Periplasmic Expression of Antibody Fragments

Abstract

This chapter focuses on the biology and practical techniques that can help in the expression of antibody fragments in E. coli. The periplasm is often preferred for expression of soluble antibody fragments and some of the factors for this choice are discussed. The periplasm is the site of expression for only ~1/10 of the soluble proteins of E. coli and does not contain any DNA or RNA that can interfere with purification. Therefore combination of periplasmic expression with carefully considered harvest and extraction regimes can result in a very useful concentration and partial purification/enrichment of the periplasmic protein of interest. An important factor for enabling useful levels of protein expression is good selection or design of the expression plasmid. The key areas considered here are: (i) plasmid copy number/ origin of replication, (ii) constitutive versus inducible promoter, (iii) resistance/selection marker, and (iv) design of the coding region. The phoA promoter along with depletion of phosphate has also been used to good effect for the production of antibody fragments. A notional advantage of periplasmic expression over extracellular expression is the ability to concentrate the product simply by harvesting the cells by centrifugation or filtration and avoiding vortex-type protein damage. The chapter describes various designs of antibody fragments along with the engineered adaptations and improvements to both the fragment and the host. The endoplasmic reticulum of eukaryotes is the natural site for the folding and assembly of antibodies.