Improving Soluble Expression Of Curcin-transferrin Receptor Binding Peptide Fusion Protein

Objective:To achieve the soluble expression of recombinant curcin-TfRBP9,three recombinant proteins,ELP-Intein-curcin-TfRBP9,SUMO-curcin-TfRBP9 and GST-SUMO-curcin-TfRBP9,were constructed by using genetic engineering technology and their ability to improve soluble expression of recombinant curcin-TfRBP9 was also determined.To explore the effectiveness of curcin-TfRBP9 on tumor cells,we evaluated its anti-tumor activity in vitro.This study provides a better purification process and an experiments basis for the application and research of curcin at cancer therapy.Methods:1.the DNA fragments coded the curcin-TfRBP9 was cloned into the elastin-like expression system to construct the recombinant exprerssion plasmid pET-ELP-Intein-curcin-TfRBP9,and then transformed into the expression host strain E.coli BLR(DE3).Colony PCR was used to screen the positive recombinant plasmid and the correctness of gene sequence ofrecombinant plasmid was determined by gene sequencing.The recombinant protein was expressed with leaky expression at 18? in Terrific Broth for 24 h.the ITC was used to purify the curcin-TfRBP9,12%SDS-PAGE analysed the soluble expression of curcin-TfRBP9.2.The expression vector pQE-30-sumo-curcin-TfRBP and pQE-30-GST-sumo-curcin-TfRBP were constructed.After inducing expression at low temprature,soluble expression of recombinant proteins was analysed by 12%SDS-PAGE.After purification through Glutathione Sepharose 4B,ubiquitin-like specific protease1(Ulp1)digestion and CM SepharoseTM Fast Flow,the MTT was used to detect untagged curcin-TfRBP9 and curcin cytotoxicity of tumor cells.Results:A recombinant plasmid,pET-ELP-Intein-curicn-TfRBP9,was constructed and transformed into E.coli BLR(DE3).After leaky expression and purification by ITC method,analysis of 12%SDS-PAGE and identification of mass spectrometry technology showed that the soluble expression of curcin-TfRBP9 in E.coli BLR(DE3)was not detected.The additional two recombinant plasmids,pQE-30-sumo-curcin-TfRBP and pQE-30-GST-sumo-curcin-TfRBP,were constructed and transformed into the expression of strain E.coli M15.The recombinant proteins were expressed by inducing by 0.5mM of isopropyl-?-D-thiogalactoside(IPTG)at low temprature,analysis of 12%SDS-PAGE showed that fusion protein,sumo-curcin-TfRBP9 expressed as a inclusion body,the expressed quantity of the GST-SUMO-curcin-TfRBP9 fusion protein was about 40% of the total protein,and the soluble expression of the recombinant protein was about 55% of the total GST-SUMO-curcin-TfRBP9 fusion protein.Purification through Glutathione Sepharose 4B,ubiquitin-like specific protease1(Ulp1)digestion and CM SepharoseTM Fast Flow yielded the untagged curcin-TfRBP9 fusion protein with a purity of more than 95%.With an increase in the concentration of the untagged curcin-TfRBP9,the inhibition rate of A549 cells gradually increased.The untagged curcin-TfRBP9 exhibited a higher cytotoxicity than did the control,the untagged curcin.The cytotoxicity of the LO-2 cells was much lower in comparison to the A549 cells Conclusion:GST and SUMO tags facilitated the soluble expression of the recombinant GST-SUMO-curcin-TfRBP9 protein.After using Glutathioneaffinity chromatography,the digestion of Ulp1 and ion-exchange chromatography,the untagged curcin-TfRBP9 was harvested.The cell inhibition assay showed that the untagged curcin-TfRBP9 had higher inhibition effects on the A549 cells over-expressing TfR versus normal cells.