Molecular cloning of a cDNA encoding ribosome inactivating protein from Amaranthus viridis and its expression in E. coli.
In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.
Kwon
S Y
SY
Korea Research Institute of Bioscience and Biotechnology, Taejon.
An
C S
CS
Liu
J R
JR
Kwak
S S
SS
Lee
H S
HS
Kim
J K
JK
Paek
K H
KH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D015723:Gene Library; D037102:Lectins / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics*; D037121:Plant Lectins; D014176:Protein Biosynthesis; D054788:Ribosome Inactivating Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D037102:Lectins; D037121:Plant Lectins; D054789:Ribosome Inactivating Proteins, Type 1; C061152:amaranthin protein, Amaranthus; D054788:Ribosome Inactivating Proteins