Construction And Expression Of Ricin A Chain And Enhanced Green Fluorescent Protein Fusion Protein To Investigate The Intracellular Pathway Taken By The Ricin A Chain

Ricin is a heterodimeric ribosome inactivating protein (RIP) composed of A and B chains joined with a disulfide bond. Ricin B-chain ( RTB ) is a galactose-specific lectin, which binds to the receptors on cell surfaces and may enhance ricin A-chain ( RTA) translocation by forming a pore in the membrane of intracellular organelles. RTA is a RNA specific N-glycosidase, which excises a specific adenine residue ( A4324 in rat) from a highly conserved loop of 26S or 28S rRNA in 60S ribosomal subunits, and causes protein synthesis inhibition and cell death. RTA can be used to conjugate with monoclonal antibodies to prepare immunotoxins for the therapy of cancer and AIDS. It develops a new way to treat the malignant tumor. However the precise pathway exploited by RTA remains unclear. The intracellular pathway taken by the toxin remains to be investigated.Green fluorescent protein (GFP) and its homolog are widely used as fluorescent markers of gene expression and for determination of protein localization and motility in living cells. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence to be utilized in nornnative organisms, wherein it can be used as areporter. It consists of 238 amino acids and its molecular mass is 27-30kDa. It can allow being fused with other proteins either in the N-terminal or in the C-terminal without loss of fluorescence. For these reasons, GFP become one of the most useful makers used in monitoring the expression of the genes in the cells or tissues and the location of the proteins. Enhanced green fluorescent protein ( EGFP ) has more fluorescence intensity than the wild, and it is more sensitive.Our goal is to construct and express EGFP-RTA fusion protein to investigate the intracellular pathway taken by the RTA. In the study the DNA sequence encoding RTA was inserted into pEGFP-Cl first to make the template sequence of the fusion protein. The fusion gene was amplified from the plasmid pEGFP-RTA by PCR, and directly subcloned into T vector. Eventually, the fusion gene was cloned into expression vector pET-28a ( + ). Expression was induced by EPTG in Kcoli BL21 (DE3 ) . The recombinant protein can be purified by metal chelate affinity chromatography ( MCAC ) on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA were evaluated by the MTT assay in Hela cells and HEP-G2 cells following fluid-phase endocytosis. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy ( LSCM ) and the immuno-gold labeling Electro Microscopy (EM). This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.From our experiments, we have demonstrated: 1 ., The plasmid DNA extracted from a single positive colony was digested withNhe I and BamH I. The agarose electrophoresis showed that the fragments werevisible. Eventually the DNA sequences of EGFP-RTA were confirmed bysequencing the desired region. 2^ Expression of EGFP-RTA was induced with IPTG . The induced bacteriashowed bright green fluorescence indicating the fusion protein was expressed.After sonication and centrifugation of the bacteria, the supematants were run on12 % SDS-PAGE. The result showed that the target proteins with aboutmolecular weight of 60 kDa could be detected in the lysates of the bacteria.3^ A Metal Chelate Affinity Chromatography ( MCAC ) was employed to purify EGFP-RTA. After purification through NTA columns, recombinant protein showed a single band on 12 % SDS-PAGE. The recombinant proteins produced at 25 "C for 3 h were purified from E. coli with yields of approximately 30~40 mg/L.4> The antigenicity of the recombinant protein EGFP-RTA analyzed by Western blotting. The results showed the rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein.5 % The purified recombinant proteins were tested by MTT assay for cytotoxicity on HeLa cells and HEP-G2 cells after fluid-phase endocytosis. The results showed that EGFP-RTA had a similar cytotoxicity of RTA indicating that EGFP tag would not influence the intracellular trafficking and translocation of RTA.6> HEP-G2 cells treated with EGFP and EGFP-RTA fusion protein were observed by the laser scanning confocal microscopy. The result indicated that EGFP-RTA was capable of avoiding being degraded inside the intracellular compartments.7^ HEP-G2 cells treated with EGFP or EGFP-RTA respectively were observed by Electro Microscopy. This result gave direct evidence that EGFP tag would not influence the intracellular trafficking and translocation of RTA and RTA were able to reach the ER of the HEP-G2 cells. This study provides a visualized way to reveal the intracellular trafficking of theRTA. Understanding the intracellular routing of RTA will help in designing moreeffective immunotoxins.