Expression, renaturation and purification of recombinant human interleukin 4 from <i>Escherichia coli</i>

Kimmenade, Anita van; Bond, Michael D.; Schumacher, J H; Laquoi, Carole; Kastelein, Rob

doi:10.1111/j.1432-1033.1988.tb13973.x

Cited by 62 publications

(28 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…HCI than with urea (60 mg versus 18 mg mutant IL-6/1 fermentation solution), in analogy with previous reports also showing that Gdn . HCI is not exchangeable for urea in the preparation from inclusion bodies of IL-6 produced as a fused protein to growth hormone (Asagoe et al, 1988) and human IL-4 (Van Kimmenade et al, 1988). Moreover, the mutant protein obtained from the Gdn HC1-solubilized pellet showed in vitru higher specific activity (EC,, = 8.0 & 1.0 pg/ml; see Materials and Methods) than the protein obtained from urea-solubilized pellet (EC,, = 46.0 2 5.0 pg/ml).…”

Section: Resultsmentioning

confidence: 99%

Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond

Breton

Fiura

Bertolero

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

A mutant species of the 185-residue chain of human interleukin-6 lacking 22-residues at its N-terminus and with a Cys+Ser substitution at positions 45 and 51 was produced in Escherichiu coli. The 163-residue protein des-(A1 -S22)-[C45S, CSISlinterleukin-6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin-6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse-phase HPLC and its structural identity was checked by N-terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin-6 gave a molecular mass of 18 695 ? 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second-derivative ultraviolet absorption spectra indicated that mutant interleukind maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild-type human interleukin-6. The urea-induced unfolding of mutant interleukin-6, monitored by circular dichroic measurements in the far-ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin-6 mediated by urea were used to calculate a value of 20.9 2 0.4 kJ . mol-' for the thermodynamic stability of the protein at 25°C in the absence of denaturant. The biological activity of mutant interleukin-6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild-type protein. Overall the results of this study show that mutant interleukin-6 is a biologically active cytokine, which could find practical use as a therapeutic agent.

show abstract

Section: Resultsmentioning

confidence: 99%

Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond

Breton

Fiura

Bertolero

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The recombinant protein had a histidine tag at its amino terminus which facilitated purification over a Ni-NTA column (Qiagen, Valencia, CA). The purified AChR ␣-chain fragment was then refolded using an oxido-shuffling method, as described by others (41,42). Briefly, the protein was treated with DTT to reduce disulfide bonds and then incubated in a renaturation buffer containing oxidized and reduced forms of glutathione, L-arginine, EDTA, and Tris-HCl (42).…”

Section: Immunizationsmentioning

confidence: 99%

Split Tolerance in a Novel Transgenic Model of Autoimmune Myasthenia Gravis

Stacy¹,

Gelb²,

Koop³

et al. 2002

The Journal of Immunology

View full text Add to dashboard Cite

Because it is one of the few autoimmune disorders in which the target autoantigen has been definitively identified, myasthenia gravis (MG) provides a unique opportunity for testing basic concepts of immune tolerance. In most MG patients, Abs against the acetylcholine receptors (AChR) at the neuromuscular junction can be readily identified and have been directly shown to cause muscle weakness. T cells have also been implicated and appear to play a role in regulating the pathogenic B cells. A murine MG model, generated by immunizing mice with heterologous AChR from the electric fish Torpedo californica, has been used extensively. In these animals, Abs cross-react with murine AChR; however, the T cells do not. Thus, to study tolerance to AChR, a transgenic mouse model was generated in which the immunodominant Torpedo AChR (T-AChR) α subunit is expressed in appropriate tissues. Upon immunization, these mice showed greatly reduced T cell responses to T-AChR and the immunodominant α-chain peptide. Limiting dilution assays suggest the likely mechanism of tolerance is deletion or anergy. Despite this tolerance, immunization with intact T-AChR induced anti-AChR Abs, including Abs against the α subunit, and the incidence of MG-like symptoms was similar to that of wild-type animals. Furthermore, evidence suggests that this B cell response to the α-chain receives help from T cells directed against the other AChR polypeptides (β, γ, or δ). This model offers a novel opportunity to elucidate mechanisms of tolerance regulation to muscle AChR and to clarify the role of T cells in MG.

show abstract

“…Of the cell suspension, 800 yl was aliquoted into the wells of a 48-well plate. IL-4 (50-100 ng/ml; prepared according to [28]) and anti-CD40 (Serotec MCA679) (1-10 yg/ml) or TCM alone in a total volume of 100 yl was added in quadruplicate to the appropriate wells [29]. Test compound was dissolved in neat dimethylsulfoxide at 10 mM, then diluted sequentially in TCM to lox the desired final concentration (total assay volume 1 ml).…”

Section: B Cell Ige Synthesis Assaymentioning

confidence: 99%

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (FcεRII)

et al. 1997

View full text Add to dashboard Cite

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

show abstract

Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli

Cited by 62 publications

References 30 publications

Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond

Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond

Split Tolerance in a Novel Transgenic Model of Autoimmune Myasthenia Gravis

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (FcεRII)

Contact Info

Product

Resources

About