Improving expression of paired antibody fragments in Escherichia coli.

The expression of antibody fragments in bacteria is often hindered by low expression yields and the propensity of the purified protein to aggregate. Furthermore, despite being constructed of immunoglobulin domains, properties tend to be sequence dependent. Addressing these issues on a case-by-case basis often requires considerable optimisation of the expression procedure.Here we investigate whether the introduction of negatively charged CDR mutations, that had been shown to improve aggregation resistance, also improve protein expression yields in Escherichia coli. These mutations were introduced into therapeutic antibodies in an scFv fragment format and assessed for protein expression. In addition, the use of framework mutations was also investigated. These experiments demonstrated considerable improvement of expression yields. Moreover, crystal structures of two scFv fragments were determined, which revealed a high degree of structural conservation, indicating compatibility of the mutations with high affinity antigen binding; this was confirmed by biosensor measurements. In addition to the scFv format, the expression of Fab fragments was also investigated. However, yields of this larger multi-chain fragment format remained poor in the E. coli system. This was observed despite systematic variation of antibody type and vector systems, indicating a more general limitation of this expression system. This was confirmed through successful production in HEK293 cells, indicating that transient expression in a eukaryotic system may be preferable as a means of expressing antibodies in an Fab format