cDNA cloning and overexpression of ribosomal protein S19 gene (RPS19) from the Giant Panda.
RPS19 is a component of the 40S small ribosomal subunit encoded by RPS19 gene. The cDNA of RPS19 was cloned successfully for the first time from the Giant Panda using RT-PCR technology. It was also sequenced, analyzed preliminarily, and expressed in Escherichia coli. The length of cDNA fragment cloned is 469 bp, and it contains an open-reading frame of 438 bp encoding 145 amino acids. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence share a high homology with those of Homo sapiens, Mus musculus, and Rattus norvegicus by 95.89%, 92.47%, and 93.61%, and 100.00%, 99.31%, and 99.31%, respectively. Topology prediction shows that there is one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one tyrosine kinase phosphorylation site, three N-myristoylation sites, one amidation site, and one ribosomal protein S19e signature in the RPS19 protein of the Giant Panda (Ailuropoda melanoleuca). The RPS19 gene was overexpressed in E. coli and expression confirmed by western blotting. The results indicated that the RPS19 gene can be readily expressed in E. coli, and the N-terminally GST-tagged protein was a 42 kDa polypeptide, in good agreement with the predicted molecular weight. The protein obtained could be purified and its function studied.
Hou
Yi-Ling
YL
College of Agriculture, Sichuan Agricultural University, Ya-an, China.
Hou
Wan-Ru
WR
Ren
Zheng-Long
ZL
Hao
Yan-Zhe
YZ
Zhang
Tian
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001503:Ursidae / Q000235:genetics*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D005982:Glutathione Transferase