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Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex

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Abstract

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

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Abbreviations

ACN:

Acetonitrile

AfCP:

Amino acid sequence corresponding to the cysteine protease asclepain f deduced from cDNA

DMSO:

Dimethyl sulfoxide

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

PFLNA:

pGlu-Phe-Leu-p-nitroanilide

PMF:

Peptide mass fingerprinting

RACE:

Rapid amplification of cDNA ends

rAfCP:

Recombinant asclepain f cysteine protease

RMSD:

Root mean square deviation

TFA:

Trifluoroacetic acid

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Acknowledgments

The present work was supported by grants from ANPCyT, CONICET, and CIC (Argentina), CYTED (Project IV.22) and BIO2007-68046-C02 (MEC, Spain). S.A. Trejo was CONICET Fellow, L.M.I. López belongs to CONICET Researcher Career, and N.O. Caffini and C.L. Natalucci belong to CIC Researcher Career. The IBB-UAB is a member of ProteoRed, funded by Genoma Spain and follows the quality criteria set up by ProteoRed standards.

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Authors and Affiliations

  1. Laboratorio de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, B1900AVW, La Plata, Argentina

    Sebastián A. Trejo, Laura M. I. López, Néstor O. Caffini & Claudia L. Natalucci

  2. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, 08193, Barcelona, Spain

    Sebastián A. Trejo, Francesc Canals & Francesc X. Avilés

  3. Medical Oncology Research Program, Proteomics Laboratory, Institut de Recerca Hospital Universitari Vall d’Hebron, Psg. Vall d’Hebron, 119-129, 08035, Barcelona, Spain

    Francesc Canals

Authors
  1. Sebastián A. Trejo
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  2. Laura M. I. López
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  3. Néstor O. Caffini
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  4. Claudia L. Natalucci
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  5. Francesc Canals
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  6. Francesc X. Avilés
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Correspondence to Claudia L. Natalucci.

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Trejo, S.A., López, L.M.I., Caffini, N.O. et al. Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex. Planta 230, 319–328 (2009). https://doi.org/10.1007/s00425-009-0942-2

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