Target cell-restricted apoptosis induction by 528scFv-TRAIL fusion protein specific for human EGFR and expressed in Escherichia coli.
We report the preparation and functional characterization of an Escherichia coli-expressed recombinant fusion protein, 528scFv-TRAIL, specific for the human epidermal growth factor receptor (EGFR) and empowered by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 528scFv-TRAIL, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified stepwise dialysis method. Treatment with 528scFv-TRAIL resulted in the specific binding to the cell surface of EGFR-positive cells with concomitant deployment of the TRAIL moiety to DR-5 receptor in a manner comparable to a commercially available form of recombinant TRAIL (cTRAIL). 528scFv-TRAIL, prepared by either of three refolding processes described herein, showed potent cytotoxic activity against EGFR-positive TFK-1 cell line and was superior to its parental 528scFv; a recombinant variable fragment with single specificity against human EGFR. Narrow variations in the cytotoxic potential of 528scFv-TRAIL were ascribed to manipulation of redox conditions during the refolding process. Together, our findings point to the potential value of 528scFv-TRAIL for treatment of EGFR-expressing cancers. Furthermore, preparation of 528scFv-TRAIL from insoluble aggregates in a prokaryotic cell based expression system by means of in vitro refolding introduces a feasible cost-benefit, time-efficient approach for industrial-scale production.
Badran
Adel
A
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan.
Asano
Ryutaro
R
Nakayama
Makoto
M
Watanabe
Yasuhiro
Y
Nakanishi
Takeshi
T
Umetsu
Mitsuo
M
Hayashi
Hiroki
H
Katayose
Yu
Y
Unno
Michiaki
M
Kumagai
Izumi
I
D000906:Antibodies / Q000737:chemistry; D017209:Apoptosis; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D013997:Time Factors
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D066246:ErbB Receptors