cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.
SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16.
Wu
Fang
F
Key Laboratory of Structural Biology, School of Life Sciences, University of Science and Technology of China, PR China.
Yan
Ming
M
Li
Yikun
Y
Chang
Shaojie
S
Song
Xiaomin
X
Zhou
Zhaocai
Z
Gong
Weimin
W
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D029905:Pachyrhizus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012639:Seeds / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship
D000817:Anilino Naphthalenesulfonates; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; C053376:pathogenesis-related proteins, plant; C027132:1-anilino-8-naphthalenesulfonate; D012260:Ribonucleases