A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm

LaVallie, Edward R.; Diblasio, Elizabeth; Kovacic, Sharlotte; Grant, Kathleen L.; Schendel, Paul F.; McCoy, John

doi:10.1038/nbt0293-187

Cited by 769 publications

(553 citation statements)

References 52 publications

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“…Because the final product has a molecular mass of 19 kDa, 10-fold enhancement of the binding signal can be expected. The reason we chose thioredoxin as a fusion partner is that thioredoxin is known to be overexpressed in bacteria without formation of inclusion bodies (28). Thioredoxin is also advantageous for biophysical experiments because the protein is stable to heat and highly soluble.…”

Section: Design Production and Characterization Of Thioredoxinfusedmentioning

confidence: 99%

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Ishino

Urbina

Bhattacharya

et al. 2005

Journal of Biological Chemistry

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The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor ␣ (IL5R␣) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N-and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556 -568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5R␣ interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide⅐receptor complex is comparable with that of the cytokine⅐receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5R␣, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its Asthma is an incurable disease characterized by eosinophil bronchial inflammation and tissue remodeling of the airway wall (1). Human interleukin (IL) 1 -5 is a key cytokine that plays a critical role in the differentiation, proliferation, migration, and activation of eosinophils and has been implicated in the pathogenesis of eosinophil-associated allergic inflammation such as asthma (2, 3). Injection of IL5 increases eosinophil numbers in the blood, bone marrow, spleen, and peritoneal cavities (4, 5). This increase can be prevented by the passive administration of anti-IL5 or anti-IL5 receptor antibodies (5, 6). One distinguishing characteristic of IL5 versus other cytokines is that IL5 functions selectively to activate eosinophils (7). These data argue that IL5 plays a central role in the development of allergen-induced eosinophils and suggest that the ability to block IL5 activity evokes the potential for alternative treatment for asthma.At the molecular level, IL5 leads to biological functions through recruitment of a cell-surface receptor composed of two polypeptide chains, ␣ and ␤ (8). The ␣ chain is IL5-specific and is called IL5 receptor ␣ (IL5R␣), whereas the ␤ chain is shared with IL3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) (9 -11) and is called the common ␤ chain (␤c). Despite a high degree of amino acid sequence similarity of the ␣ chains for IL5, IL3, and GM-CSF, their interaction with cognate cytokine is strictly specific, and no cross-reactivity is found among these cytokines. Previous observations argue that receptor subunit recruitment occurs stepwise, with initial formation of the IL5⅐IL5R␣ complex required for ␤c binding to induce cytoplasmic signal transduction (12). IL5R␣ alone binds IL5 with an equilibrium dissociation constant of 0.8 nM when expressed in COS cells, and this binding affinity is increased by only 1.5-fold when ␣ and ␤ chains a...

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Section: Design Production and Characterization Of Thioredoxinfusedmentioning

confidence: 99%

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Ishino

Urbina

Bhattacharya

et al. 2005

Journal of Biological Chemistry

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“…In the strategy described here, the engineered protein is synthesized as a fusion with the B1 immunoglobulin binding domain of streptococcal protein G (GB]; 56 residues) (Gronenborn et al, 1991). As is true for other N-terminal fusion proteins, such as those containing domains from glutathione S-transferase (Smith & Johnson, 1988), maltose binding protein (di Guan et al, 1988), protein A (Nilsson et al, 1985;Jansson et al, 1996), or thioredoxin (LaVallie et al, 1993), the presence of the GB1 domain results in very high expression levels of the fusion protein and limits the need to optimize expression conditions. For most fusion proteinbased expression strategies, the N-terminal domain is rather large and is removed by proteolysis prior to NMR analysis.…”

mentioning

confidence: 99%

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

et al. 1997

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Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB 1 domain to allow direct NMR spectroscopic analysis of the fusion protein by ' H-l5N correlation spectroscopy.Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, "N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using iSN-labeled GBI-peptide fusions and unlabeled target.Keywords: domain structure; GBI-fusion protein; intermolecular interactions; NMR When investigating whether a protein or protein-DNA complex is suitable for NMR structural analysis, several technical goals must be met. First, the size, solubility, and stability of the protein domain must be optimized. Second, the protein should be expressed in a host (usually bacteria), allowing for easy and inexpensive labeling with "C and "N. A third challenge pertains to the preparation of protein-DNA complexes where both the protein and DNA sequences must be optimized to achieve a complex that is most amenable to structural investigation; that is, the system should not exhibit intermediate exchange on the NMR time scale. The practical size limit for structure determination by current solution NMR methodology is approximately 40 kDa, so that, for many proteins, subcloning of a smaller domain that retains functional qualities of the full-length protein is required. All too frequently, however, engineering of protein domains results in limited solubility and/or low stability, often to the point where many engineered proteins are partially or predominately unfolded. In practice, many variations of a protein sequence may be evaluated by NMR

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“…Das (2012) carried out an experiment with Trx·tag by IPTG induced method to increase the solubility of hGM-CSF, another colony stimulating factor which is useful to maintain post transplantation leukocytes level. In addition, Trx has the ability to be spesifically released from the E.coli cytoplasma by freezethaw methods or osmotic shock in the presence of EDTA, which facilitates to isolate the protein from the cells (LaValielli, 1993).…”

Section: Resultsmentioning

confidence: 99%

SOLUBLE EXPRESSION OF SYNTHETIC CSF3syn GENE FUSED WITH THIOREDOXIN IN Escherichia coli BL21(DE3) THROUGH AUTOINDUCTION METHOD AND PURIFICATION

Pratiwi

Fuad

2015

Indonesian J. Pharm.

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A synthetic human gene of CSF3 (CSF3syn.Ec3), coding for hG-CSF was succesfully subcloned into pET32a(+) expression vector and fused with thioredoxin (Trx) at its N-terminal as fusion partner. The obtained fusion gene of Trx-CSF3syn within the recombinant plasmid pET32a(+)_CSF3yn.Ec3 was verified by PCR, plasmid restriction, and DNA sequencing analysis. In order to investigate the fusion gene expression, we transformed Escherichia coli BL21(DE3) as the host with the recombinant plasmid. The gene was succesfully expressed within the cytosol as fusion protein of Trx·tag, His·tag, S·tag, EK-site, and hG-CSF moieties. By the auto induction method, 49% of the protein was found in the soluble fraction and the other 51% was found in the insoluble fraction. The soluble fraction was subsequently purified by IMAC method (Ni-NTA) and characterized.

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A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm

Cited by 769 publications

References 52 publications

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

SOLUBLE EXPRESSION OF SYNTHETIC CSF3syn GENE FUSED WITH THIOREDOXIN IN Escherichia coli BL21(DE3) THROUGH AUTOINDUCTION METHOD AND PURIFICATION

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