Polypeptide compositions and methods

Abstract

A method of preparing a bioactive polypeptide in a stable, inactivated form, the method comprising the step of treating the polypeptide with ozonated water in order to oxidize and/or stabilize the cysteine residues, and in turn, prevent the formation of disulfide bridges necessary for bioactivity. The method can involve the use of ozonated water to both oxidize the disulfide bridges in a bioactive polypeptide, and to then stabilize the resultant cysteine residues. Optionally, and preferably, the method can involve the use of ozonated water to stabilize the cysteine residues, and thereby prevent the formation of disulfide bridges, in a polypeptide produced by recombinant means in a manner that allows the polypeptide to be recovered with the disulfide bridges unformed.

What is claimed is:  
     
         1 . A method of preparing a composition comprising an inactivated bioactive polypeptide, the method comprising the steps of: 
 a) identifying a polypeptide having a biological activity dependent on the presence of one or more disulfide bridges in its tertiary structure,    b) preparing a cDNA strand encoding the polypeptide,    c) expressing the cDNA under conditions in which the polypeptide is recovered in an inactive form due to the failure to form one or more disulfide bridges, and    d) recovering the inactive polypeptide and formulating it into an aqueous composition.    
     
     
         2 . A method according to  claim 1 , wherein the composition is suitable for parenteral administration to a host.  
     
     
         3 . A method according to  claim 1  wherein the bioactive polypeptide is selected from the group consisting of toxins affecting the presynaptic neurojunction, toxins affecting the postsynaptic neurojunction, toxins those affecting ion channels, and toxins that damage the cell membrane.  
     
     
         4 . A method according to  claim 3  wherein the toxins affecting the presynaptic neurojunction toxins are selected from the group consisting of notexin, β-bungarotoxin, crotoxin, taipoxin, textilotoxin and α-latrotoxin.  
     
     
         5 . A method according to  claim 3  wherein the toxins affecting the postsynaptic neurojunction are selected from the group consisting of α-conotoxins, α-cobrotoxin, erabutoxin, α-cobratoxin and α-bungarotoxin.  
     
     
         6 . A method according to  claim 3  wherein the toxins affecting ion channels are selected from the group consisting of dendrotoxins, scorpion toxins, μ-conotoxins, and sea anemone toxins.  
     
     
         7 . A method according to  claim 3  wherein the toxins that damage the cell membrane are membrane-damaging toxin selected from the group consisting of myotoxins, cariotoxins, mellitin, and phospholipases.  
     
     
         8 . A method according to  claim 1  wherein the toxin is produced by expressing cDNA under conditions that allow the polypeptide to be recovered with one or more disulfide bridges unformed.  
     
     
         9 . A method according to  claim 8  wherein the cDNA is expressed in an expression system selected from the group consisting of bacteria, yeast or higher eucaryotic cell lines.  
     
     
         10 . A method according to  claim 9  wherein the expression system is a yeast expression system.  
     
     
         11 . A method according to  claim 10  wherein the yeast expression system is selected from the group consisting of  Saccharomyces cerevisiae  and  Pichia pastoris  expression systems.  
     
     
         12 . A method according to  claim 11  wherein the expression system is a Pichia expression system and the polypeptide is cytoplasmically produced in a manner that allows the product to be recovered in inactive form, without the formation of disulfide bridges.  
     
     
         13 . A method according to  claim 12  comprising the further step of treating the recovered polypeptide with ozonated water in order to stabilize the cysteine residues and prevent the formation of disulfide bridges.  
     
     
         14 . A method according to  claim 9  wherein the polypeptide is recovered in active form subjected to ozone treatment in order to break the disulfide bridges and render the polypeptide inactive and atoxic.  
     
     
         15 . A composition comprising an atoxic polypeptide prepared according to the method of  claim 1 .  
     
     
         16 . A composition according to  claim 15  wherein the composition is provided in sterile form suitable for parenteral administration to a host.  
     
     
         17 . A method for preparing a bioactive polypeptide in an inactivated form, the method comprising the step of treating the polypeptide with ozonated water under conditions suitable to both oxidize any disulfide bonds in order to form corresponding pairs of cysteine residues, and to then stabilize the resultant cysteine residues and prevent the reformation of disulfide bonds.  
     
     
         18 . An inactivated bioactive polypeptide prepared by a method that comprises the step of treating a bioactive polypeptide with ozonated water under conditions suitable to both oxidize any disulfide bonds in order to form corresponding pairs of cysteine residues, and to then stabilize the resultant cysteine residues and prevent the reformation of disulfide bonds.