Overexpression of human transforming growth factor-β1 using a recombinant CHO cell expression system

doi:10.1016/j.pep.2003.06.001

Protein Expression and Purification

Volume 37, Issue 2, October 2004, Pages 265-272

https://doi.org/10.1016/j.pep.2003.06.001 Get rights and content

Abstract

Transforming growth factor-β1 (TGF-β1) is secreted by most cells as a high molecular weight latent complex, which consists of latent TGF-β1 disulfide bonded to latent TGF-β1-binding protein (LTBP). Current recombinant expression systems yield less than 1–2 mg of the mature TGF-β1 per liter of cell culture medium. In an effort to produce large quantities of the recombinant cytokine for structural studies, we have constructed a mammalian expression system based on a modified pcDNA3.1(+) vector with a glutamine synthetase gene inserted for gene amplification. The leader peptide of TGF-β1 was replaced with that of rat serum albumin, and an eight-histidine tag was inserted immediately after the leader sequence to facilitate protein purification. In addition, Cys 33 of TGF-β1, which forms a disulfide bond with LTBP, was replaced by a serine residue. The resulting expression construct produced a stable clone expressing 30 mg of mature TGF-β1 per liter of spent medium. Purified TGF-β1 bound with high affinity to its type II receptor with a solution dissociation constant of ∼70 nM, and was fully active in both a Mv1Lu cell growth inhibition assay and in a PAI-1 luciferase reporter assay. Owing to similarities in the synthesis, secretion, and structure of TGF-β family members, this recombinant expression system may also be applied to the overexpression of other TGF-β isomers and even to members of the TGF-β superfamily to facilitate their preparation.

Section snippets

Enzymes, media, and chemicals

XmaI and Bsp 119I were from Fermentas (Hanover, MD). BamHI, XhoI, and T4 DNA ligase were from New England Biolabs (Beverly, MA). Pfu DNA polymerase was from Stratagene (La Jolla, CA). DMEM/F12 medium, fetal bovine serum (FBS), CHO-S-SFM II serum-free medium, OPTI-MEM I medium, and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Glutamine-free GMEM-S medium and GS supplement were from JRH Biosciences (Lenexa, KS). Ni–NTA agarose was from Qiagen (Valencia, CA). Chemicals were from

Construction of a TGF-β1 expression plasmid

The expression process of TGF-β1 is distinct from those of other proteins, as it is synthesized and secreted as a homodimeric latent form. Mature TGF-β1 can be released from the noncovalently associated LAP through an activation process by either extreme pH or other methods [20]. In addition to TGF-β1, most cells also secrete LTBP disulfide bonded with LAP through Cys 33. The absence of LTBP in the recombinant CHO TGF-β1 expression system, however, may complicate the release of mature TGF-β1

Discussion

TGF-β was first purified from human platelets, with extremely low yields [24]. Subsequently, several groups have successfully expressed recombinant TGF-β1 in CHO cells using wild-type TGF-β1 cDNA [12], [15]. The expression levels of these recombinant constructs varied from less than one to several milligram/liter, even with use of roller bottles or bioreactors. In addition, due to the lack of a cost-effective affinity purification method, previous recombinant systems necessitated lengthy,

Acknowledgements

We thank C. Hammer for mass spectrometry measurements, M. Garfield for N-terminal peptide sequencing, S.-J. Kim for assistance with the TGF-β1 biological activity assays, Z. Lu for assistance with surface plasmon resonance measurements, K. Johnson for her involvement in early experiments, and C. Foster for help with the manuscript.

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Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

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Protein Expression and Purification

Abstract

Section snippets

Enzymes, media, and chemicals

Construction of a TGF-β1 expression plasmid

Discussion

Acknowledgements

References (25)

Genealogy, expression, and cellular function of transforming growth factor-beta

Pharmacol. Ther

Mechanisms of TGF-beta signaling from cell membrane to the nucleus

Cell

Processing of transforming growth factor beta 1 precursor by human furin convertase

J. Biol. Chem

TGF-beta: from latent to active

Microbes Infect

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins

J. Biol. Chem

Development of a recombinant bacterial expression system for the active form of a human transforming growth factor beta type II receptor ligand binding domain

Protein Expr. Purif

Recombinant human transforming growth factor-beta 1: expression by Chinese hamster ovary cells, isolation, and characterization

Protein Expr. Purif

An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct

Anal. Biochem

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

FEBS Lett

Efficient expression and secretion of recombinant hirudin III in E. coli using the l-asparaginase II signal sequence

Protein Expr. Purif

Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

J. Biol. Chem

New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues

Proc. Natl. Acad. Sci. USA

Cited by (36)

Protein expression and purification of bioactive growth factors for use in cell culture and cellular agriculture

Recombinant production of growth factors for application in cell culture

Current status, and the developments of hosts and expression systems for the production of recombinant human cytokines

Structures of activin ligand traps using natural sets of type I and type II TGFβ receptors

High level stable expression of recombinant HIV gp120 in glutamine synthetase gene deficient HEK293T cells

Cryo-EM Reveals Integrin-Mediated TGF-β Activation without Release from Latent TGF-β