Molecular cloning and biochemical characterization of a novel cystatin from Hevea rubber latex.
A novel cDNA encoding a cysteine proteinase inhibitor or phytocystatin was isolated from Hevea brasiliensis RRIM600 rubber latex cDNA library. The full-length HbCPI obtained from rapid amplification of cDNA ends contains 588 bp. An open reading frame of 306 bp encodes for a protein of 101 amino acids with the typical inhibitory motifs of phytocystatin superfamily, namely the central signature motif QXVXG, a GG doublet and LARFAV-like motifs in the N-terminal part, and conserved A/PW residues in the C-terminal region. Sequence comparison showed that the deduced amino acid sequence was similar to that of cysteine protease inhibitor from Manihot esculenta (84% identity). The HbCPI was subcloned into expression vector pQE-40 and then overexpressed in Escherichia coli M15 strain (pREP4) as a His-tagged recombinant protein with molecular mass approximately 13 kDa. The purified HbCPI showed thermal stable property and efficiently inhibited the protease activity of papain by non-competitive inhibition with K(i) value of 15.4 nM. Beside latex, HbCPI also transcripted in leaf and young seed. The HbCPI message accumulation was induced by phytopathogenic fungi Phytophthora palmivora infection. These data suggest that HbCPI might play crucial roles in defense mechanism against biotic stimuli.
School of Science, Walailak University, 222 Thasala, Nakhon Si Thammarat, Thailand. bphuwado@wu.ac.th
Phuwadol
P
Bangrak
Wilaiwan
W
Chotigeat
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D017343:Genes, Plant; D005822:Genetic Vectors; D028482:Hevea / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007840:Latex / Q000378:metabolism; D002365:Manihot / Q000235:genetics; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors*; D010838:Phytophthora; D010935:Plant Diseases / Q000382:microbiology; D057865:Plant Immunity / Q000235:genetics / Q000502:physiology*; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000378:metabolism
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D007840:Latex; D010940:Plant Proteins; D011994:Recombinant Proteins; D010206:Papain