ABSTRACT
The typical protein to be expressed in gram-negative bacteria comprises 100-300 amino acids, has no posttranslational modications, and possesses a restricted number of cysteine residues to make in vitro refolding possible. Whereas this chapter pertains mainly to gram-negative bacteria, a discussion of how the gram-positive bacteria are viewed with great interest should be made. The problems of methionine blocking the N-terminus and the intracellular formation of inclusion bodies which add host cell impurities in the use of gram-negative bacteria can be resolved by using gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis. Also, the presence of endotoxin is of little concern in grampositive bacteria. On the negative side, while using Bacillus, endogenous proteolytic degradation can be signicant, a drawback not found in Lactococcus. The extracellular expression of the folded target protein in the use of gram-positive bacteria additionally eliminates signicant problems in the use of Escherichia coli expression system: the requirements of in vitro folding and performing cleavage of the N-terminal tag. This results in simpler process design comprising of harvest, capture, intermediate purication, polishing, concentration, and nishing as described below for gram-negative bacteria. The rest of the chapter refers to expression system comprising gram-negative bacteria, particularly E. coli.
