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Purification of recombinant high molecular weight two-partner secretion proteins from Escherichia coli

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Abstract

This protocol describes the purification of a recombinant high molecular weight (HMW) two-partner secretion exoprotein (generically referred to as TpsA proteins) from Escherichia coli using methods developed recently to obtain highly purified flagellin-free recombinant EtpA (rEtpA) glycoprotein. The protocol addresses problems frequently encountered with the expression of these HMW proteins, namely plasmid instability and protein degradation, as well as a recently recognized issue of flagellin contamination. Briefly, the TpsA protein of interest is expressed with its outer membrane transporter (TpsB) protein in a flagellin-minus recombinant E. coli background. Culture supernatants are collected, concentrated through high molecular weight cutoff filters, followed by purification by size exclusion column chromatography. Details are included for the expression of HMW TpsA glycoproteins as polyhistidine-tagged molecules, which can be further purified by metal affinity chromatography (MAC). Using this protocol, it is possible to obtain highly purified microgram–milligram quantities of the TpsA protein of interest within 2–3 days.

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Figure 1: Two-partner secretion (TPS) operons.
Figure 2: General outline for purification of two-partner secretion of exoprotein (TpsA proteins).
Figure 3: Recombinant plasmids for the co-expression of Tps genes in Escherichia coli.
Figure 4: Pilot expression of TpsA exoprotein.
Figure 5: Purification of two-partner secretion (TPS) exoprotein by size-exclusion (gel filtration) or affinity chromatography.
Figure 6: Purified recombinant two-partner secretion exoprotein (TpsA proteins) retain functional activity.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (National Center for Research Resources) RR16190-05, the Department of Veterans Affairs and funds from the University of Tennessee Microbial Pathogenesis Research Center.

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  1. Department of Veterans Affairs Medical Center Research (151), Memphis, Tennessee, USA

    James M Fleckenstein & Koushik Roy

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  1. James M Fleckenstein
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Correspondence to James M Fleckenstein.

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The University of Tennessee Research corporation has filled for a patent on the use of EtpA and flagellin in the treatment/prevention of Gram-negative infections.

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Fleckenstein, J., Roy, K. Purification of recombinant high molecular weight two-partner secretion proteins from Escherichia coli. Nat Protoc 4, 1083–1092 (2009). https://doi.org/10.1038/nprot.2009.87

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