©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
High Level Expression and Refolding of Mouse Interleukin 4 Synthesized in Escherichia coli(*)

(Received for publication, January 13, 1995)

Alan D. Levine (§) Shaukat H. Rangwala Nancy A. Horn (¶) Michelle A. Peel Brian K. Matthews Richard M. Leimgruber (1) Julia A. Manning Bruce F. Bishop (2) Peter O. Olins

From the  (1)From Searle Discovery Research,Monsanto Corporate Research, and (2)The Agricultural Group, Monsanto, St. Louis, Missouri 63198

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 times 10^7 units/mg. This recombinant IL-4 contained a threeamino-acid NH(2)-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH(2) terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.


INTRODUCTION

Murine Interleukin 4 (IL-4) (^1)is a potent mediator of an immune response, affecting both the growth and differentiation of a wide variety of cells in the hematopoietic lineage (Ohara and Paul, 1987). This cytokine is expressed by activated T lymphocytes (Howard et al., 1982) and mast cells (Brown et al., 1987; Burd et al., 1989; Plaut et al., 1989) as a 20-kDa glycoprotein (Ohara et al., 1987). The cDNA for IL-4 was initially isolated by two laboratories, using expression vectors and screening for either a IgG-inducing factor (Noma et al., 1986) or a mast cell growth factor (Lee et al., 1986). The derived amino acid sequence from the cDNA clones was used to predict a protein backbone for IL-4 of 14 kDa. This is consistent with the observation that N-glycanase treatment of natural IL-4, to remove N-linked carbohydrates, yields a protein core of 14 kDa (Ohara et al., 1987). Initial experiments with deglycosylated native IL-4 and with deglycosylated recombinant IL-4, expressed initially in yeast as a heterogeneous, hyperglycosylated molecule, suggested that the carbohydrate modifications of IL-4 do not affect its ability to bind to receptor and to stimulate T and B cell growth (Grabstein et al., 1986; Park et al., 1987; Paul and Ohara, 1987). Characterization of the role of the carbohydrate moieties on IL-4 using in vivo assays has not been rigorously examined. The Escherichia coli-derived recombinant, non-glycosylated IL-4 described in this report retains biological activity in vitro and is being used in vivo to characterize the role of IL-4 in the synthesis of IgE, stimulating erythropoiesis, and regulating autoimmune T cells (Racke et al., 1994).

We have previously described an inducible, efficient expression vector, which was designed to optimize the transcriptional and translational efficiency of foreign gene expression in E. coli (Olins et al., 1988). Transcriptional regulation of recombinant IL-4 expression is mediated by the DNA repair/recombination promoter rec A which can be induced with nalidixic acid. Translational efficiency is increased by using the T7 phage g10-L translational enhancer which has been shown to be one of the most effective ribosome-binding sites for the initiation of translation in E. coli (Olins and Rangwala, 1989, 1990).

In this report we describe the construction of an inducible expression vector for the synthesis of recombinant mouse IL-4 in E. coli. The IL-4 molecule was expressed as a 14-kDa protein which, after disruption of aggregates, refolding, and purification, retained all of the biological activities tested that have been ascribed in vitro to both natural and recombinant glycosylated mouse IL-4. The ability to purify milligram quantities of this protein has allowed us to examine the immunoregulatory properties of IL-4 in vivo (Racke et al., 1994) and has aided the effort to characterize the biochemical properties of this molecule (Carr et al., 1991).


EXPERIMENTAL PROCEDURES

Bacteria and Cell Lines

E. coli strain JM 101 (Messing, 1979) was used for the subcloning and bench top expression of the cDNA coding for mouse IL-4. The hematopoietic precursor IL-3-dependent cell line FDC-P1 was kindly supplied by Dr. J. McKearn, Searle, St. Louis, MO, and was maintained in Iscove's modified Dulbecco's medium, supplemented with 10% fetal calf serum, 1 mM pyruvate, 2 mM glutamate, and 10% Wehi 3B conditioned media as a source of IL-3 (London and McKearn, 1988).

Monoclonal Antibodies and Polyclonal Sera

11B11 is a rat-mouse hybridoma which secretes an IgG2b neutralizing antibody for mouse IL-4 (Ohara and Paul, 1985) that was grown as an ascites tumor in pristine-primed nude mice (Charles River). The ascites fluid was filter sterilized and used directly. Polyclonal antisera for immunoblot assays consisted of a mixture at optimal titers of five rabbit sera directed against synthetic mouse IL-4 peptides. Rabbit 101 (anti-peptide 47-66), 110 (anti-peptide 21-39), and 113 (anti-peptide 38-54) sera were diluted 1/100,000; rabbit 109 (anti-peptide 114-140) to 1/5,400; and rabbit 112 (anti-peptide 79-95) to 1/25,000. Peptides were synthesized by the Merrifield solid-phase method on an Applied Biosystems model 430A peptide synthesizer at an 0.5 mmol scale. A p-methylbenzhydrylamine resin was employed for peptide amides and a phenylacetamidomethyl resin for peptide acids. Coupling of appropriate Boc-amino acids was performed using dicyclohexyl-carbodiimide-hydroxybenzotriazole coupling cycles as recommended by the manufacturer. Peptides were removed from the resin and treated with hydrogen fluoride-anisole-dimethyl sulfide. Purification to >90% purity was accomplished by high pressure liquid chromatography, using either a Vydac C-18 reverse-phase column (Separations Group) or a µBondapak column (Waters) with gradients of 0-30% acetronitrile containing 0.05% trifluoroacetic acid. Peptide-protein conjugates were prepared by coupling cysteinyl-peptides to thyroglobulin (Sigma) with N-succinimidyl 3-(2-pyridyldithio)propionate (Pharmacia LKB Biotechnol.) according to the manufacturer's instructions. Amino acid numbering follows the convention of Noma et al.(1986).

Plasmid Constructions

A cDNA clone (pCB1) isolated from the murine thymoma EL-4, subcloned into the HincII site of pGEM 3, was kindly provided by Dr. W. Paul (Brown et al., 1987). Standard methods for DNA manipulation were employed (Maniatis et al., 1982). Plasmid vector pMON 5743 was used for expression of mIL-4 in E. coli (Olins and Rangwala, 1990). The coding region for the mature mIL-4 protein was obtained as an RsaI fragment and was cloned into the NcoI site of pMON 5743, which had previously been filled-in using dNTPs and DNA polymerase. The resulting plasmid, pMON 5738, is shown in Fig. 1. The coding region comprises the amino acid sequence Met-Thr-Arg-Ser, followed by the mature mIL-4 coding sequence.


Figure 1: Plasmid vector for expression of mIL-4 in E. coli. The plasmid is based on pBR 327 (Soberon et al., 1980) (from the SalI to EcoRI sites). Inducible transcription is from the E. coli recA promoter (Feinstein et al., 1983; Olins et al., 1988), and efficient translation is provided by the g10-L ribosome-binding site (Olins et al., 1988; Olins and Rangwala, 1989). The NcoI and EcoRI sites which were filled-in with DNA polymerase are denoted by NcoI-X and EcoRI-X, respectively. The plasmid includes the pBR 327 origin of replication (ori-327) and the F1 bacteriophage origin of single-stranded replication (ori-f1). The selectable marker is beta-lactamase (bla). The diagram is not to scale.



Bacterial Culture Conditions

Plasmid pMON 5738 was transformed into E. coli strain JM 101 (Messing, 1979) and selected in the presence of 200 µg/ml ampicillin. Cultures were grown at 37 °C in M9 medium (Maniatis et al., 1982), supplemented as described in Obukowicz et al.(1988). When the cultures reached OD = 0.5, transcription from the recA promoter was induced by the addition of 50 µg/ml nalidixic acid, and growth was continued for a further 4 h. Hourly aliquots of cells were taken, and cells were harvested by centrifugation.

Cell Lysis

The total cell pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and sonicated on ice for three 20-s bursts. The soluble supernatant fraction and insoluble pellet were separated by centrifugation at 10,000 rpm (16, 900 times g) for 10 min.

Fermentation Protocol for IL-4 Production

Fermentations were run in 15-liter LSL Biolafitte fermentors containing 10 liters of M9 minimal salts medium supplemented with 20 g/liter casamino acids, as described previously (Padgette et al., 1987). The fermentors were inoculated with a 1-liter overnight shake flask culture. The fermentations were run at 37 °C with an air sparge rate of 15 liters/min and 5 pounds/square inch backpressure. The pH was controlled at 7.0 with ammonium hydroxide, and dissolved oxygen was maintained at 30% saturation by increasing agitation rate from 500 to 1000 rpm. Glucose was added to the fermentor at an initial concentration of 10 g/liter and allowed to decrease to 5.0 g/liter after which the glucose concentration was maintained at 5.0 ± 1.0 g/liter by controlled feed of a 50% (w/v) glucose solution. A Gilson Stasar II spectrophotometer was used to monitor optical density of the culture at 550 nm. At OD = 20, induction was initiated by the addition of nalidixic acid to a final concentration of 50 µg/ml. Cells were harvested at 3-h post-induction by concentration in an Amicon DC10L using an HP100 hollow fiber cartridge. The cell slurry was then harvested by centrifugation in a Beckman J2-21 centrifuge. The cell paste was frozen at -80 °C.

Purification of E. coli-derived Mouse IL-4

Cell Disruption and Isolation of Inclusion Bodies

10 g of frozen cell paste were resuspended in 1.0 liter of 0.05 M Tris-HCl, pH 7.5 (buffer A). The suspension was homogenized in one pass at 8000 pounds/square inch with a Gaulin 15 M homogenizer equipped with a knife-edged valve. The homogenized suspension was separated by centrifugation in a Sorvall RC5C centrifuge with a GS-3 rotor at 6500 rpm (7140 times g) for 30 min. The supernatant was discarded. The pellet was fully resuspended in 1.0 liter of 0.05 M Tris-HCl and precipitated again by centrifugation at 6500 rpm in a Sorvall RC5C centrifuge with a GS-3 rotor. This washing step was repeated twice.

Formation of Intramolecular Disulfide Bonds

Denaturation, Reduction, and Oxidation Conditions

All steps were performed at room temperature. The washed inclusion bodies were dissolved (15-20 g pellet/liter) in buffer B (4 M guanidine, 0.5 M NaCl, 0.05 M DTT, and 0.05 M CHES, pH 9.5, for 4-6 h. This solution was loaded into Spectra/Por dialysis membrane tubing (molecular weight cutoff, 3500) previously washed in Milli-Q H(2)O and dialyzed against 15 volumes of 4 M guanidine, 0.5 M NaCl, 0.02 M cysteine, 0.05 M CHES, pH 9.5 (buffer C) to begin slow disulfide formation. After 12-15 h of dialysis in buffer C, the dialysis buffer was changed to 4 M guanidine, 0.5 M NaCl, 0.05 M CHES, pH 9.5 (buffer D) to initiate fast disulfide formation. Buffer D was changed after 8-12 h. Oxidation was complete within 24 h after dialysis against buffer D.

HPLC Analytical

Oxidation of IL-4 to monomeric isoforms was monitored by size exclusion HPLC on a TSK SW3000 column with a 7-cm precolumn. The mobile phase was 0.1 M Na(2)HPO(4), pH 7.6. The flowrate was 0.5 ml/min, and the run time was 60 min. DTT-treated monomeric IL-4 had a retention time of approximately 30.5 min while oxidized monomeric IL-4 eluted at approximately 28 min. Oxidation was judged complete when no additional IL-4 had accumulated in the 28-min peak.

Refolding and Partial Purification of IL-4 by Negative Precipitation

Oxidized IL-4 was refolded, partially purified, and prepared for ion-exchange chromatography by two negative precipitation steps. First, the guanidine was removed by dialysis against 15 volumes of buffer D with two buffer changes (8-12 h apart). The salt was then removed by dialysis against 15 volumes of 0.05 M CHES, pH 9.5 (buffer E). A heavy cream-colored precipitate formed in the dialysis bag. The refold solution was clarified by centrifugation in a Sorvall RC5C centrifuge with a GS-3 rotor at 10,000 rpm (16,900 times g) for 30 min in preparation for the second precipitation step. The supernatant was loaded into Spectra/Por dialysis membrane tubing (molecular weight cutoff, 3500) previously washed in Milli-Q H(2)O and dialyzed against 15 volumes buffer F (0.05 M acetic acid adjusted to pH 5.0 with NaOH) at 4-10 °C with two exchanges (8-12 h apart). A white precipitate formed in the dialysis bag. The acidified refold solution was clarified by centrifugation in a Sorvall RC5C centrifuge with a GS-3 rotor at 10,000 rpm (16,900 times g) for 30 min. The supernatant was filtered through a 0.22-µm cellulose acetate membrane and stored at 4-10 °C.

Chromatographic Purification of IL-4

Analytical

Purification beyond refold and precipitation of IL-4 was monitored by reverse-phase HPLC on a 4.6 mm times 15 cm Vydac 214TP column. At the initiation of the run, the column was equilibrated with 30% acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. For the first 3 min, the column was in the isocratic mode. From minute 3 to minute 35 the column was developed with a linear gradient of 30% acetonitrile to 55% acetonitrile in 0.05% trifluoroacetic acid. The column remained in 55% acetonitrile, 0.05% trifluoroacetic acid for 5 min.

S-Sepharose Fast Flow Ion-exchange Chromatography

Partially purified refolded mIL-4 was further purified on Pharmacia S-Sepharose Fast Flow. The sterile filtered sample was applied to a 1 times 5-cm column packed with S-Sepharose Fast Flow and equilibrated in buffer F with a flow rate of 0.5 ml/min. Following loading, the column was washed with 0.2 M acetic acid, pH 5.0, until the absorbance at 280 nm returned to base line. The column was developed with a linear gradient from 0.2 M acetic acid to 0.2 M acetic acid, 1 M NaCl, pH 5.0. Biologically active IL-4 eluted at a conductivity between 57 and 62 milliSiemens.

Toyopearle HW-55 Size Exclusion Chromatography

The pooled biologically active fractions from the ion-exchange column were applied to a 4.4 times 80-cm column packed with Toyopearle HW-55 (fine grade) equilibrated in 0.05 M Na(2)HPO(4), 0.15 M NaCl, 0.01% Tween 80 (w/v), pH 7.5. The flow rate was 1.0 ml/min, and the sample was injected into the bottom of the column. Monomeric biologically active IL-4 eluted in the 125-135-ml fractions.

Polyacrylamide Gel Electrophoresis

Cell lysates and purified proteins were analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970; Olins et al., 1988). Two-dimensional gel electrophoresis was performed using non-equilibrium pH gradient electrophoresis for the first dimension essentially as described by O'Farrell(1975). Western blots were performed as described by Renart et al.(1979). Silver staining was performed using a Rapid Ag Stain Kit obtained from ICN. Development of the stain was terminated with 7% acetic acid.

Biological Assays for Mouse Interleukin 4

A detailed description of the biological assays which were used to characterize the in vitro properties of recombinant IL-4 is described elsewhere (Conrad et al., 1989; Carr et al., 1991; Snapper et al., 1991). Refolding efficiency and purification of biologically active mouse rIL-4 was monitored in a hematopoietic precursor cell proliferation assay, using the IL-3/IL-4/granulocyte macrophage colony-stimulating factor-dependent cell line FDC-P1 (London and McKearn, 1988). Cells were maintained in Iscove's modified Dulbecco's medium, 10% fetal calf serum, 1% glutamine and sodium pyruvate, and 10% supernatant from Wehi 3B cells as a source of mouse IL-3. Briefly, cells were washed three times to remove IL-3 and resuspended at 2 times 10^5 cells/ml in the same media lacking Wehi supernatant. 50 µl of cells were mixed in a 96-well flat-bottomed plate (CoStar) with 50 µl of 3-fold serially diluted samples. The culture was incubated for 16 h at 5% CO(2) at 37 °C and was then pulsed with 1 µCi of [^3H]thymidine (DuPont New England Nuclear) per well for 4 h. Cells are harvested using a Skatron cell harvester, and [^3H]thymidine incorporation is measured with a liquid scintillation beta counter (Micromedics). One unit of IL-4 is defined as the mass of IL-4 required to half-maximally stimulate [^3H]thymidine uptake in the 100-µl culture. Natural murine IL-4 was obtained from phorbol myristic acid-induced EL-4 thymona cells and was purified as described by Ohara et al. (1987).


RESULTS

Expression of Mouse IL-4 in E. coli

The coding region for the mature mouse IL-4 protein was cloned downstream of the ATG initiator codon of plasmid pMON 5743, as described under ``Experimental Procedures.'' The resulting expression plasmid, pMON 5738, is shown in Fig. 1. It contains the recA promoter of E. coli and the g10-L ribosome-binding site (Olins and Rangwala, 1990). JM 101 cells carrying plasmid pMON 5738 were grown as described under ``Experimental Procedures.'' When a mid-logarithmic phase of growth was reached, an aliquot of cells was taken, and the culture was induced by the addition of nalidixic acid. Total cellular protein was analyzed by SDS-polyacrylamide gel electrophoresis, as illustrated in Fig. 2. As can be seen, nalidixic acid induction resulted in the high level accumulation of a new protein, with a molecular mass of approximately 14 kDa. This corresponds to the predicted molecular weight for full-length mouse IL-4. Further cell fractionation, described in Fig. 2, showed that the 14-kDa protein was insoluble and accumulated in inclusion bodies. This property aided considerably in subsequent purification of the protein. The expression level of IL-4 was quantitated by densitometric scanning of a Coomassie Blue-stained SDS-polyacrylamide gel and enzyme-linked immunoassay for mouse IL-4. IL-4 represented 17% of total cellular protein. Verification that the major band denoted as IL-4 in Fig. 2was obtained by immunoblotting using a mixture of five rabbit anti-sera raised against five different peptides synthesized from the mouse IL-4 sequence, as described under ``Experimental Procedures'' (data not shown). No biological activity was found associated with the large mass of IL-4 in the insoluble fraction. Although a substantial level of biological activity was detected in the soluble fraction, a reliable estimate of specific activity was not possible due to the high level of contamination from bacterial proteins (data not shown). While the initial transformants selected for the expression of recombinant IL-4 grew slowly on solid medium, there was no evidence of toxicity to the host cell in the uninduced state in larger shake flasks or fermentations.


Figure 2: SDS-polyacrylamide showing the induction and expression of E. coli-derived IL-4. E. coli cells bearing plasmid pMON 5738 were grown to an OD = 0.5 in M9 media supplemented with casamino acids and were induced with 50 µg/ml nalidixic acid. Cells from a 1-ml culture were harvested at 1-h time points after induction, lysed in 100 µl of Laemmli sample buffer (Laemmli, 1970), and the proteins were fractionated by electrophoresis on a 15% polyacrylamide gel. At 4 h after induction, the cells were also lysed by sonication in 1 ml of PBS and soluble (Supernatant) and insoluble (Pellet) fractions were isolated by centrifugation at 10,000 rpm in an Eppendorf microcentrifuge and analyzed by gel electrophoresis. The mIL-4 standard is E. coli-derived IL-4 purified as described in Fig. 3. The Coomassie Blue-staining pattern is shown. Molecular mass markers are ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; beta-lactoglobulin, 18 kDa; lysozyme, 14 kDa; bovine trypsin inhibitor, 6 kDa; and insulin (A and B chain), 3 kDa.




Figure 3: Summary of the refold protocol and purification scheme of E. coli-derived IL-4. Inclusion bodies were isolated as described under ``Experimental Procedures.'' Recombinant mouse IL-4 was refolded and purified as depicted by the cartoon and described in detail under ``Experimental Procedures.''



Refolding of Inclusion Body-associated IL-4

While the biological activity of the rIL-4 was found associated with the soluble fraction of the cellular lysate, the majority of IL-4 protein mass remained in the inclusion bodies (Fig. 2, lanes Supernatant and Pellet). The inclusion bodies were purified from 10 g of cell paste as described under ``Experimental Procedures'' and the protein solubilized in 4 M guanidine, 0.5 M NaCl, 0.05 M DTT, and 0.05 M CHES, pH 9.5. The protocol of refolding is described in Fig. 3. Excess DTT was added prior to refolding to eliminate any interpolypeptide disulfide bonds which might have formed during lysis. Identification of the elution pattern of monomeric, oxidized IL-4 on a size exclusion TSK SW3000 HPLC column was initially determined by biological assay. All of the fractions shown in panel A of Fig. 4were assayed for their ability to stimulate the proliferation of an IL-4 responsive prehematopoietic cell line. As is shown with the dashed line, IL-4 activity eluted from the TSK SW3000 column with a retention time of 28 min and represented a very small percentage of total protein present in the inclusion bodies. Oxidation of IL-4 was accomplished by sequential slow and fast intramolecular disulfide formation. Slow disulfide bond formation was attained by replacing the DTT reducing agent with cysteine, to retard the rate of oxidation. The oxidation process was accelerated by removal of the cysteine by dialysis. As shown in Fig. 4, the appearance of oxidized IL-4 increased with the time of dialysis and was complete within 24 h. Oxidation efficiency was 15% as measured by the analytic HPLC size elution profile (Fig. 4D). From 1 g of inclusion bodies, containing 500 mg IL-4, the yield of oxidized monomer is 50-75 mg. The refolding process was initiated by the slow removal of the guanidine denaturant by dialysis.


Figure 4: HPLC-TSK analytic chromatography detailing the time course of oxidation and refolding of IL-4. Oxidation and refolding efficiency of IL-4 was monitored by size exclusion chromatography on a 25-cm TSK SW3000 column with a 7-cm precolumn. The column was run at 0.5 ml/min with a mobile phase of 0.1 M Na(2)HPO(4), pH 7.6, for 60 min. Absorbance at 220 nm was monitored continuously. The initiation of disulfide bond formation coincided with the removal of the DTT during dialysis (see Fig. 3). The appearance of oxidized IL-4 can been seen at the elution time of 28 min. The dashed line represents the distribution of IL-4 proliferation units, as measured by biological assay of fractions collected from the TSK column. Samples were diluted into PBS, sterilized with a Co-Star 0.22-µm Spin-X centrifuge filtration unit, and added to the proliferation assay. The solid line is absorbance at 220 nm (A) at the start of the dialysis, 1 h after removal of the cysteine (B), 4 h later (C), and after 24 h (D). At the end of the formation of intramolecular disulfide bonds, renaturation and refolding were initiated when the guanidine was removed by dialysis against 0.05 M CHES, pH 9.5. The refolded solution was clarified by centrifugation at 16,900 times g for 30 min. The supernatant was dialyzed against 0.05 M acetic acid, pH 5.0. The solution was clarified at 16,900 times g for 30 min and the soluble, refolded protein (E) analyzed by chromatography on the TSK column.



Purification of Refolded, Biologically Active IL-4

As shown schematically in Fig. 3, monomeric, oxidized IL-4 required further purification. The preparation was acidified with acetic acid to pH 5.0 and clarified by centrifugation at 10,000 rpm for 10 min in a GS-3 rotor (Fig. 4E). The soluble fraction, which contained a mixture of biologically active and inactive monomeric, oxidized IL-4, was then applied to an S-Sepharose Fast Flow ion-exchange column to remove the majority of cationic proteins that did not bind to the column. IL-4 was eluted with an NaCl gradient from 0.1 to 0.55 M, as shown in Fig. 5. Elution of biologically active, refolded IL-4 was followed with the proliferation assay as denoted by Pool in Fig. 5. Active IL-4 eluted late from this cationic exchange resin (Fig. 5) and represented only 15% of the total IL-4 applied to the column. Since there were aggregates of IL-4 present in this pooled fraction, final purification involved an HPLC TSK sizing column with a mobile phase of buffer G, applied isocratically. The majority of IL-4 protein mass eluted at 14 kDa, as predicted (Fig. 6). In addition, the minor high molecular weight peak also contained IL-4 protein as judged by gel electrophoresis, immunoblotting, and amino acid sequence analysis (data not shown). This aggregated IL-4 material retained a moderate level of biological activity.


Figure 5: Cation-exchange chromatography of refolded IL-4. After the precipitation of the preparation by lowering the pH to 5.0, IL-4 was further purified on a S-Sepharose Fast Flow column. The sterile filtered sample was applied to a 1 times 5-cm column equilibrated in 0.05 M acetic acid, pH 5.0, with a flow rate of 0.5 ml/min. The column was washed with 0.2 M acetic acid until the absorbance at 280 nm returned to base line (NaCl arrow). Elution was performed with a gradient of NaCl from 0-1 M in 0.02 M acetic acid, pH 5.0. The biologically active fractions are indicated by the Pool notation.




Figure 6: Purification of IL-4 by TSK sizing matrix HW55 fine. The pooled biologically active fractions from the ion-exchange column were applied to a 4.4 times 80-cm Toyopearle HW-55 size exclusion column equilibrated in 0.05 M Na(2) HPO(4), 0.15 M NaCl, 0.01% Tween 80 (w/v), pH 7.5. Further details are described in the text. The major absorbance peak of pooled fractions contains monomeric, active IL-4.



Biological Activity of Purified rIL-4 Compared to Natural EL-4-derived IL-4

The relative potency and specific activity of purified recombinant IL-4 from the TSK column was compared to purified, natural IL-4 secreted by phorbol myristic acid-stimulated EL-4 thymona. As shown in Fig. 7, rIL-4 has an identical ED (5 pM), a similarly shaped concentration response curve, and can stimulate FDC-P1 cells to an identical level of proliferation as natural IL-4. In both cases the protein concentration was determined by amino acid composition and purity by NH(2)-terminal sequencing (see Table 2). The variation in quantitating protein mass by a variety of techniques is summarized in Table 1. Purified, E. coli-derived IL-4 has a specific activity of 4-7 times 10^7 units/mg.


Figure 7: Refolded E. coli-derived recombinant IL-4 shares an identical dose-response curve with natural IL-4. Purified E. coli-derived (TSK HW55 monomeric fraction) and natural IL-4 were diluted into cell culture media and added to FDC-P1 cells at half-log dilutions for an overnight incubation. After a 4-h pulse with [^3H]thymidine, the cells were harvested and counted. Protein concentrations were determined by amino acid composition. , recombinant E. coli-derived IL-4; circle, natural EL-4-derived IL-4.







Biochemical and Biophysical Characterization of Purified IL-4

The biochemical properties of recombinant IL-4 were characterized by two-dimensional gel electrophoresis. The non-glycosylated, bacterially synthesized IL-4 exhibited insolubility in an isoelectric focusing gel (first dimension) at sample loads of greater than 1 µg. This form of IL-4 is also insoluble if a non-ionic detergent such as Nonidet P-40 was present (data not shown). Optimal results were obtained when 0.5 µg of IL-4 was focused in a gel containing urea and the zwitterionic detergent CHAPS. The pH gradient of the first dimension gel spanned the range of 3.6-9.0. Since rIL-4 exhibited a pH greater than 9.0, non-equilibrium conditions were necessary (Fig. 8), which precluded the determination of a precise pI.


Figure 8: Two- dimensional gel electrophoresis analysis of purified, monomeric, biologically active IL-4. The IL-4 was focused under non-equilibrium conditions for 1050 V-hrs. The gel was oriented with the acidic end of the gel to the left. A, Coomassie Blue R-250 stained, 1.0 µg of IL-4; B, immunoblot of A, 2-h exposure; C, silver stained, 0.5 µg of IL-4. Molecular mass markers are phosphorylase b, 98 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa, carbonic anhydrase, 29 kDa; soybean trypsin inhibitor, 21 kDa; lysozyme, 14 kDa. Further details are described in the text and under ``Experimental Procedures.''



Purified, biologically active IL-4 consisted of at least three isoforms, each with an apparent molecular mass of approximately 14 kDa (Fig. 8A). All three isoforms are immunoreactive with the anti-IL-4 peptide anti-sera (Fig. 8C). A minor amount of 20-kDa material is detected by silver staining. However, this material was not detected by either Coomassie Blue staining nor by the anti-IL-4 peptide anti-sera (Fig. 8, B and C), and probably represents a minor contaminant, which does not contain the epitopes recognized by the anti-sera mixture. The purity of the rIL-4 was also assessed by chromatographic separation on a microbore reverse phase C-8 column. While two-dimensional gel electrophoresis revealed three isoforms of rIL-4, the reverse-phase column was unable to resolve these structures (Fig. 9). Experiments to determine if these three isoforms exist in deglycosylated native IL-4 have not been performed.


Figure 9: Reverse-phase microbore HPLC of IL-4. Purified E. coli-derived IL-4 was applied to an Applied Biosystems C8-microbore column run at 1 ml/min at room temperature. The protein was eluted with a gradient from 0 to 60% (v/v) acetonitrile in 0.05% (v/v) trifluoroacetic acid. The protein elution was monitored with a UV detector at 280 nm.



E. coli contains a methionine aminopeptidase whose activity is dependent on the second encoded amino acid (Ben-Bassat et al., 1987). As shown in Table 2, NH(2)-terminal amino acid sequence analysis of purified IL-4 revealed that approximately half the protein lacked the NH(2)-terminal methionine. This NH(2)-terminal microheterogeneity may explain the two major bands of IL-4 revealed by non-equilibrium pH gradient electrophoresis (Fig. 8). Since our biological assay is not sensitive within a factor of 2, we are unable to compare the relative potency of each of these species. A trace amount (4%) of IL-4 lacks the first four amino acids (Table 2).


DISCUSSION

IL-4 regulates the growth and differentiation of a variety of cells of hematopoietic lineage, when it is assayed in in vitro using cell culture models. While studies to examine the effects of this regulatory protein in vivo are difficult to undertake directly, three indirect approaches have been used. Two series of transgenic mice have been constructed. One set expresses IL-4 constitutively by both B and T cells under the regulation of the immunoglobulin enhancer (Tepper et al., 1990). The other line of transgenic mice expresses IL-4 locally within thymocytes using the lck promoter (Lewis et al., 1990). The data revealed thus far from these transgenic studies suggest an important in vivo role for IL-4 in IgE regulation and thymocyte differentiation. A second approach to define the function of IL-4 in regulating an immune response in vivo has been to neutralize its activity with blocking monoclonal antibodies (Finkelman et al., 1990). These studies demonstrated that IL-4 is necessary for the synthesis of IgE in a variety of models (Finkelman et al., 1986, 1988, 1989), but is not necessary to stimulate an IgG1 response, as is predicted from the in vitro data (Noma et al., 1986; Snapper et al., 1987; Finkelman et al., 1987). In addition, transgenic mice homozygous for a mutation that inactivates the IL-4 gene were generated. Studies with these mice confirmed the critical role played by IL-4 in regulating an IgE response and the Th2 phenotype (Kuhn et al., 1991; Kopf et al., 1993). Each of these approaches have provided important clues toward unraveling the complex regulatory network associated with the biology of IL-4, but each has its limitation.

A complementary approach to this problem is to administer IL-4 protein directly to an animal in controlled doses and at specific times. We describe in this report the synthesis, purification, and characterization of recombinant IL-4 from a prokaryotic host. A scale up of the procedure described here generates 100 mg of purified rIL-4/liter of fermentation, which has already lead to the evaluation of IL-4 in vivo in murine models of immune dysfunction (Racke et al., 1994). This correctly folded, purified protein was also used for studying its biochemical properties (Carr et al., 1991) and in preparing monoconal and polyclonal antibodies.

The majority of foreign proteins produced in E. coli accumulate in inclusion bodies (Olins and Rangwala, 1990). Since inclusion bodies can be rapidly removed from the total cell lysate by centrifugation, IL-4 can be readily purified as the major protein component in the sample (Fig. 1, Pellet). However, since IL-4 can potentially form three disulfide bonds and at least one of these is required for biological activity (Ohara et al., 1987; Carr et al., 1991), the use of inclusion bodies for purification is only attractive if conditions for efficient refolding of the protein can be identified. Other immunomodulatory proteins, such as macrophage colony-stimulating factor (Halenbeck et al., 1989; Taylor et al., 1994) and interferon- (Kleman et al., 1990) have also been purified by refolding from inclusion bodies.

It was not possible to determine the biological activity of IL-4 in the inclusion body lysate due to the toxicity of the guanidine on the target cells. Instead the efficiency of refolding was monitored by following the conversion of the reduced monomer to oxidized monomer on the size exclusion column (Fig. 4). Surprisingly, the oxidized form eluted from the TSK column first. We do not have an explanation, since oxidized proteins normally assume a globular conformation which reduces the Stokes' radius (Freifelder, 1982). We propose that the reduced monomer may have interacted more directly with the surface of the column and been retarded by the matrix.

An important observation that led to the first successful isolation of active IL-4 was made during analytical size exclusion chromatography of the reduced refractile bodies dissolved in 4 M guanidine. The apparent molecular mass of the crude refractile body preparation was predominantly greater than 100 kDa, and on-line diode array scanning of these high molecular mass peaks revealed a high 260-280-nm absorbance ratio. When the pH was changed to 9.0 and 0.5 M NaCl added to the buffer, 90% of the protein previously observed in several high molecular weight peaks coalesced into a single peak with a retention time consistent with monomeric IL-4. Scanning of this peak showed a much lower 260-280-nm ratio. We suspect that aggregation with nucleic acids may have inhibited previous refolding efforts. The negative precipitation step documented in Fig. 4E selectively removed only inactive monomeric proteins, as expected, since we have observed that mammalian cell culture-derived mouse IL-4 is soluble at pH 5. While the loss of biologically active material during this acidic precipitation was not monitored directly, the amount of oxidized, monomeric IL-4 as followed by analytic HPLC was not reduced.

The three isoforms of IL-4 revealed by two-dimensional gel electrophoresis are confirmed in the immunoblot to be the same protein species with different net charges. Further confirmation that the final preparation of IL-4 is a mixture of three isoforms is shown by the NH(2)-terminal sequencing data presented in Table 2. The preparation of IL-4 described here is a mixture containing NH(2)-terminal heterogeneity represented as 49% full-length protein, 47% des-Met IL-4, and 4% amino-terminal proteolyzed protein. We propose that the three isoforms revealed by two-dimensional gel electrophoresis (Fig. 8) are the three amino-terminal variants, but have not matched each spot with its corresponding sequence. It is formally possible that deglycosylated natural IL-4 may also electrophorese as distinct isoforms and that the variants identified in this report represent natural folding isoforms. This possibility has not yet been addressed. One important technical note of caution from this data is that the finding of a single, symmetric, homogeneous peak on a reverse phase microbore column is no guarantee that a single species of protein is present (contrast Fig. 8with Fig. 9).

The observation that modification of the amino terminus of mouse IL-4 does not effect its biological activity is supported by the data presented in this report and by the research of others. As shown in Fig. 7and Table 1, recombinant mouse IL-4 containing either a four- or three-amino-acid [(M)TRS] extension has an identical specific activity (4-7 times 10^7 units/mg) and dose-response curve as native IL-4. When Park et al.(1987) derivatized the NH(2) terminus of IL-4 synthesized in yeast to create an affinity column for purification of the IL-4 receptor, the binding of receptor to immobilized IL-4 was not altered. These data suggest that the amino terminus of mouse IL-4 does not play a role in receptor binding or signaling.

Regulation of antibody isotype selection, thymocyte differentiation, and T cell maturation is a complex process, dependent on the timing between immunization and challenge, route of antigen exposure, age, and immunologic history of the animal. The availability of milligram quantities of recombinant, murine IL-4 will permit an exploration of the role of this cytokine in vivo. The data presented in this report demonstrate how bacterially expressed IL-4 can be refolded to full biological activity and provide useful techniques to aid in the expression and refolding of other important biological mediators.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Immunology, Searle Discovery Research, Monsanto AA4G, 700 Chesterfield Parkway North, St. Louis, MO 63198. Tel.: 314-537-6862; Fax: 314-536-6862.

Present address: Amylin Corp., 9373 Towne Centre, Suite 250, San Diego, CA 92121.

(^1)
The abbreviations used are: IL-4, interleukin 4; rIL-4, recombinant interleukin 4; mIL-4, murine interleukin 4; IL-3, interleukin 3; g10, gene 10; dNTPs, deoxynucleotide triphosphates; CHES, 2-[N-cyclohexylamino]ethanesulfonic acid; HPLC, high performance liquid chromatography; RP-HPLC, reverse phase HPLC; CHAPS, (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); DTT, dithiothreitol; rpm, revolutions/min.


ACKNOWLEDGEMENTS

We thank Dan Conrad, William Paul, and David Tiemeier for providing commentary and Pat Kallaos and Carla Campbell for patiently typing this manuscript. Arnie Hershman provided encouragement through this project. We thank Joseph W. Bulock who synthesized and conjugated the peptides and Edwin Rowold for injecting and bleeding the rabbits.


REFERENCES

  1. Ben-Bassat, A., Bauer, K., Chang, S.-Y., Myambo, K., Boosman, A., and Chang, S. (1987) J. Bacteriol. 169, 751-757 [Medline] [Order article via Infotrieve]
  2. Brown, M. A., Pierce, J. H., Watson, C. J., Falco, J., Ihle, J. N., and Paul, W. E. (1987) Cell 50, 809-818 [CrossRef][Medline] [Order article via Infotrieve]
  3. Burd, P. R., Rogers, H. W., Gordon, J. R., Martin, C. A., Jayaraman, S., Wilson, S. D., Dvorak, A. M. Galli, S. J., and Dorf, M. E. (1989) J. Exp. Med. 170, 245-257 [Abstract]
  4. Carr, C., Aykent, S., Kimack, N. M., and Levine, A. D. (1991) Biochemistry 30, 1515-1523 [Medline] [Order article via Infotrieve]
  5. Conrad, D. H., Keegan, A. D., Kalli, K. R., Van Dusen, R., Rao, M., and Levine, A. D. (1988) J. Immunol. 141, 1091-1097 [Abstract/Free Full Text]
  6. Finkelman, F. D., Katona, I. M., Urban, J. F., Jr., Snapper, C. M., Ohara, J., and Paul, W. E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9675-9679 [Abstract]
  7. Finkelman, F. D., Katona, I. M., Urban, J. F., Jr., Holmes, J., Ohara, J., Tung, A. S., Sample, J. V. G., and Paul, W. E. (1988) J. Immunol. 141, 2335-2341 [Abstract/Free Full Text]
  8. Finkelman, F. D., Holmes, J., Urban, J. F., Jr., Paul, W. E., and Katona, I. M. (1989) J. Immunol. 142, 403-408 [Abstract/Free Full Text]
  9. Finkelman, F. D., Holmes, J., Katona, I. M., Urban, J. F., Jr., Beckmann, M. P., Park, L. S., Schooley, K. A., Coffman, R. L., Mosmann, T. R., and Paul, W. E. (1990) Ann. Rev. Immunol. 8, 303-331 [CrossRef][Medline] [Order article via Infotrieve]
  10. Freifelder, D. (1982) Physical Biochemistry: Applications to Biochemistry and Molecular Biology, 2nd Ed., W. H. Freeman and Company, San Francisco
  11. Grabstein, K., Eisenmar, J., Mochinzuki, D., Shanebech, K., Conlon, P., Hopp, T., March, C., and Gillis, S. (1986) J. Exp. Med. 163, 1405-1414 [Abstract]
  12. Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, K., Hamoaka, T., and Paul, W. E. (1982) J. Exp. Med. 155, 914-923 [Abstract]
  13. Kopf, M., Le Gros, G., Bachmann, M., Lamers, M. C., Bluethmann, H., and Kohler, G. (1993) Nature 362, 245-248 [CrossRef][Medline] [Order article via Infotrieve]
  14. Kuhn, R., Rajewsky, K., and Muller, W. (1991) Science 254, 707-710 [Medline] [Order article via Infotrieve]
  15. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  16. Lee, F., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., Coffman, R., Mosmann, T., Rennick, D., Roehm, N., Smith, C., Zlotnick, A., and Arai, K. (1986) Proc. Natl. Acad. Sci., U. S. A. 83, 2061-2065 [Abstract]
  17. Lewis, D. B., Yu, C. C., Forbush, K. A., Carpenter, J., Sato, T. A., Grossman, A., Liggitt, D. H., and Perlmutter, R. M. (1991) J. Exp. Med. 173, 89-100 [Abstract]
  18. London, L., and McKearn, J. P. (1987) J. Exp. Med. 166, 1419-1435 [Abstract]
  19. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Messing, J. (1979) in Recombinant DNA Technical Bulletin, NIH publication no. 79-99, Vol. 2, pp. 43-48, Bethesda, MD _
  21. Noma, Y., Sideras, T., Naito, T., Bergstedt-Lindqvist, S., Azuma, C., Severinson, E., Takabe, T., Kinashi, T., Matsuda, F., Yaoita, Y., and Honjo, T. (1986) Nature 319, 640-646 [CrossRef][Medline] [Order article via Infotrieve]
  22. Obukowicz, M., Turner, M. A., Wong, E. Y., and Tacon, W. C. (1988) Mol. & Gen. Genet. 215, 19-25
  23. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021 [Abstract]
  24. Ohara, J., and Paul, W. E. (1985) Nature 315, 333-336 [Medline] [Order article via Infotrieve]
  25. Ohara, J., Coligan, J., Zoon, K., Maloy, W. L., and Paul, W. E. (1987) J. Immunol. 139, 1127-1134 [Abstract/Free Full Text]
  26. Olins, P. O., and Rangwala, S. H. (1989) J. Biol. Chem. 264, 16973-16976 [Abstract/Free Full Text]
  27. Olins, P. O., and Rangwala, S. H. (1990) Methods Enzymol. 185, 115-119 [Medline] [Order article via Infotrieve]
  28. Olins, P. O., Devine, C. S., Rangwala, S. H., and Kavka, K. S. (1988) Gene (Amst.) 73, 227-235 [CrossRef][Medline] [Order article via Infotrieve]
  29. Padgette, S. R., Huynh, Q. K., Borgmeyer, J., Shah, D. M., Brand, L. A., Re, D. B., Bishop, B. F., Rogers, S. G., Fraley, R. T., and Kishore, G. M. (1987) Arch. Biochem. Biophys. 258, 564-573 [Medline] [Order article via Infotrieve]
  30. Park, L. S., Friend, D., Grabstein, K., and Urdal, D. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1669-1673 [Abstract]
  31. Paul, W. E., and Ohara, J. (1987) Ann. Rev. Immunol. 5, 429-459 [CrossRef][Medline] [Order article via Infotrieve]
  32. Plaut, M., Pierce, J. H., Watson, C. J., Hanley-Hyde, J., Nordon, R. P., and Paul, W. E. (1989) Nature 339, 64-67 [CrossRef][Medline] [Order article via Infotrieve]
  33. Racke, M. K., Bonomo, A., Scott, D. E., Cannelle, B., Levine, A., Raine, C. S., Shevach, E. M., and Rocken, M. (1994) J. Exp. Med. 180, 1961-1966 [Abstract]
  34. Renart, J., Reiser, J., and Stark, G. R. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3116-3120 [Abstract]
  35. Snapper, C. M., Finkelman, F. D., and Paul, W. E. (1988) J. Exp. Med. 167, 183-196 [Abstract]
  36. Snapper, C. M., Pechanha, L. M. T., Levine, A. D., and Mond, J. J. (1991) J. Immunol. 147, 1163-1170 [Abstract/Free Full Text]
  37. Soberon, X., Covarrubias, L., and Bolivar, F. (1980) Gene (Amst.) 9, 287-305 [Medline] [Order article via Infotrieve]
  38. Tepper, R. I., Levinson, D. A., Stanger, B. Z., Campos-Torres, J., Abbas, A. K., and Leder, P. (1990) Cell 62, 457-467 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.