[Construction and expression of ricin A chain and green fluorescent protein fusion gene in E. coli].
To study the expression and purification of a fusion protein of ricin A chain (RTA) and green fluorescent protein (GFP).



The DNA sequence encoding ricin A chain was inserted into pEGFPC1 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. The fusion gene then was cloned into expression vector pET-28a(+), and the sequence was confirmed by sequencing. Expression was induced by IPTG in E. coli BL21(DE3). The fusion protein was purified by metal chelated affinity chromatography. The cytotoxicity of fusion protein was analyzed by the MTT assay in HepG2 and Hela cells.



The fusion protein of ricin A chain and GFP could be produced in E. coli transformed with the expression plasmid of pET-28a(+)-GFP-RTA. The molecular weight of the recombinant protein was measured by SDS-PAGE. The fusion protein showed a green fluorescence and had a similar cytotoxicity of RTA.



A recombinant fusion protein of RTA and GFP expressed in E. coli is possessed of similar biological activity of individual GFP and RTA, which could be used in study of the intracellular trafficking and translocation of RTA.
Chen
Xin-hong
XH
Department of Biochemistry and Molecular Biology, College of Medicine, Zhejiang University, Hangzhou 310006, China.
Liu
Qiong
Q
Zhan
Jin-biao
JB
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012276:Ricin / Q000235:genetics*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012276:Ricin