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Gene selection and cloning approaches for co-expression and production of recombinant protein–protein complexes

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Journal of Structural and Functional Genomics

Abstract

Multiprotein complexes play essential roles in all cells and X-ray crystallography can provide unparalleled insight into their structure and function. Many of these complexes are believed to be sufficiently stable for structural biology studies, but the production of protein–protein complexes using recombinant technologies is still labor-intensive. We have explored several strategies for the identification and cloning of heterodimers and heterotrimers that are compatible with the high-throughput (HTP) structural biology pipeline developed for single proteins. Two approaches are presented and compared which resulted in co-expression of paired genes from a single expression vector. Native operons encoding predicted interacting proteins were selected from a repertoire of genomes, and cloned directly to expression vector. In an alternative approach, Helicobacter pylori proteins predicted to interact strongly were cloned, each associated with translational control elements, then linked into an artificial operon. Proteins were then expressed and purified by standard HTP protocols, resulting to date in the structure determination of two H. pylori complexes.

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Acknowledgments

The authors wish to thank members of the Structural Biology Center at Argonne National Laboratory for their help with data collection at the 19-ID beamline. This work was supported by the National Institutes of Health Grants GM074942 and GM094585, and by the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Author contributions

GB, R.J., M.D. and A.J. designed the experiments. R.J. and W.E. performed the cloning and small scale expression of the ‘Eps-RBS-fusion-strategy’ and ‘Operon-strategy’ targets. L.S., N.M., A.W. and R.W. purified and crystallized complexes, B.N and A.S. determined the structure of the protein complexes. All authors read and revised the manuscript.

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    1. Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, 9700 S Cass Ave., Argonne, IL, 60439, USA

      György Babnigg, Robert Jedrzejczak, Boguslaw Nocek, Adam Stein, William Eschenfeldt, Lucy Stols, Norman Marshall, Alicia Weger, Ruiying Wu, Mark Donnelly & Andrzej Joachimiak

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    Correspondence to György Babnigg or Andrzej Joachimiak.

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    György Babnigg and Robert Jedrzejczak have contributed equally to this work.

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    Babnigg, G., Jedrzejczak, R., Nocek, B. et al. Gene selection and cloning approaches for co-expression and production of recombinant protein–protein complexes. J Struct Funct Genomics 16, 113–128 (2015). https://doi.org/10.1007/s10969-015-9200-y

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