VPB 321: Animal Biotechnology (2+1)

INDUCIBLE EXPRESSION VECTORS

• It is usually advantageous to keep a cloned gene represed until ready to express it. 
• One reason is that eukaryotic proteins produced in large quantities in bacteria can be toxic. 
• Even if these proteins are not actually toxic, they can build up to such great levels that they interfere with bacterial growth. 
• In either case, if the cloned gene were allowed to remain turned on constantly, the bacteria bearing the gene would never grow to a great enough concentration to produce meaningful quantities of protein product. 
• The solution is to keep the cloned gene turned off by placing it downstream of an inducible promoter that can be turned off.
• The lac promoter is inducible to a certain extent, presumably remaining off until stimulated by the synthetic inducer isopropylthiogalactoside (IPTG). 
• Another strategy is to use a tightly controlled promoter as the λ phage promoter PL.
• Expression vectors with this promoter/operator system are cloned into host cells bearing a temperature-sensitive λ repressor gene (c1857).
• As long as these cells kept relatively low (32°C ), the repressor functions, and no expression takes place.  However, when the temperature is raised to the nonpermissive level (42ºC), the temperature-sensitive repressor can no longer function and the cloned gene is induced.