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Applied and Environmental Microbiology, December 2005, p. 8451-8459, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8451-8459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

Jae-Young Kim,1 Elizabeth A. Fogarty,1 Franklin J. Lu,1 Hui Zhu,2 Geoffrey D. Wheelock,2 Lee A. Henderson,2 and Matthew P. DeLisa1*

School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853,1 Viral Therapeutics, Inc., Ithaca, New York 148502

Received 19 July 2005/ Accepted 26 August 2005

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.

* Corresponding author. Mailing address: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853. Phone: (607) 254-8560. Fax: (607) 255-9166. E-mail: md255{at}cornell.edu.

Applied and Environmental Microbiology, December 2005, p. 8451-8459, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8451-8459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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