Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli

doi:10.1016/j.pep.2004.10.012

Protein Expression and Purification

Volume 39, Issue 2, February 2005, Pages 189-198

https://doi.org/10.1016/j.pep.2004.10.012 Get rights and content

Abstract

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-β-d-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14–22 and 21–28 mg highly purified and biologically active protein was obtained from 1 L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

Section snippets

Cloning of chimeric constructs for IL-13PE38QQR and IL-13PE38 in pET24a expression vector

Mature human interleukin-13 was cloned from total RNA isolated from human peripheral blood mononuclear cells (PBMCs), which were stimulated with 10 μg/ml of lipopolysaccharide for 24 h. Total RNA was extracted from the PBMCs using the RNeasy RNA extraction kit (Qiagen, Valencia, CA) and reversed transcribed with Moloney murine leukemia virus reverse transcriptase. The primers amplify the amino acids serine (19) to aspargine (133) that include matured form of human IL-13 (pMPL13) with NdeI

Construction, expression, and purification of the recombinant cytotoxins

The recombinant plasmids IL-13PE38QQR and IL-13PE38 were constructed in a pET24a prokaryotic expression system (Fig. 1) and transformed into E. coli BL21(DE3) or BL21(DE3)pLysS. The pLysS plasmid, which expresses T7 lysozyme in the bacterial cytoplasm, strongly represses protein expression from the pET vector in the absence of induction, thus, enabling expression of toxic proteins. We found that colonies could easily be produced at 37 °C. However, growth of the cells containing this plasmid was

Acknowledgments

We thank Dr. S. Rafat Husain for his help in FPLC purification of the proteins. We also thank Dr. Raj K. Singh of University of Alabama, Bermingham, Alabama for critical discussions and helpful suggestions, and Ms. Pamela Dover and Niti Behari for their help in experiments. Our thanks are due to Drs. Andrew Byrnes and Nancy Markovitz of the Division of Cellular and Gene Therapies for critical reading of the manuscript and providing suggestions. We also thank the members of Laboratory of

References (40)

S. Bagga et al.
Cytotoxicity of ribosome-inactivating protein saporin is not mediated through alpha2-macroglobulin receptor
FEBS Lett.
(2003)
S. Bagga et al.
The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities
J. Biol. Chem.
(2003)
D. Rathore et al.
Overproduction of fungal ribotoxin alpha-sarcin in Escherichia coli: generation of an active immunotoxin
Gene
(1997)
D. Rathore et al.
Generation of active immunotoxins containing recombinant restrictocin
Biochem. Biophys. Res. Commun.
(1996)
J.K. Batra et al.
TGF alpha-anti-Tac(Fv)-PE40: a bifunctional toxin cytotoxic for cells with EGF or IL2 receptors
Biochem. Biophys. Res. Commun.
(1990)
J.K. Batra et al.
Anti-Tac (Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interleukin 2 receptor bearing cells
J. Biol. Chem.
(1990)
S. Liu et al.
Potent antitumor activity of a urokinase-activated engineered anthrax toxin
Proc. Natl. Acad. Sci. USA
(2003)
T. Murata et al.
Structure of IL-13 receptor: analysis of subunit composition in cancer and immune cells
Biochem. Biophys. Res. Commun.
(1997)
S.R. Husain et al.
Interleukin-13 fusion cytotoxin as a potent targeted agent for AIDS-Kaposi’s sarcoma xenograft
Blood
(2000)
W. Debinski et al.
A novel chimeric protein composed of interleukin 13 and Pseudomonas exotoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4
J. Biol. Chem.
(1995)

J.R. Sinacola et al.

Rapid refolding and polishing of single-chain antibodies from Escherichia coli inclusion bodies

Protein Expr. Purif.

(2002)

B. Matthey et al.

A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins

Gene

(1999)

M.J. Aman et al.

cDNA cloning and characterization of the human interleukin 13 receptor alpha chain

J. Biol. Chem.

(1996)

H. Lilie et al.

Advances in refolding of proteins produced in E. coli

Curr. Opin. Biotechnol.

(1998)

J. Buchner et al.

A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies

Anal. Biochem.

(1992)

P.H. Tan et al.

Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency

Biophys. J.

(1998)

K. Tsumoto et al.

Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent—application to a human single-chain Fv fragment

J. Immunol. Methods

(1998)

A. Goyal et al.

Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins

Biochem. J.

(2000)

R.J. Kreitman et al.

Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: relative importance of the two toxin disulfide bonds

Bioconjug. Chem.

(1993)

J.K. Batra et al.

Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin

Proc. Natl. Acad. Sci. USA

(1992)

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Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli

Abstract

Section snippets

Cloning of chimeric constructs for IL-13PE38QQR and IL-13PE38 in pET24a expression vector

Construction, expression, and purification of the recombinant cytotoxins

Acknowledgments

FEBS Lett.

J. Biol. Chem.

Gene

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Proc. Natl. Acad. Sci. USA

Biochem. Biophys. Res. Commun.

Blood

J. Biol. Chem.

Protein Expr. Purif.

Gene

J. Biol. Chem.

Curr. Opin. Biotechnol.

Anal. Biochem.

Biophys. J.

J. Immunol. Methods

Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins

Biochem. J.

Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: relative importance of the two toxin disulfide bonds

Bioconjug. Chem.

Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin

Proc. Natl. Acad. Sci. USA