Biosynthesis Of A Plant Toxin Gelonin With Tumor Acidic Micronenvironment Targeting Ability

Gelonin is a plant toxin isolated from the seeds of Euphorbiaceae plant Gelonium multiflorum.It belongs to type I ribosome-inactivating protein(RIP)that inhibits irreversible inactivation of ribosomes by the cleavage of of eukaryotic 28 S ribosomal RNA adenine at the 4324 site,thereby inhibiting protein synthesis.Gelonin lacks the cell membrane binding domain,which makes it relatively non-toxic to the intact cells.A small amount of Gelonin can enter into cells by endocytosis,inhibit protein synthesis and promote cell apoptosis.However,the cellular uptake of Gelonin is very low and lacks of tissue specificity.Therefore,the cellular internalization efficiency and tissue specificity of Gelonin are of immense importance for improving its antitumor activity.Tumor microenvironment is more acidic than normal tissues due to the Warberg effect of tumor cells.p H low insertion peptide(p HLIP)can form transmembrane alpha helix under acidic conditions and insert its C-terminal into cell membrane without destroying the permeability of cell membrane.In this study,we successfully prokaryotically expressed the recombinant Gelonin(r Gel)and Trx-p HLIP-Gelonin(Tp G),which is composed of thioredoxin(Trx)tag,p HLIP and Gelonin.This paper mainly includes the following three sections.(1)Recombinant plasmids p ET28a-p HLIP-Gelonin,p ET28a-Gelonin,p GEX-4T-1-p HLIP-Gelonin,p ET32a-p HLIP-Gelonin were constructed by traditional double digestion,whole plasmid PCR and seamless cloning.These recombinant plasmids were successfully constructed as verified by colony PCR and gene sequencing.(2)The constructed recombinant plasmid was transformed into the expression host E.coli BL21(DE3)or E.coli BL21.After exploring the expression conditions,we finally employed p ET28a-Gelonin and p ET32a-p HLIP-Gelonin for the expression of r Gel and Tp G,respectively.The recombinant proteins r Gel and Tp G tag with His tag were purified by nickel affinity chromatography.The purity of r Gel and Tp G was 95% and 90%,respectively,as measured by densitometry analysis of the SDS-PAGE gels.MALDI-TOF-MS assay indicated that the molecular weights of r Gel and Tp G are 20613 Da and 50923 Da,respectively,and sequence coverage rates were 48% and 37%,respectively,indicating these two proteins were expressed correctly..The secondary structure and thermal stability of r Gel were determined by circular dichroism(CD).The main secondary structure of r Gel was β-sheet and random coil,and it has a good thermal stability in the range of 20-60 °C.(3)Finally,the MTT assay was performed to determine the cytotoxicity of r Gel against two colon cancer cells HCT116 and HCT-8,r Gel significantly inhibited the cell growth and proliferation in a dose-and time-dependent manner.After 72 h of treatment with 10 μM r Gel,the cell inhibitory rates of HCT116 and HCT-8 were 52.50% and 44.31%,respectively.The N-glycosidase activity of r Gel was examined by measuring the cellular protein level.Consistent with the MTT results,r Gel inhibited cellular protein synthesis in a dose-and time-dependent pattern.After 72 h of treatment with 10 μM r Gel,the relative cellular protein levels in HCT116 and HCT-8 cells were 14.56% and 35.62% of control cells,respectively.In summary,we have successfully constructed the expression vectors p ET28a-Gelonin and p ET32a-p HLIP-Gelonin,and successfully expressed the recombinant proteins r Gel and Tp G.In vitro cell experiments showed that r Gel can enter into cells by endocytosis,inhibit the synthesis of intracellular proteins and induce tumor cell apoptosis.