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Science 20 February 2004:
Vol. 303. no. 5661, pp. 1185 - 1189
DOI: 10.1126/science.1092612
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Reports

An Engineered Pathway for the Formation of Protein Disulfide Bonds

Lluis Masip,1 Jonathan L. Pan,3,4 Suranjana Haldar,5 James E. Penner-Hahn,5 Matthew P. DeLisa,1 George Georgiou,1,2* James C. A. Bardwell,3,4* Jean-François Collet3

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.

1 Department of Chemical Engineering and Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA.
2 Department of Biomedical Engineering, University of Texas, Austin, TX 78712, USA.
3 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
4 Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.
5 Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

* To whom correspondence should be addressed. E-mail: gg{at}che.utexas.edu (G.G.); jbardwel{at}umich.edu (J.C.A.B.)

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