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Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum

Paper ID Volume ID Publish Year Pages File Format Full-Text
3648 180 2012 6 PDF Available
Title
Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum
Abstract

Human Fas ligand (hFasL) is a type II membrane protein that induces apoptosis in the Fas-bearing cells. Its special biological activity has the potential for the therapeutic use as an anti-cancer agent directed at enhancing apoptosis in tumor cells. In this study Escherichia coli and eukaryotic Dictyostelium discoideum were used to produce a soluble form of hFasL in large amounts. An expression vector for hFasL production in E. coli was constructed based on plasmid pET32a(+). By cultivation of the hFasL-producing E. coli clone on LB medium and induction with IPTG, a hFasL concentration of 1.0 mg L−1 was achieved. D. discoideum strain AX3-hFasL-H was cultured in a conventional stirred bioreactor on an improved synthetic medium using a simple fed-batch strategy, and cell densities of up to 8.3 × 107 cells/mL and a maximum hFasL concentration of 420 μg/L were obtained. Using Ni-NTA affinity chromatography purification, two kinds of recombinant hFasLs from E. coli and D. discoideum were purified with a purity of 94% and 90%, respectively. They showed similar biological activities in inducing apoptosis in Fas-expressing cells.

► We expressed soluble form of human Fas ligand (shFasL) in Escherichia coli and Dictyostelium discoideum, respectively. ► We developed a newly completely synthetic medium for high cell density cultivation of D. discoideum. ► We used Ni-NTA affinity chromatography to purify the recombinant shFasLs expressed by the two different expression systems. ► The two kinds of recombinant shFasLs showed similar biological activities in inducing apoptosis in Fas-expressing cells.

Keywords
Human Fas ligand; Escherichia coli; Dictyostelium discoideum; Soluble production; Affinity chromatography; Cytotoxic activity
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Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 62, 15 March 2012, Pages 86–91
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us