Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli.
A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).
Begam
Mehbuba
M
Division of Biochemistry, Indian Agricultural Research Institute, New Delhi 110012, India.
Kumar
Sushil
S
Roy
Sribash
S
Campanella
James J
JJ
Kapoor
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D034762:Celosia / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D035264:Flowers; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics / Q000821:virology*; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014027:Tobacco Mosaic Virus / Q000502:physiology*
D018076:DNA, Complementary; D010940:Plant Proteins; D006026:Glycoside Hydrolases