Active modified hedgehog proteins

Abstract

A post-translationally processed hedgehog protein mutant which exhibits a molecular weight of 22±1 kDa under alkylating conditions, exhibits a molecular weight of 24±1 kD under reducing conditions, is stabilized with respect to its activity by suramin is inactivated when 8 or more amino acids are cleaved N-terminally is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37° C., induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin, is not modified by cholesterol, exhibits an activity that is increased many times.

1 . Post-translationally processed hedgehog protein mutant which is obtainable by expressing a gene which codes for a hedgehog protein in a baculovirus expression system in a fermentation for a period of up to 30 hours, purifying the cell supernatant in the presence of a protease inhibitor and a non-ionic detergent and isolating the hh mutant which binds to heparin-Sepharose and hydroxylapatite and is characterized in that this hh mutant 
 exhibits a molecular weight of 22+1 kDa under alkylating conditions,    exhibits a molecular weight of 24+1 kD under reducing conditions,    is stabilized with respect to its activity by suramin    is inactivated when 8 or more amino acids are cleaved N-terminally    is inactivated by 90% or more when incubated with 10 mmol/1 DTE for 2.5 hours at 37° C.,    induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,    is not modified by cholesterol.    
     
     
         2 . Process for the production of a post-translationally processed hedgehog protein mutant by expressing a gene which codes for a hedgehog protein in a baculovirus expression system in a fermentation for a period of 24 to 27 hours, purifying the cell supernatant in the presence of a protease inhibitor and a non-ionic detergent and isolating the hh mutant which binds to heparin-Sepharose and hydroxylapatite and is characterized in that this hh mutant 
 exhibits a molecular weight of 22+1 kDa under alkylating conditions,    exhibits a molecular weight of 24+1 kD under reducing conditions,    is stabilized with respect to its activity by suramin    is inactivated when 8 or more amino acids are cleaved N-terminally    is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37° C.,    induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,    is not modified by cholesterol.    
     
     
         3 . Process as claimed in  claim 2 , wherein, after chromatography on heparin-Sepharose, it is dialysed against lower ionic strengths.  
     
     
         4 . Process as claimed in  claim 3 , wherein the dialysis is carried out in the presence of 10-100 mmol/l sodium chloride.  
     
     
         5 . Pharmaceutical composition containing a hh mutant as claimed in  claim 1 .  
     
     
         6 . Pharmaceutical composition as claimed in  claim 5 , containing suramin, a biocompatible matrix and/or a sequestering agent.  
     
     
         7 . Process for the production of a pharmaceutical composition by combination of a hh mutant as claimed in  claim 1  with a pharmaceutical auxiliary substance or with suramin.  
     
     
         8 . Process for the production of a pharmaceutical composition by combination of a hh mutant as claimed in  claim 1  with a biocompatible matrix and/or a sequestering agent.