Production Of CPD Glycosylases

Abstract

Recombinant forms of DNA sequences for CPD glycosylases, including the bacteriophage T4 gene denV, are described that are capable of expression at high levels. Active CPD glycosylases can be recovered from inclusion bodies resulting from the high expression using, for example, a homogenization process which employs stream mixing, and the active proteins can be used in, for example, topical formulations for treatment of photosensitive diseases. Stream mixing can also be used to solubilize inclusion bodies containing proteins other than CPD glycosylases.

1 . A culture of cells that produces a CPD glycosylase protein wherein for at least one period of at least ten minutes, at least 25 weight percent of the total protein produced by the culture during the period is the CPD glycosylase protein. 
     
     
         2 . The culture of  claim 1  wherein the CPD glycosylase protein is T4 endonuclease V. 
     
     
         3 . A method for producing a CPD glycosylase protein comprising:
 (a) culturing cells which produce the CPD glycosylase protein; and   (b) collecting the CPD glycosylase protein;   wherein the CPD glycosylase protein comprises at least 25 weight percent of the total protein produced by the cells during step (a).   
     
     
         4 . The method of  claim 3  wherein the CPD glycosylase protein is T4 endonuclease V. 
     
     
         5 . A method for producing a protein comprising:
 (a) culturing cells which produce the protein, at least some of said protein being in inclusions bodies; and   (b) collecting the protein;   wherein in step (b), at least part of the protein produced by the cells in step (a) is subjected to a mixing process that comprises: (i) forming two or more flowing streams at least one of which contains inclusion bodies which contain the protein, and (ii) impinging the streams on one another to disrupt at least some of the inclusion bodies.   
     
     
         6 . The method of  claim 5  wherein the at least one flowing stream which contains inclusion bodies containing the protein comprises intact cells. 
     
     
         7 . The method of  claim 5  wherein the at least one flowing stream which contains inclusion bodies containing the protein comprises disrupted cells. 
     
     
         8 . The method of  claim 5  wherein:
 (i) at least one of the flowing streams has a velocity V1 prior to the impingement, and   (ii) the impingement produces a stream that has a velocity V2, where V1 minus V2 is greater than or equal to 90 meters/second.   
     
     
         9 . The method of  claim 5  wherein:
 (i) at least one of the flowing streams has a velocity greater than or equal to 100 meters/second prior to the impingement, and   (ii) the impingement produces a stream that has a velocity which is less than 10 meters/second.   
     
     
         10 . The method of  claim 5  wherein the protein is a CPD glycosylase protein.