yeast or fungal cell, and in which the released recombinant POI is recovered from the membrane extracting composition. 
     
     
         10 . A method for screening for mutated cells or transformed cells producing elevated levels of a soluble or membrane associated intracellular recombinant POI comprising the steps of:
 (a) growing the mutated cells at 30° C.;   (b) incubating the mutated cells or transformed cells with the membrane extracting composition comprising a quaternary ammonium compound at a concentration of between 0.05% to 0.6% by weight;   (c) recovering the cell free medium   (d) screening the cell free medium for elevated levels of the intracellular recombinant POI;   
       such that the presence of the intracellular recombinant POI in the cell free medium is indicative that the intracellular recombinant POI has been released. 
     
     
         11 . A membrane extracting composition suitable for releasing a soluble or membrane associated intracellular recombinant POI, wherein the composition is contacted with the cell under the following conditions:
 (a) a percentage by weight of quaternary ammonium compound from about 0.05% to about 0.6%;   (b) a pH of from about 2.0 to about 11.0   (c) a temperature of from about 4° C. to about 40° C.;   
       Such that the intracellular recombinant POI substantially free of contaminating proteins is obtained. 
     
     
         12 . A method of using a membrane extracting composition comprising a quaternary ammonium compound to extract a soluble or membrane associated intracellular recombinant POI from a bacterial, yeast or fungal cell, the POI being released from the bacterial, yeast or fungal cell, in which the membrane extracting composition comprises a quaternary ammonium compound at a concentration of between 0.05% to 0.6% by weight and is contacted with the bacterial, yeast or fungal cell and in which the released recombinant POI is recovered from the membrane extracting composition. 
     
     
         13 . The method according to  claim 1 , in which the recombinant POI is a glucan lyase. 
     
     
         14 . The method according to  claim 9 , in which the recombinant POI is a glucan lyase. 
     
     
         15 . The method according to  claim 10 , in which the recombinant POI is a glucan lyase. 
     
     
         16 . The method according to  claim 12 , in which the recombinant POI is a glucan lyase. 
     
     
         17 . The method according to  claim 1 , in which the recombinant POI is an interleukin-1 receptor antagonist (IL-1ra). 
     
     
         18 . The method according to  claim 9 , in which the recombinant POI is an interleukin-1 receptor antagonist (IL-1ra). 
     
     
         19 . The method according to  claim 10 , in which the recombinant POI is an interleukin-1 receptor antagonist (IL-1ra). 
     
     
         20 . The method according to  claim 12 , in which the recombinant POI is an interleukin-1 receptor antagonist (IL-1ra). 
     
     
         21 . The method according to  claim 1 , in which the recombinant POI is hexose oxidase (HOX) enzyme. 
     
     
         22 . The method according to  claim 9 , in which the recombinant POI is hexose oxidase (HOX) enzyme. 
     
     
         23 . The method according to  claim 10 , in which the recombinant POI is hexose oxidase (HOX) enzyme. 
     
     
         24 . The method according to  claim 12 , in which the recombinant POI is hexose oxidase (HOX) enzyme. 
     
     
         25 . The method according to  claim 21 , in which the hexose oxidase (HOX) enzyme comprises the amino acid sequence set out in SEQ ID No 22. 
     
     
         26 . The method according to  claim 22 , in which the hexose oxidase (HOX) enzyme comprises the amino acid sequence set out in SEQ ID No 22. 
     
     
         27 . The method according to  claim 23 , in which the hexose oxidase (HOX) enzyme comprises the amino acid sequence set out in SEQ ID No 22. 
     
     
         28 . The method according to  claim 24 , in which the hexose oxidase (HOX) enzyme comprises the amino acid sequence set out in SEQ ID No 22. 
     
     
         29 . The method according to  claim 21 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence set out in SEQ ID No 22. 
     
     
         30 . The method according to  claim 22 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence set out in SEQ ID No 22. 
     
     
         31 . The method according to  claim 23 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence set out in SEQ ID No 22. 
     
     
         32 . The method according to  claim 24 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence set out in SEQ ID No 22. 
     
     
         33 . The method according to  claim 21 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence capable of hybridising to the nucleotide sequence set out in SEQ ID No 22. 
     
     
         34 . The method according to  claim 22 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence capable of hybridising to the nucleotide sequence set out in SEQ ID No 22. 
     
     
         35 . The method according to  claim 23 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence capable of hybridising to the nucleotide sequence set out in SEQ ID No 22. 
     
     
         36 . The method according to  claim 24 , in which the hexose oxidase (HOX) enzyme is encoded by a nucleotide sequence capable of hybridising to the nucleotide sequence set out in SEQ ID No 22. 
     
     
         37 . A POI producible by a method according to  claim 1  wherein said POI is glucan lyase. 
     
     
         38 . A POI producible by a method according to  claim 1  wherein said POI is interleukin-1 receptor antagonist. 
     
     
         39 . A POI producible by a method according to  claim 1  wherein said POI is hexose oxidase (HOX). 
     
     
         40 . A POI producible by the method according to  claim 1  wherein said POI is hexose oxidase enzyme and said HOX enzyme is encoded by a nucleotide sequence, and wherein the nucleotide sequence set out in SEQ ID NO: 22, or a sequence complementary to the hybridisable sequence, and wherein the nucleotide sequence is synthesised by the oligonucleotides as set out in SEQ ID NOs: 2-22. 
     
     
         41 . A POI as defined in  claim 1 , wherein the POI is released in a substantially non-glycosylated form from a eukaryotic host organism. 
     
     
         42 . A substantially non-glycosylated POI released from a eukaryotic host organism. 
     
     
         43 . A substantially non-glycosylated POI released from a eukaryotic host organism, wherein the POI is released by the method of  claim 1 .