Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.
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 pPICZalpha 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.
Trejo
Sebastián A
SA
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.
López
Laura M I
LM
Caffini
Néstor O
NO
Natalucci
Claudia L
CL
Canals
Francesc
F
Avilés
Francesc X
FX
D000595:Amino Acid Sequence; D031108:Asclepias / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D010940:Plant Proteins; D003546:Cysteine Endopeptidases; C018647:asclepain