PLASMID STABILITY IN RECOMBINANT SACCHAROMYCES-CEREVISIAE

Because of many advantages, the yeast Saccharomyces cerevisiae is incr easingly being employed for expression of recombinant proteins. Usuall y, hybrid plasmids (shuttle vectors) are employed as carriers to intro duce the foreign DNA into the yeast host. Unfortunately, the transform ed host often suffers from some kind of instability, tending to lose o r alter the foreign plasmid. Construction of stable plasmids, and main tenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discusse d. The focus is on S. cerevisiae, but where relevant, examples From th e better studied Escherichia coli system are discussed. Compared to fr ee suspension culture, immobilization of cells is particularly effecti ve in improving plasmid retention; hence, immobilized systems are exam ined in some detail. Immobilized cell systems combine high cell concen trations with enhanced productivity of the recombinant product, thereb y offering a potentially attractive production method, particularly wh en nonselective media are used. Understanding of the stabilizing mecha nisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.