Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP.

TitleOverproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP.
Publication TypeJournal Article
Year of Publication2010
AuthorsVan der Henst, C., C. Charlier, M. Deghelt, J. Wouters, J-Y. Matroule, J-J. Letesson, and X. De Bolle
JournalBMC Microbiol
Volume10
Pagination248
Date Published2010 Sep 28
ISSN1471-2180
KeywordsBacterial Proteins, Brucella abortus, Escherichia coli, Histidine Kinase, Inclusion Bodies, Molecular Chaperones, Protein Folding, Protein Kinases, Recombinant Proteins, Solubility
Abstract

BACKGROUND: When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.RESULTS: We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.CONCLUSION: The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

DOI10.1186/1471-2180-10-248
Alternate JournalBMC Microbiol.
PubMed ID20920169
PubMed Central IDPMC2957392
Research group: