title: L-cysteine-enhanced renaturation of bioactive soluble tumor necrosis factor ligand family member LIGHT from inclusion bodies in Escherichia coli.
abstract: LIGHT is a membrane-bound protein that belongs to the tumor necrosis factor (TNF) superfamily ligands. In this study, we established an effective strategy for producing a bioactive soluble form of LIGHT (sLIGHT), an extracellular region (Ile⁸⁴-Val²⁴⁰) of human LIGHT. Because sLIGHT was expressed as inclusion bodies in Escherichia coli, we investigated reagents that enhance the renaturation of sLIGHT from the inclusion bodies. Interestingly, L-cysteine in the denaturation buffer containing 3.5 M guanidine hydrochloride significantly improved the renaturation efficiency of sLIGHT. The effect of L-cysteine was synergistically enhanced by L-arginine in the refolding buffer. The optimal concentrations of L-cysteine and L-arginine in the denaturation and refolding buffers were 8 mM and 0.8 M, respectively. With these buffers, approximately 90 mg of sLIGHT was purified from 200 g of frozen E. coli cells. sLIGHT thus obtained significantly induced apoptosis in the WiDr human colon adenocarcinoma cell line at nanomolar concentrations, the same amount of sLIGHT that was produced by Sf9 insect cells. These results suggest that L-cysteine in the denaturation buffer enhances the renaturation of recombinant proteins from inclusion bodies in E. coli.
authors: Biology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan.
authors: Isamu
authors: I
authors: Tsuji
authors: Hideki
authors: H
authors: Mastui
authors: Tatsuo
authors: T
authors: Ito
authors: Tomofumi
authors: T
authors: Kurokawa
authors: Yasushi
authors: Y
authors: Shintani
mesh_terms: D000409:Alanine / Q000494:pharmacology; D017209:Apoptosis; D001681:Biological Assay; D002021:Buffers; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002021:Buffers; D011994:Recombinant Proteins; C505914:TNFSF14 protein, human; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D019791:Guanidine; D003545:Cysteine; D000409:Alanine