ABSTRACT
This chapter focuses on the genetic and molecular biological approaches which has generated high-level expression of toxin and toxin fragments. Escherichia coli was first used for high-level expression of tox because of the successful expression of many heterologous genes and the amount of knowledge concerning transcriptional and translational regulation in that organism.Gram-negative bacteria contain an inner cytoplasmic membrane and an outer membrane both of which bound the periplasmic space. The process of secretion, transport of proteins from their site of synthesis to the periplasmic space, is quite complex. Proteins which are rapidly degraded include incomplete polypeptides, complete proteins containing amino acid substitutions, free subunits of large multimene complexes, posttranslationally damaged proteins, and certain polypeptides synthesized through recombinant DNA technology. To ensure safety, cloning of tox has been limited to hypotoxic peptide fragments or inactive mutants. Both classic genetics and current molecular biological approaches have been applied to the study and manipulation of the gene for diphtheria toxin.
