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Engineering Ecotin for Identifying Proteins with a Trypsin Fold

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Abstract

Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.

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Acknowledgments

We thank the support of Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal Docente (CAPES), and Programa de Pós-Graduação da Universidade Federal Fluminense (UFF) for the financial support and H.C.C. and P. S. fellowships. TT and CSC were supported by NIH grant CA072006. We also thank Dr. Sushma Selvarajan for donating the mammary gland extracts.

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Authors and Affiliations

  1. LaBioMol, Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, 24001-970, Brazil

    Plínio C. Sathler & Helena C. Castro

  2. Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA, 94158, USA

    Charles S. Craik & Toshihiko Takeuchi

  3. LabHemoVen, Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-590, Brazil

    Russolina B. Zingali

Authors
  1. Plínio C. Sathler
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  2. Charles S. Craik
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  3. Toshihiko Takeuchi
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  4. Russolina B. Zingali
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  5. Helena C. Castro
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Correspondence to Helena C. Castro.

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Sathler, P.C., Craik, C.S., Takeuchi, T. et al. Engineering Ecotin for Identifying Proteins with a Trypsin Fold. Appl Biochem Biotechnol 160, 2355–2365 (2010). https://doi.org/10.1007/s12010-009-8711-z

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  • DOI: https://doi.org/10.1007/s12010-009-8711-z

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