Planta

, Volume 230, Issue 2, pp 319–328

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

  • Sebastián A. Trejo
  • Laura M. I. López
  • Néstor O. Caffini
  • Claudia L. Natalucci
  • Francesc Canals
  • Francesc X. Avilés
Original Article

DOI: 10.1007/s00425-009-0942-2

Cite this article as:
Trejo, S.A., López, L.M.I., Caffini, N.O. et al. Planta (2009) 230: 319. doi:10.1007/s00425-009-0942-2

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.

Keywords

Asclepias fruticosa Gomphocarpus fruticosus subsp. fruticosus Plant latex Cysteine endopeptidase Cloning Overexpression in Pichia pastoris 

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

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Sebastián A. Trejo
    • 1
    • 2
  • Laura M. I. López
    • 1
  • Néstor O. Caffini
    • 1
  • Claudia L. Natalucci
    • 1
  • Francesc Canals
    • 2
    • 3
  • Francesc X. Avilés
    • 2
  1. 1.Laboratorio de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias ExactasUniversidad Nacional de La PlataLa PlataArgentina
  2. 2.Institut de Biotecnologia i de BiomedicinaUniversitat Autònoma de BarcelonaBarcelonaSpain
  3. 3.Medical Oncology Research Program, Proteomics LaboratoryInstitut de Recerca Hospital Universitari Vall d’HebronBarcelonaSpain

Personalised recommendations