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Recombinant expression of selectively sulfated proteins in Escherichia coli

Nature Biotechnology volume 24, pages 1436–1440 (2006)Cite this article

Abstract

Although tyrosine sulfation is a post-translational modification widespread across multicellular eukaryotes1, its biological functions remain largely unknown. This is in part due to the difficulties of synthesizing selectively sulfated proteins. Here we report the selective incorporation of sulfotyrosine into proteins in bacteria by genetically encoding the modified amino acid in response to the amber nonsense codon TAG. Moreover, we show that this strategy enables direct expression in Escherichia coli of sulfo-hirudin, previously inaccessible through recombinant methods. The affinity of sulfo-hirudin toward human thrombin is enhanced more than tenfold over that of desulfo-hirudin, suggesting that sulfo-hirudin may offer clinical advantages for use as an anticoagulant2. This general approach to the biosynthesis of sulfated proteins should facilitate further study and application of tyrosine sulfation.

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Figure 1
Figure 2: Desulfo-hirudin and sulfo-hirudin migration on a denaturing PAGE gel stained with Coomassie blue.
Figure 3: Representative plots of thrombin inhibition with their respective fitted progress curves superimposed on the raw data points.

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Acknowledgements

We thank Jianming Xie for providing synthetase libraries for this project and Youngha Ryu for providing the pSup vector. C.C.L. thanks Jianming Xie, Eric Brustad, Vaughn Smider, Wenshe Liu, Jacob Pinnas and Michael Jahnz for invaluable discussions and advice. C.C.L. gratefully acknowledges the Fannie and John Hertz Foundation and the National Science Foundation for predoctoral fellowships. This research was generously supported by the USNational Institutes of Health (GM62159).

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Authors and Affiliations

  1. Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Chang C Liu & Peter G Schultz

  2. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121, California, USA

    Peter G Schultz

Authors
  1. Chang C Liu
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  2. Peter G Schultz
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Correspondence to Chang C Liu or Peter G Schultz.

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Supplementary information

Supplementary Fig. 1

Sulfotyrosine dependent expression of Z-domain. (PDF 1726 kb)

Supplementary Fig. 2

Mass spectra characterization of sulfo-hirudin. (PDF 301 kb)

Supplementary Fig. 3

Sulfotyrosine dependent expression of sulfo-hirudin. (PDF 403 kb)

Supplementary Table 1

Thrombin inhibition by hirudin kinetic constants. (PDF 50 kb)

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Liu, C., Schultz, P. Recombinant expression of selectively sulfated proteins in Escherichia coli. Nat Biotechnol 24, 1436–1440 (2006). https://doi.org/10.1038/nbt1254

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