Basic Biotechnology

Introduction

Bacteria and yeasts have been the organisms of choice for the industrial production of heterologous recombinant proteins for many years. The ability to cultivate bacterial strains to high cell density at large scale has become an increasingly important technique throughout the field of biotechnology, from basic research programmes (structural or kinetic studies) to large-scale pharmaceutical production processes. Escherichia coli remains one of the most attractive organisms for the production of recombinant proteins (see Chapters 4, 5, and 21 for examples) where no complex post-translational modifications (e.g. glycosylation or disulphide bond formation) are required for biological activity and because its genetics and physiology are well understood. However, there are important drawbacks associated with the use of prokaryotic organisms. The low percentage of GC nucleotides in their genomes, when compared to mammalian genes, and the existence of rare codons that often result in low expression levels or inactive truncated forms and, in many cases, proteins are expressed as insoluble inclusion bodies in the bacterial periplasmic space. Bacteria are also incapable of carrying out any post-translational modifications, which strongly influences protein stability, folding, solubility and, hence, its biological activity. Yeasts (e.g. Saccaromyces cerevisiae or Pichia pastoris), though, can perform some post-translational modifications similar to those of the more complex eukaryotic cells (see Chapter 5). However N-glycosylation of mammalian proteins in yeast seems to be very inefficient. Additionally, both bacteria and yeast cells are surrounded by a mechanically strong cell wall that may hinder recovery of any non-secreted proteins.