Molecular cloning, functional expression in Escherichia coli and enzymatic characterisation of a cysteine protease from white clover (Trifolium repens).
This paper presents the cloning and biochemical characterisation of the cysteine protease Tr-cp 14 from white clover (Trifolium repens). The predicted amino acid sequence of Tr-cp 14 is 71%, 74% and 74% identical to the cysteine proteases XCP1 and XCP2 from Arabidopsis thaliana, and p48h-17 from Zinnia elegans, respectively. These cysteine proteases have previously been shown to be involved in programmed cell death during tracheary element differentiation. The precursor polypeptide of Tr-cp 14 was expressed in Escherichia coli, purified from inclusion bodies and refolded. The precursor polypeptide could be processed to its active mature form autocatalytically at pH 5.0 and had a requirement for 20 mM l-cysteine for optimal activity. Mature Tr-cp 14 showed a preference for synthetic aminomethylcoumarin substrates with either Leu or Phe in the P2 position when tested with Arg in P1. A substrate with Arg in both the P1 and P2 position was not accepted as substrate.
Asp
Torben
T
Danish Institute of Agricultural Sciences, Department of Plant Biology, Research Centre Flakkebjerg, 4200 Slagelse, Denmark.
Bowra
Steve
S
Borg
Søren
S
Holm
Preben Bach
PB
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000166:cytology / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D029921:Trifolium / Q000166:cytology / Q000201:enzymology*
D003374:Coumarins; C030123:coumarin; D003546:Cysteine Endopeptidases