Commercial Scale Production of Cvi Endonucleases

Chlorella virus (Cvi) restriction nucleases are a large, untapped source of gene mapping tools that have considerable commercial potential. Unlike bacterial restriction endonucleases, which recognize 4 to 8 base pair (bp) DNA target sequences, Cvi endonucleases define unique 2 to 4 bp cleavage sites and lend themselves to a variety of novel fine structure gene mapping applications. However, at the present time technology transfer from laboratory-scale enzyme identification and purification has not proceeded to large-scale commercial production. We will begin the development of large-scale production of three chlorella virus DNA restriction endonucleases (R.CviJI cleaves PuG/CPY, R.CviRI cleaves TG/CA, and R.CviAII cleaves C/ATG) for which there is a clearcut demand and cannot be isolated from bacteria. Two independent approaches toward large-scale (> 100,000 enzyme units) commercial production will be examined. (i) Increased production will be sought from virus-infected algae. (ii) The DNA restriction endonuclease gene will be cloned and overexpressed in E. coli. These studies will serve as an instructive model for the commercial scale production of a major new class of gene mapping enzymes.