Cloning and soluble expression of mature alpha-luffin from Luffa cylindrica and its antitumor activities in vitro.
Luffin-a, a single-chain Type I ribosome-inactivating protein, which is known to be the most toxic of the luffin family and apparently possesses antitumor activity, was isolated from Luffa cylindrica seeds. In the present study, mature alpha-luffin was cloned from L. cylindrica and it was found that mature alpha-luffin shared 96% amino acid similarity with luffin-a. The recombinant mature alpha-luffin was successfully expressed in a partly soluble form in Escherichia coli after optimization of expression conditions. The effects of the recombinant protein on bacterial growth and its in vitro protein synthesis inhibition activity were tested. Then, its antitumor activities against different human cancer cell lines were evaluated by CCK-8 assay and flow cytometry. The results indicated that the recombinant alpha-luffin was slightly toxic to E. coli. It could inhibit protein synthesis in the rabbit reticulocyte lysate system. At the same time, it inhibited the growth of the tumor cell lines in a dose- and time-dependent manner. Additionally, recombinant alpha-luffin was able to induce cell death by apoptosis. The cytotoxicity of alpha-luffin towards tumor cells makes it a potential antitumor agent.
Liu
Liling
L
Department of Dermatology, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
Wang
Rupeng
R
He
Wei
W
He
Fengtian
F
Huang
Gang
G
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D056945:Hep G2 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D029776:Luffa / Q000235:genetics* / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012995:Solubility
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C555599:alpha-luffin protein, Luffa cylindrica