Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase.
The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3 (1) with unit-cell dimensions a = b = 72.5 A, c = 141.5 A. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.
Gasparian
Marine E
ME
Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, Moscow, 117997 GSP, Russia. marine_gasparian@yahoo.com
Ostapchenko
Valeriy G
VG
Yagolovich
Anne V
AV
Tsygannik
Igor N
IN
Chernyak
Boris V
BV
Dolgikh
Dmitry A
DA
Kirpichnikov
Mikhail P
MP
D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003461:Crystallography; D004765:Enteropeptidase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D013879:Thioredoxins; D004765:Enteropeptidase