The goal of this proposal is to discover, improve, and make available new bacterial hosts for expressing foreign proteins. E. coli K-12, the most commonly used host strain, is inadequate for expression of many proteins of commercial or medical importance. Indications are that other bacterial hosts may be far superior to E. coli K-12 for expressing a number of human proteins. Moreover, there is no need to alter the existing clone, saving time, effort, and resources. These strains (TOPPTM cells) have already been made commercially available. They are a rapid alternative to subcloning or vector manipulation for over-expressing recombinant proteins when E. coli K-12 is incapable. Continuing to improve these strains, and screening for new hosts will augment this already commercially successful endeavor. In addition, developing the technique called cointegrate conversion for """"""""nonsubcloning cloning,"""""""" and expanding it to both prokaryotic and eukaryotic expression systems, represents a rapid and inexpensive method for manipulating genes.
