Complex Reconstitution and Characterization by Combining Co-expression Techniques in Escherichia coli with High-Throughput.

Abstract

Single protein expression technologies have strongly benefited from the Structural Genomics initiatives that have introduced parallelization at the laboratory level. Specifically, the developments made in the wake of these initiatives have revitalized the use of Escherichia coli as major host for heterologous protein expression. In parallel to these improvements for single expression, technologies for complex reconstitution by co-expression in E. coli have been developed. Assessments of these co-expression technologies have highlighted the need for combinatorial experiments requiring automated protocols. These requirements can be fulfilled by adapting the high-throughput approaches that have been developed for single expression to the co-expression technologies. Yet, challenges are laying ahead that further need to be addressed and that are only starting to be taken into account in the case of single expression. These notably include the biophysical characterization of the samples at the small-scale level. Specifically, these approaches aim at discriminating the samples at an early stage of their production based on various biophysical criteria leading to cost-effectiveness and time-saving. This chapter addresses these various issues to provide the reader with a broad and comprehensive overview of complex reconstitution and characterization by co-expression in E. coli.