Method for screening restriction endonucleases

Abstract

A method is provided for identifying a restriction endonuclease that includes: screening a target DNA sequence for the presence of known methylase sequence motifs, identifying any open reading frames which lie close to the screened methylase sequence motif and assaying the protein products of the open reading frames for restriction endonuclease activity.

What is claimed is:  
     
         1 . A vector suitable for cloning a DNA sequence encoding a cytotoxic protein wherein the vector comprises at least a first and a second transcription promotor and is adapted to accept the DNA sequence insert and wherein the first and second transcription promoters are independently controllable.  
     
     
         2 . The vector of  claim 1 , wherein the first transcription promotor enables anti-sense strand transcription and the second transcription promotor enables sense strand transcription.  
     
     
         3 . The vector of  claim 1 , wherein the first transcription promotor comprises λ phage promotor and the second transcription promotor comprises T7 RNA polymerase promotor.  
     
     
         4 . The vector of  claim 1 , wherein the vector is pLT7K.  
     
     
         5 . The vector of  claim 1 , wherein the independent control of the first and second transcription promoters is achieved by temperature, IPTG addition, or RNA polymerase inhibition.  
     
     
         6 . The vector of  claim 5  wherein the RNA polymerase inhibition is achieved by bacteriophage T7 lysozyme expression, or utilization of a T7 RNA polymerase negative  E. coli  strain.  
     
     
         7 . An  E. coli  host cell transformed by the vector of any one of claims  1 ,  2 ,  3 ,  4 ,  5  or  6 .  
     
     
         8 . A method for producing a recombinant cytotoxic protein, the method comprising the steps of: 
 (1) inserting a DNA sequence encoding the cytotoxic protein into the vector of any one of claims  1 ,  2 ,  3 ,  4 ,  5 , or  6 ;    (2) transforming a host cell with the vector of step (1) under conditions which disallow the expression of the sense strand;    (3) culturing the transformed host cell of step (2) under conditions which disallow the expression of the sense strand;    (4) inducing the selective expression of the sense strand; and    (5) producing the recombinant cytotoxic protein.    
     
     
         9 . The method of  claim 8 , wherein step (4) further comprises inducing the selective expression of the sense strand by temperature, IPTG addition, or RNA polymerase inhibition.