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Adv Exp Med Biol. 2016;896:43-58. doi: 10.1007/978-3-319-27216-0_4.

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

Author information

1
Architecture et Fonction des Macromolécules Biologiques (A.F.M.B), UMR7257 CNRS, Université Aix-Marseille, Case 932, 163 Avenue de Luminy, 13288, Marseille cedex 9, France. renaud.vincentelli@afmb.univ-mrs.fr.
2
Département de Biologie Structurale Intégrative, Centre de Biologie Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404, Illkirch Cedex, France. romier@igbmc.fr.

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.

KEYWORDS:

Automation; Biophysics; Co-expression; Escherichia coli; Expression parameters; Expression vectors; Protein complexes; Purification; Structural characterization

PMID:
27165318
DOI:
10.1007/978-3-319-27216-0_4
[Indexed for MEDLINE]

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