Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca).
Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.
College of Life Science, China West Normal University, Nanchong 637009, China.
Xiu-Lan
XL
Su
Yi-Ling
YL
Hou
Xiang-Hui
XH
Yan
Xiang
X
Ding
Wan-Ru
WR
Hou
Bing
B
Sun
Si-Nan
SN
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056945:Hep G2 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000494:pharmacology* / Q000502:physiology*; D017385:Sequence Homology; D001503:Ursidae
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C050985:ribosomal protein L31