(Received for publication, January 13, 1995)
From the
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 10
units/mg. This recombinant IL-4 contained a threeamino-acid
NH
-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
terminus of mouse IL-4 play a critical role in
contributing to its in vitro biological activity.
Murine Interleukin 4 (IL-4) ()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).
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 -lactamase (bla). The diagram is not to
scale.
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;
-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.''
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 NaHPO
, 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
g for 30 min. The supernatant
was dialyzed against 0.05 M acetic acid, pH 5.0. The solution
was clarified at 16,900
g for 30 min and the soluble,
refolded protein (E) analyzed by chromatography on the TSK
column.
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 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 80-cm Toyopearle
HW-55 size exclusion column equilibrated in 0.05 M Na
HPO
, 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.
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 [H]thymidine, the cells were
harvested and counted. Protein concentrations were determined by amino
acid composition.
, recombinant E. coli-derived IL-4;
, natural EL-4-derived IL-4.
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-terminal amino acid sequence analysis of purified IL-4
revealed that approximately half the protein lacked the
NH
-terminal methionine. This NH
-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).
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-terminal sequencing data
presented in Table 2. The preparation of IL-4 described here is a
mixture containing NH
-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 10
units/mg) and dose-response curve as
native IL-4. When Park et al.(1987) derivatized the NH
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.