Recombinantly produced neutral protease originating from paenibacillus polymyxa

Abstract

The present disclosure provides the sequence of a Paenibacillus polymyxa preproenzyme which is the precursor of a neutral protease, expression thereof in a transformed host organism, and methods for production of the neutral protease, by recombinant means. Further, use of the recombinantly produced neutral protease is disclosed in the field of cell biology, particularly for the purpose of tissue dissociation. The disclosure also includes blends with other proteases. Further disclosed are nucleotide sequences encoding the neutral protease.

1 . A method for recombinantly producing a neutral protease, the method comprising the steps of
 (a) providing in an expression vector a DNA with a sequence encoding a preproenzyme according to SEQ ID NO:5, and transforming a host organism with the expression vector, thereby obtaining a transformed host organism, wherein the host organism is a gram-positive prokaryotic species; followed by   (b) expressing the DNA in the transformed host organism, wherein the transformed host organism secretes the neutral protease; followed by   (c) isolating the secreted neutral protease;   thereby recombinantly producing the neutral protease.   
     
     
         2 . The method according to  claim 1 , wherein the DNA comprises the sequence of position 34 to position 1896 of SEQ ID NO:6. 
     
     
         3 . The method according to  claim 1 , wherein the host organism is a gram-positive eubacterial species. 
     
     
         4 . The method according to  claim 3 , wherein the gram-positive bacterial species is selected from the group consisting of  Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus  and  Streptococcus.    
     
     
         5 . The method according to  claim 4 , wherein the gram-positive bacterial species is  Bacillus amyloliquefaciens.    
     
     
         6 . The method according to  claim 1 , wherein step (b) comprises culturing the transformed host organism in a liquid medium, wherein the transformed host organism secretes the neutral protease into the liquid medium. 
     
     
         7 . The method according to  claim 6 , wherein step (c) comprises isolating the secreted neutral protease from the liquid medium. 
     
     
         8 . The method according to  claim 5 , wherein the host organism is deficient of an extracellular protease selected from Npr and Apr. 
     
     
         9 . A method of isolating living cells from animal tissue in vitro, comprising the steps of (a) providing a recombinantly produced neutral protease obtained by performing a method according to  claim 1 , and (b) incubating the tissue in vitro with the neutral protease of step (a), wherein protein components of the extracellular matrix of the tissue are proteolytically degraded, and wherein a layer of cells or a suspension of individual living cells is obtained, thereby isolating living cells from animal tissue in vitro. 
     
     
         10 . The method according to  claim 9 , wherein the animal tissue originates from a vertebrate animal. 
     
     
         11 . The method according to  claim 9 , wherein in step (b) the tissue is additionally incubated with a collagenase. 
     
     
         12 . A kit of parts comprising in a sealed compartment a lyophilizate of a neutral protease obtained by performing a method according to  claim 5 . 
     
     
         13 . The kit according to  claim 15 , wherein the kit further comprises in a separate sealed compartment a lyophilized preparation of a collagenase. 
     
     
         14 . The kit according to  claim 12 , wherein the sealed compartment further contains a collagenase, the collagenase being blended with the neutral protease. 
     
     
         15 . A method for making a blend of a plurality of proteases, comprising the steps of (a) providing a recombinantly produced neutral protease obtained by performing a method according to  claim 1 , and (b) mixing the neutral protease of step (a) with a further protease. 
     
     
         16 . The method according to  claim 15 , wherein the further protease is selected from a collagenase and thermolysin. 
     
     
         17 . A nucleotide sequence encoding a polypeptide comprising the amino acid sequence of position 289 to position 592 of SEQ ID NO:5, the nucleotide sequence being selected from the group consisting of
 (a) a nucleotide sequence having the sequence of position 898 to position 1811 in SEQ ID NO:6;   (b) nucleotide sequences derived from the nucleotide sequence of position 898 to position 1811 of SEQ ID NO:6 as a result of the degenerated code.   
     
     
         18 . The nucleotide sequence according to  claim 17 , wherein the nucleotide sequence is the sequence of position 34 to position 1811 of SEQ ID NO:6.