Expression, purification, and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. Here, a recombinant form of the extracellular domain of the TRAIL (sTRAIL) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded sTRAIL was purified by anion-exchange chromatography. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 19.2 kDa, which equalled the theoretically expected mass. N-terminal sequencing of refolding sTRAIL showed the sequence which corresponded to the designed protein. The renatured protein displayed its immunoreactivity with the antibodies to TRAIL protein by Western blotting. The purified sTRAIL had a strong cytotoxic activity against human cervical cancer HeLa cells with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with beta-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.
Lin
Zhihua
Z
Department of Biology, School of Chemistry and Life Sciences, Lishui University, Lishui, 323000 Zhejiang, PR China. zhihualin1015@126.com
Lei
Huanzong
H
Cao
Peng
P
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human