THE JOURNAL or BIOLOGICAL CHEMISTRY
`© 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 267, N0. 13, Issue of May 5, pp. 8770-3777, 1992
`Printed in U.S.A.
`
`Folding and Oxidation of Recombinant Human Granulocyte Colony
`Stimulating Factor Produced in Escherichia coli
`CHARACTERIZATION or THE DISULFIDE-REDUCED INTERMEDIATES AND CYSTEINE .) SERINE
`ANALOGS*
`
`Hsieng S. Lu:l:, Christi L. Clogston, Linda 0. Narhi, Lee Anne Merewether, Wayne R. Pearl, and
`Thomas C. Boone
`
`From Amgen Incorporated, Amgen Center, Thousand Oaks, California 91320
`
`(Received for publication, October 14, 1991)
`
`The folding and oxidation of recombinant human
`granulocyte colony-stimulating factor solubilized from
`Escherichia coli inclusion bodies was investigated.
`During the folding process, two intermediates, I1 and
`I2, were detected by kinetic studies using high perform-
`ance liquid chromatography. I1 exists transiently and
`disappears quickly with the concomitant formation of
`12. In contrast, 12 requires a longer time to fold into the
`final oxidized form, N. CuSO4 catalysis increases the
`folding rate of 12 from 11, while CuSO4 and elevated
`temperature (37 °C) have a dramatic effect on the fold-
`ing rate of N from 12. These observations suggest the
`following sequential oxidative folding pathway.
`11 :—)I2 ?:—)N
`fast
`rate-limiting
`
`Peptide map analysis of the iodoacetate-labeled in-
`termediates revealed that I1 represents the fully re-
`duced granulocyte colony-stimulating factor contain-
`ing 5 free cysteines; I2 is the partially oxidized species
`containing a single Cys“-Cys” disulfide bond; and N,
`the final folded form, has two disulfide bonds. The
`physicochemical properties and biological activities of
`I1_ 12, N, and several Cys —> Set analogs made by site-
`directed mutagenesis were further investigated. In
`guanidine hydrochloride-induced denaturation stud-
`ies, the disulfide-reduced intermediates and the ana-
`logs missing either of the disulfide bonds are confor-
`mationally less stable than those of the wild type mol-
`ecule or the analog with the free Cys at position 17
`changed to Set. Recombinant human granulocyte col-
`ony stimulating factor lacking either disulfide bond or
`both has overall secondary and tertiary structures dif-
`ferent from those of the wild type molecule and exhibits
`lower biological activity. These studies show that di-
`sulfide bond formation is crucial for maintaining the
`molecule in a properly folded and biologically active
`form.
`
`The mechanistic study of protein folding is important in
`understanding the structure and function of proteins (1, 2).
`The difficulty in elucidating a protein folding pathway lies in
`measuring the structural properties of intermediate protein
`
`species, since they are usually short-lived. As described by
`Creighton and others (3—7), a model system that includes an
`oxidative refolding of a disulfide-reduced protein allows one
`to investigate the pathway of protein folding in detail. There
`are two possible advantages in investigating a disulfide-con-
`taining protein. First, the intrachain S—S bond is a natural
`covalent cross-link which is closely correlated with protein
`conformation, and the intramolecular disulfide bond forma-
`tion reflects the proximity of two relevant sulfhydryls in
`intermediate forms (3-5). Second, proteins in disulfide-re-
`duced states are not short lived allowing the intermediates to
`be trapped by chemical modification during folding.
`Human granulocyte colony-stimulating factor (hG-CSF)‘ is
`one of the hemopoietic growth factors which plays an impor-
`tant role in stimulating proliferation, differentiation, and
`functional activation of blood cells (8). Human G-CSF is
`capable of supporting neutrophil proliferation in vitro and in
`vivo (9, 10). The human and murine G-CSF genes have been
`cloned and characterized and are about 75% homologous (11,
`12). Large quantities of recombinant hG-CSF (rhG-CSF) have
`been produced in genetically engineered Escherichia coli and
`have been successfully used in human clinical studies to treat
`cancer patients suffering from chemotherapy-induced neutro-
`penia (13-15). E. coli-produced rhG-CSF is a 175-amino acid
`polypeptide chain containing an extra Met (at position -1)
`at its NH2 terminus. The molecule also contains a free cys-
`teine at position 17 and two intramolecular disulfide bonds,
`Cys“-Cys“ and Cys““—Cys“ (16). The two disulfide bonds
`form two small loops which are separated by 21 amino acids.
`Like other bacteria-derived recombinant proteins, rhG-CSF
`produced in E. coli requires an oxidative folding procedure in
`order to recover its biological activity (17). In this paper, we
`describe the kinetic study of a folding and oxidation procedure
`for the reduced rhG-CSF solubilized from the inclusion bodies
`as well as the isolation and characterization of the disulfide-
`reduced intermediates. To establish the role of disulfide bond
`
`formation in the folding of biologically active rhG-CSF, we
`also describe the biological and physicochemical characteriza~
`tion of intermediates and analogs made by site-directed mu-
`tagenesis at the Cys residues.
`
`* The costs of publication of this article were defrayed in part by
`the payment of page charges. This article must therefore be hereby
`marked “aduertisement" in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`El: To whom correspondence should be addressed.
`
`‘ The abbreviations used are: hG-CSF, human granulocyte colony-
`stimulating factor; rhG-CSF, recombinant human granulocyte col-
`ony-stimulating factor; Gdnl-lCl, guanidine HCI; RP-HPLC, reverse-
`phase high performance liquid chromatography; DTT, dithiothreitol;
`TFA, trifluoroacetic acid; DTNB, 5,5’-dithiobis(nitrobenzoic acid).
`
`8770
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`Amgcn Exhibit 2005
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`Page 1
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`Oxidative folding of hG—CSF
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`AboorbnnooIt215nm(1.2AUF‘S)—>
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`Retention Time (Minutu)
`
`FIG. 2. Folding of rhG-CSF at 25 °C in CuSO. monitored by
`RP-HPLC at different times. Chromatograms 1-6, 20 min, 1, 2, 4,
`8, and 12 h, respectively. Approximately 50 pg of rhG-CSF in folding
`mixture was injected. Intermediates I1 and I2 and the final oxidized
`form N are indicated. Note that retention times shown in Fig. 2 are
`slightly different from those in Fig. 1 due to the use of different C-4
`columns.
`
`MATERIALS AND METHODSZ
`
`RESULTS
`
`Folding Intermediates and Folding Kinetics of rhG-CSF-
`Fig. 1 shows the RP-HPLC elution profiles of the native and
`the fully denatured and reduced rhG-CSFs (chromatograms 1
`and 2, respectively). Their retention times differ by approxi-
`mately 2.1-2.5 min. Fully denatured and reduced rhG-CSF in
`6 M GdnHCl is retained slightly longer and elutes as a single
`sharp peak. Fig. 1 also shows that rhG-CSF present in the
`crude E. coli lysate solubilized in either GdnHCl or Sarkosyl
`at 2-5 °C elutes as broader peaks (chromatograms 3, and 4,
`respectively) at slightly earlier retention times than the fully
`denatured and reduced rhG-CSF. The difference in the chro-
`matographic elution times among these rhG-CSF forms has
`allowed us to detect folding intermediates and to study folding
`kinetics by RP-HPLC.
`Shown in Fig. 2 are the RP-HPLC chromatograms of the
`solubilized rhG-CSF samples prepared at different refolding
`times during incubation at 25 “C in the presence of CuSO.1.
`The populations of the three major rhG-CSF-related species
`which elute at retention times around 45 to 48 min change
`dramatically as a function of time. Intermediate I1 as depicted
`in Fig. 2 (chromatogram 1) is the starting reduced rhG-CSF.
`At the 20-min incubation time, 12 has already accumulated to
`a level of 33% of the total rhG-CSF. At 1, 2, and 4 h, the
`generation of I2 has proceeded further with the concomitant
`disappearance of 11 and appearance of the final oxidized form
`N (Fig. 2, chromatograms 2-4). The folding into form N
`reaches its maximum in 12 h and is greater than 95% complete
`(Fig. 2, chromatogram 6).
`The initial first order folding rate for conversion of I1 into
`12 was estimated to be 1.9 X 10'2 s“ and the initial rate for
`conversion of 12 into the final oxidized rhG-CSF (form N) to
`be approximately 3.1 X 10'3 s"‘ (Table 1). The half-maximal
`conversion of I1 to I2 takes approximately 30 min, while the
`
`2 Portions of this paper (including “Materials and Methods," Figs.
`1, 3-7, and 9, and Tables 1-4) are presented in miniprint at the end
`of this paper. Miniprint is easily read with the aid of a standard
`magnifying glass. Full size photocopies are included in the microfilm
`edition of the Journal that is available from Waverly Press.
`
`half-maximal conversion of I2 to N takes almost 4.5 h. Kinetic
`studies also indicate that the oxidative folding of form N from
`I2 is biphasic. The folding rate from I2 to N during the first
`phase is faster. The second phase of folding starts at approx-
`imately 8 h with a much slower rate.
`The folding kinetics of rhG-CSF at 25 and 37 “C without
`addition of copper sulfate were also investigated. As listed in
`Table 1, the initial rates for I2 formation at both 25 and 37 “C
`are similar (1.0 X 10” s“) and slightly slower than the folding
`of rhG-CSF in the presence of copper sulfate (1.9 X 10‘2 S‘).
`However, at 25 “C the generation of completely oxidized rhG-
`CSF (form N) is relatively slow (rate = 6.6 X 10“ s“). In this
`case,
`I2 persists much longer and approximately 20 h are
`required to reach half-maximal folding of form N from I2
`versus 4.5 h in the presence of copper sulfate at 25 “C. At
`37 °C the folding of form N from 12 without CuS0., is faster,
`but the biphasic kinetics becomes apparent. The first phase
`of oxidation takes about 5 h, while the second phase takes
`place at approximately 8 h. After 23 h, the recovery of com-
`pletely oxidized rhG-CSF reaches only about 80%.
`Isolation and Structural Characterization of Intermediates—
`An alkylating agent, iodoacetic acid, was used to trap the
`intermediates that may be disulfide-reduced. The resulting
`carboxymethylated derivatives contain more negatively
`charged carboxymethyl groups than the oxidized rhG-CSF
`and are separable by ion—exchange HPLC using a sulfoethyl
`polyaspartamide silica-based column (data not shown).
`To estimate the stoichiometry of labeling, the modification
`was also performed using [3H]C2-iodoacetic acid. Table 2 lists
`the labeling results for rhG-CSF and the purified intermedi-
`ates. In the absence and presence of 6 M GdnHCl, native rhG-
`CSF gives 0 and 1 mol of label/mol of protein, respectively,
`consistent with our previous observation (16). The fully re-
`duced and denatured rhG-CSF gives 5 mol of label. The
`trapped I1 gives 3.74 mol of label and 12 1.9 mol of label/mol
`of protein. These results indicate that 4 and 2 free cysteinyl
`residues are available for the labeling in 11 and I2, respectively.
`To further characterize the structure of intermediates, the
`“H-labeled I1 and I2 were reduced with DTT in 6 M GdnHCl
`and then carboxymethylated with non-radioactive iodoacetate
`to generate fully denatured and alkylated derivatives, which
`were then subjected to HPLC peptide mapping. Fig. 3 shows
`a typical peptide map derived from the Staphylococcus aureus
`V-8 protease digestion of the 12 derivative. Peptide fractions
`were pooled and aliquots were analyzed for determination of
`peptide concentration, radioactivity counting, and NH2-ter-
`minal sequence analysis. Table 3 summarizes the isolation
`and characterization of the labeled and unlabeled peptides
`derived from I1 and I2. For form 12, only peptides 7 and 8
`(Leu“7 to Glu” and Leu“ to Glu”, respectively) containing
`Cys“ and Cys“ were radioactively labeled. Sequence analysis
`confirmed that both Cys“ and Cys“ were labeled, supporting
`the quantitative data that 2 labeled cysteines are present in
`12 (Table 2). For form I1, Cys“ and Cys”, found in peptides 7
`and 8, as well as Cys“ and Cys” in peptide 2 (Lys3“ to Glu“)
`were radioactively labeled. This confirms that the 4 cysteines
`at positions 36, 42, 64, and 74 in I1 are not involved in disulfide
`bonding.
`As indicated in Table 3, peptide 4 is the NH2-terminal
`peptide of rhG-CSF containing Cys at position 17. Analysis
`of peptide 4 derived from both intermediates I1 and I2indicated
`that no radioactive label was present. The data suggest that,
`like the native rhG-CSF, both intermediates contain an in-
`accessible free cysteine at position 17.
`Preparation and RP-HPLC Analysis of rhG-CSF Analogs-
`The Cys —> Ser analogs made by site-directed mutagenesis of
`
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`8772
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`Oxidative folding of hG-CSF
`
`the rhG-CSF gene were recovered by the procedures developed
`for rhG-CSF including folding and chromatographic separa-
`tion. rhG-CSF[Cys" —> Ser”] exhibited oxidation and folding
`similar to those of the wild type rhG-CSF and could be isolated
`with equivalent recovery. In contrast, rhG-CSF[Cys‘°""“2 —>
`Ser3“'“] exhibited very slow folding in the absence of CuSO1
`(greater than 4 days) and was recovered in low yield. The
`[Cys“ —> Ser7“] analog lacking the Cys6“-Cys“ disulfide bond
`also folded moderately slowly but correctly in the absence of
`CuSO..
`(more rapidly but
`incorrectly in the presence of
`CuSO..), but recovered in low yield.
`Fig. 4A shows the elution of purified rhG-SCF[Cys7“ —>
`Ser7“] from a C-4 reverse—phase HPLC column. It elutes 2.20
`min (chromatograms 2 and 3 at pH 3 and 7, respectively) later
`than the wild type rhG-CSF (chromatogram 1). I1 lacking the
`Cys“-Cys74 disulfide bond elutes essentially at the same re-
`tention time as the Cys“ —> Ser“ analog, suggesting that the
`molecules have similar hydrophobicities. Recombinant hG-
`CSF[Cys"""“ —> Ser3“'”] (Fig. 4B, chromatogram 4) elutes only
`0.33 min later than wild type rhG-CSF.
`Biological Activity and Physicochemical Properties of the
`rhG-CSF, Intermediates and Analogs-—Table 4 lists the in
`vitro biological activities of rhG-CSF, folding intermediates,
`and analogs. The wild type rhG-CSF standard has an activity
`of approximately 1.0 X 10” units/mg. Carboxymethylated
`intermediates I1 and I2 have only approximately 3—5% activity
`of the wild type rhG-CSF. Both rhG—CSF[Cys36'42 —> Ser36'42]
`and rhG-CSF[Cys7“ —> Ser7“] also exhibit very low activity (1
`and 3%, respectively relative to the wild type molecule). In
`contrast, rhG-CSF[Cys" ——> Ser”] exhibits full in vitro biolog-
`ical activity.
`Conformational stabilities of rhG-CSF intermediates and
`analogs were compared by denaturation with GdnHCl at pH
`7.2. Fig. 5A shows the absorbance spectra of native and
`denatured rhG-CSFs. It appears that GdnHCl denaturation
`results in a blue shift of the UV spectrum, reflecting increased
`exposure of the aromatic amino acids to the polar aqueous
`solvent. The absorbance difference at 290 nm can thus be
`used to estimate the effect of a denaturing agent on the
`conformational stability of rhG-CSF. From the results shown
`in Fig. 5B, rhG-CSF appears to stay in the native state below
`2 M GdnHCl concentration, denatures quickly above 2.5 M
`denaturant, and is completely unfolded at 3.5 M GdnHCl.
`This denaturation profile approximates a simple two-state
`transition. The midpoint of denaturation is at approximately
`3 M GdnHCl (Table 4). Also indicated in Fig. 5B is the
`accessibility of the free Cys” at different GdnHCl concentra-
`tions as determined by DTNB reaction. The increased acces-
`sibility of Cys” is coincident with the denaturation of rhG-
`CSF detected by the change in absorbance at 290 nm.
`As shown in Fig. 6, similar GdnHCl denaturation studies
`were also performed on I1, 12, rhG-CSF[Cys7‘ —> Ser7“], and
`rhG-CSF[Cys“‘**“2 -—> Ser““"2]. The denaturation transition oc-
`curs at lower GdnHCl concentration for the intermediates
`and analogs. A further decrease in absorbance was observed
`to proceed at higher GdnHCl concentration. The concentra-
`tions of GdnHCl that are required to achieve a midpoint
`denaturation for the intermediates and analogs range from
`1.4 to 2.1 M (Table 4). The thermodynamic constant for rhG-
`CSF is 5.4 Kcal/mol, a value typical of a folded globular
`protein while all of the disulfide-reduced intermediates and
`analogs have values below 3 kcal/mol.
`Fig. 7A shows the far UV CD spectra of the native molecule,
`the rhG-CSF[Cys7“ —-> Ser”] analog, the rhG-CSF[Cys““"‘2 —->
`Ser"‘“"2] analog, and the folding intermediates at pH 7.5. rhG-
`CSF is rich in oz-helix, as evidenced by the minima at 222 and
`
`208 nm. All of the molecules tested exhibit or-helical structure,
`but the native molecule and the rhG-CSF[Cys3“"2 —> Seraw]
`molecule contain substantially higher helical content than the
`other species examined, Le. 64% helix versus 38% helix at
`neutral pH, calculated using the Greenfield-Fasman equation
`(19). The native and rhG-CSF[Cys3""2 —> Ser36"‘2] are the only
`species that show an increase in helicity (up to 75%) at pH
`3.5 (Fig. 7B). The far UV CD spectra of I1 and I2 with or
`without iodoacetate modification are identical.
`As seen in Fig. 8A, the fluorescence emission spectrum of
`the rhG-CSF molecule at neutral pH is characterized by a
`single peak with a maximum at 344 nm, typical of a somewhat
`solvent-exposed Trp (20). There is no detectable Tyr fluores-
`cence (around 300 nm). The spectra of the two analogs are
`similar to that of the native rhG-CSF, although the intensities
`are greater, indicating that the Trp fluorescence might be
`somewhat less quenched by the surrounding environment.
`The spectra of 11 and I2 differ slightly from those of the
`respective labeled derivatives; the peaks are broader, again
`reflecting a difference in the environment of the 2 Trp resi-
`dues.
`
`Fig. 8B shows the fluorescence spectra at pH 3. The Trp
`peak of native rhG-CSF is still evident, but is greatly de-
`creased in intensity (the absolute scale of this figure is less
`than that in A), and a peak at 304 nm, attributable to Tyr, is
`also present. This suggests that the molecule has undergone
`a reversible change in conformation so that energy transfer
`from Tyr to Trp no longer occurs. However, such change
`occurs only for the native molecule, since the other species
`show a decrease in the intensity of the Trp fluorescence, but
`no change in the Tyr fluorescence. The spectrum of the rhG-
`CSF[Cys36"2 -—> Ser““"“"] has a shoulder at 304 nm, indicating
`a slight acid-induced conformational change.
`Fig. 9 shows the hydrodynamic behavior of the native
`molecule, the Cys“ —-> Ser7‘analog, and Cys36'” —> Ser3°"2
`analog as determined by gel filtration. Recombinant hG-CSF
`is a very compact molecule (M, = 15,000 versus the expected
`18,800). While both analogs still elute with an apparent
`molecular weight smaller than expected, they both elute ear-
`lier than the native molecule, indicating that without either
`disulfide bond, the molecule behaves somewhat larger, prob-
`ably due to an increase in flexibility.
`
`DISCUSSION
`
`The present study demonstrates that the folding of reduced
`rhG-CSF proceeds through identifiable intermediates I1 and
`I2 (Fig. 2). The originally fully reduced (I1), partially oxidized
`(I2), and the final oxidized (N) forms can be separated and
`quantitated by HPLC for detailed kinetic studies. The disul-
`fide-reduced intermediates as well as the disulfide-unpaired
`analogs appear to behave more hydrophobically than the
`native rhG-CSF.
`Copper ion and other trace metals have been reported to
`catalyze air oxidation of proteins due to their ability to accel-
`erate thiol oxidation at concentrations ranging from 0.1 and
`10 uM (21-24). The optimal Cu“ concentration for rhG-CSF
`oxidation is in the range of 20-40 }lM (17). The kinetic data
`show that copper ion increases the folding rate of intermediate
`I2 from I1 approximately 2-fold and increases the folding rate
`of N from I2 approximately 5-fold at 25 °C. Cu“ also promotes
`oxidation to greater than 95% completion and shortens the
`second phase folding time (see “Results”). Increasing the
`temperature to 37 °C can also accelerate folding and oxidation
`of the final oxidized form N but does not appear to increase
`the rate of the second phase of folding. The kinetic studies
`and the detection of different intermediate folding forms
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`Oxidative folding of hG-CSF
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`FluorescenceIntensity(ArbitraryUnits)
`
`FIG. 8. Fluorescence spectra of
`rhG-CSF species at pH 7.5 (panel
`A) and at pH 3.2 (panel B). Spectrum
`1, rhG-CSF standard; spectrum 2, rhG-
`CSF[Cys7" —> Ser7‘]; spectrum 3, rhG-
`CSF[Cys3“"2 -—-> Ser3“"2]; spectrum 4, in-
`termediate I2 derivative; spectrum 5, re-
`duced rhG-CSF (or intermediate I] de-
`rivative).
`
`(280
`
`300
`
`320
`
`340
`
`360
`
`380
`
`400
`
`420>
`
`B
`
`
`
`
`
`
`
`
`
`
`FluorescenceIntensity(ArbitraryUnits)
`
`<280
`
`300
`
`320
`
`340
`
`nm
`
`360
`
`Emission Wavelength
`
`380
`
`400
`
`420>
`
`suggest that the mechanistic pathway for rhG-CSF folding
`under the described conditions is sequential: I1 —> I2 —> N.
`The folding from I, to I2 is fast, while the folding from 12 to
`N is rate-limiting (Table 1).
`I, and 12 were the two predominant intermediates observed
`during folding and no other species containing intermolecular
`disulfide forms were detected. The absence of such species
`was supported by other experimental evidence. For example,
`a small amount (<5%) of interdisulfide-linked rhG-CSF dimer
`or aggregate forms is detected by non-reducing sodium dodecyl
`sulfate-polyacrylamide gel electrophoresis (data not shown).
`Since levels of these species do not increase during folding,
`they may be unrelated to the described folding pathway.
`Moreover, by HPLC analysis, intermediates that contain non-
`native disulfide bonds were also not evident, indicating that
`disulfide formation does not occur randomly.
`
`For structural characterization, I1 and I2 have to be trapped
`as stable derivatives at an early time point during folding;
`these derivatives do not represent the final folded forms.
`Characterization of the rhG—CSF[Cys3“""" —-> Ser3“'”] and rhG-
`CSF[Cys7“ -—> Ser“] analogs lacking a single disulfide bond
`corroborates the findings obtained from physicochemical
`analysis of the isolated intermediate derivatives since they
`can be folded and oxidized to their respective stable oxidized
`states and isolated by rhG-CSF purification procedures.
`In the folding studies, the Cys36“2 —> Ser3“""’ analog under-
`goes a sequential oxidation and folding pathway similar to
`that observed for the wild type rhG-CSF going from I1 (data
`not shown). This analog is distinct from any of the folding
`intermediates and exhibits HPLC retention time similar to
`that of the wild type rhG-CSF (Fig. 4). Since the folding and
`Cys“-Cys“ disulfide bond formation of the Ser3“'” analog is
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`Oxidative folding of hG-CSF
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`slower than the wild type G-CSF, the formation of Cys“-
`Cys" disulfide in the folding of wild type G-CSF seems to be
`important for providing the initial stabilization force for the
`conformational integrity of the molecule, and for allowing
`moderately rapid formation of the Cyss‘-Cys“ bond. More-
`over, it is interesting to note that the far UV CD spectra of
`rhG-CSF[Cys3“"” —> Ser36'”] at neutral and acidic pH values
`resemble those obtained for native rhG-CSF (>60% helix),
`suggesting that formation of the rhG-CSF secondary structure
`is independent of formation of the Cys“-Cys“ disulfide bond.
`The rhG-CSF[Cys7‘ —> Ser7“] analog and 12 that do not
`contain the Cys“-Cys" bond have some common properties.
`They have similar HPLC elution times and exhibit structural
`similarity as detected by CD spectropolarimetric analyses.
`Since the observed magnitude of the far UV CD spectrum for
`rhG-CSF[Cys7“ -> Ser7‘] is only half that of the native rhG-
`CSF, and is similar to that of the isolated 12 (~35% a-helix),
`it appears that both I2 and rhG-CSF[Cys"“ —> Ser7"] contain
`partial secondary (or helical) structure. The low a-helical
`content observed for I2 and rhG-CSF[Cys7“ —> Ser7“] analog
`suggests that the formation of Cys“-Cys“ disulfide bond may
`be critical for allowing complete secondary structure forma-
`tion of rhG-CSF.
`Effects of the presence or absence of disulfide bonds on the
`tertiary structural folding and conformational stability of
`rhG-CSF were studied by fluorescence spectrometry and
`GdnHCl denaturation. Under acidic pH the wild type rhG-
`CSF exhibits a characteristic Tyr and Trp fluorescence spec-
`trum (Fig. 8B ); this was clearly not observed in any of the
`rhG-CSF analogs and the trapped intermediates. These data
`appear to suggest that both disulfide bonds (Cys'“““Cys‘2 and
`Cys“-Cys“) are essential in maintaining a properly folded
`tertiary structure of the rhG-CSF molecule. In GdnHCl-
`induced denaturation, it is clear that rhG—CSF[Cys3“""’ —>
`Ser36"‘2], rhG-CSF[Cys7‘ —> Ser“], and the trapped I1 and I2
`are conformationally less stable than the wild type rhG-CSF
`or rhG-CSF‘[Cys” —-> Ser"]. Interestingly, AGE“) determined
`for both Cys“-‘Z —> Ser3°"2 and Cys“ —> Set“ analogs are
`similar, suggesting that the two disulfide bonds may contrib-
`ute equally to the stabilization of rhG-CSF.
`Chemical labeling of Cys” is an excellent conformational
`probe for intermediates I1 and I2. The inaccessibility of Cys”
`by iodoacetate modification suggests that both intermediates
`may be partially folded near the NH2-terminal region of the
`G-CSF molecule and contain a somewhat native-like struc-
`ture. The predicted secondary structure of rhG-CSF revealed
`that an NH;-terminal 27 amino acid peptide segment, Gln“
`to Ala", has a high propensity to form an as-helical structure
`(16). The formation of this helical structure suggests that
`Cys” may be located at the end of the second helical turn and
`oriented in a hydrophobic environment, thus becoming un-
`available to chemical labeling. Moreover, during denaturation
`studies of rhG-CSF (Fig. 5), the DTNB reactivity of Cys" is
`coincident with the absorbance change at 290 nm, suggesting
`that the NH2-terminal helix (Gln“ to Ala”) may have been
`unfolded simultaneously upon denaturation of rhG-CSF.
`Like the free thiols in other proteins (25), the free Cys at
`position 17 in G-CSF may be prone to several undesired
`modifications such as formation of mispaired disulfide bond
`via thiol-disulfide exchange, covalent dimerization, and ag-
`gregation. These modifications can lead to structural changes
`and pose difficult stability problems (26). By site-directed
`mutagenesis, G-CSF variants lacking the reactive cysteine
`can be made to prevent the occurrence of these reactions. As
`described in this report, a Ser" variant was expressed in E.
`coli at high yield and purified after proper folding. This analog
`
`resembles the wild type rhG-CSF in overall secondary and
`tertiary structures? immunogenicity,‘ and retains full biolog-
`ical activity (Table 4). Preliminary studies“ show that the
`analog has increased stability, suggesting that it could also
`serve as a replacement therapeutic agent because of its added
`stability.
`In contrast to E. coli-derived rhG-CSF used in the present
`folding studies, natural G-CSF or rhG-CSF produced in mam-
`malian expression system is glycosylated. Glycosylated G-
`CSF contains a simple tri- or tetrasaccharide moiety (M, ~
`700) which is linked to Thr at position 133.5 Detailed carbo-
`hydrate structure of rhG-CSF glycoform has been determined
`by nuclear magnetic resonance spectrometric studies (27).
`Preliminary studies indicated that both glycosylated and non-
`glycosylated G-CSFs have similar biological activity and im-
`munological properties.‘ By GdnHCl denaturation/renatura—
`tion studies under non-reducing conditions, the glycoform of
`rhG-CSF exhibits the same conformational stability as the
`unglycosylated rhG—CSF.3 However,
`the oligosaccharide
`chains have been reported to influence the ability of proteins
`to fold properly (28). Further studies are thus required to
`reveal the effect of the carbohydrate moiety on the folding
`rate and folding pathway for the reduced and denatured rhG-
`CSF glycoform.
`
`Acknowledgments—We are indebted to Dr. Keith Langley, Depart-
`ment of Protein Chemistry, Amgen Inc., for his critical reading of the
`manuscript and to Jean Bennett for her help in typing the manu-
`script.
`
`REFERENCES
`
`
`
`9°?‘.°"$":".°-"
`
`10.
`
`11.
`
`1. Fhelis, C., and Yon, J. (1982) Protein Folding, Academic Press,
`New York
`2. Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51,
`459-489
`Creighton, T. E. (1986) Methods Enzymol. 131, 83-106
`Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293
`Creighton, T. E. (1977) J. Mol. Biol. 113, 295-312
`Creighton, T. E., and Goldenberg, D. P. (1984) J. Mol. Biol. 179,
`497-526
`Thornton, J. M. (1981) J. Mol. Biol. 151, 261-287
`Metcalf, D. (1984) The Hemopoietic Colony Stimulating Factors,
`Elsevier, Amsterdam
`9. Zsebo, K. M., Cohen, A. M., Murdock, D. C., Boone, T. C., Inoue,
`H., Chazin, V. R., Hines, D., and Souza, L. M. (1986) Immu-
`nobiology 172, 175-134
`Cohen, A. M., Zsebo, K. M., Inoue, H., Hines, D., Boone, T. C.,
`Chazin, V. R., Tsai, L., Ritch, T., and Souza, L. M. (1987) Proc.
`Natl. Acad. Sci. U. S. A. 84, 2484-2488
`Souza, L. M., Boone, T. C., Gabrilove, J., Lai, P. H., Zsebo, K.
`M., Murdock, D. C., Chazin, V. R., Bruszewski, J., Lu, H.,
`Chen, K. K., Barendt, J., Platzer, E., Moore, M. A. S., Mertels-
`mann, R., and Welte, K. (1986) Science 232, 61-65
`Tsuchiya, M., Asano, S., Kaziro, Y., and Nagata, S. (1986) Proc.
`Natl. Acad. Sci. U. S. A. 83, 7633-7637
`Bronchud, M. H., Scarffe, J. H., Thatcher, N., Crowther, D.,
`Souza, L. M., Alton, N. K., Testa, N. G., and Dexter, T. M.
`(1987) Br. J. Cancer 56, 809-813
`Morstyn, G., Souza, L. M., Keech, J., Sheridan, W., Campbell,
`L., Alton, N. K., Green, M., Metcalf, D., and Fox, R. (1988)
`Lancet 1, 667-672
`Gabrilove, J. L., Jakubowski, A., Scher, H., Sternberg, C., Wong,
`G., Grous, J ., Yagoda, A., Fain, K., Moore, M. A. S., Clarkson,
`B., Oettgen, H., Alton, N. K., Welte, K., and Souza, L. M.
`(1988) New Engl. J. Med. 318, 1414-1422
`Lu, H. S., Boone, T. C., Souza, L. M., and Lai, P.-H. (1989) Arch.
`Biochem. Biophys. 268, 81-92
`Souza, L. M. (March 7, 1989) U. S. Patent 4,810,643
`Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`18.
`
`3 L. Narhi and T. Arakawa, personal communication.
`“ H. Lu and D. Chang, personal communication.
`5 H. Lu, C. Clogston, and T. Boone, unpublished data.
`
`Page 5
`
`

`
`19. Greenfield, N., and Fasman, G. D. (1969) Biochemistry 8, 4108-
`4116
`20. Narhi, L. 0., Kenney, W. C., and Arakawa, T. (1991) J. Protein
`Chem. 10, 359-367
`21. Cecil, R., and McPhee, J. R. (1959) Adv. Protein Chem. 14, 255-
`389
`22. Takagi, T., and Isemura, T. (1963) Biochem. Biophys. Res. Com-
`murl. 13, 353-359
`23. Yutani, K., Yutani, A., Imanishi, A., and Isemura. T. (1968) J.
`Biochem. (Tokyo) 64, 449-455
`
`25.
`
`8775
`
`26.
`
`27.
`
`28.
`
`Oxidative folding of hG-CSF
`24.
`Saxena, P., and Wetlaufer, D. B. (1970) Biochemistry 9, 5015-
`5023
`Fernandez-Diez, M. J., Osuga, D. T., and Feeney, R. E. (1964)
`Arch. Biochem. Biophys. 107, 449458
`Manning, M. C., Patel, K., and Borchardt, R. T. (1989) Pharm.
`Res. 6, 903-918
`Oheda, M., Hase, S., Ono, M., and Ikenaka, T. (1988) J. Biochem.
`(Tokyo) 103, 544-546
`Berger, E. G., Buddeke, E., Kamerling, J. P., Kobata, A., Paulson,
`J. C., and Vliegenthart, J. F. G. (1982) Experientia 38, 1129-
`1162
`
`Supplementary Material to: FOLDING AND OXIDATION OF RECOMBINANT HUMAN
`GRANULOCYTE COLONY STIIIULATING FACTOR PRODUCED lN ESCHERICHIA COLI:
`CHARACTERIZATION OF THE DISULFIDE-REDUCED INTEHMEDIATES AND CV5-—>SEli
`ANALOGS. Hsieng 5. Lu‘. Christi L. clogston. Linda 0. Narhi, Leo Ann Merewether, Wayne Pean
`and Thomas 0. Boone. Amgan Inc., Amgen Center. Thousand 0316. CA 91320
`To determine the thermodynamic constants tor denaturatlon, a two-slate transition approximation was
`used and the tree energy change. AGD. was calculated tor the reaction. F (lolded) —i U (unfolded). at a
`given GdnHCI concentration using the equation,
`A .
`(1)
`AGD=-FlTlnA.AU
`where A is the observed absorbance at 290 rim, and AF and Au are the absortiances ol the lolded and
`.
`H
`.
`.
`unlolded states. respetnively. AGDZO was the tree energy for dsnaturation tn the absence oi the
`denaturant determined by a linear relation method (see equation 2).
`(2)
`AGO . Acg2° — m[GdnHC|]
`.
`in E .
`Samples obtained from denaturatron ot native rhG-CSF at ditteront GdnHC| concentrations were added
`to 50 ul ol treshly prepared DTNB (Sigma Chem. Co.) solution (4 mg/ml) in 0.1 M sodium phosphate. pH
`7.0. Alter incubation at 25°C tor 15 min, the absorbance of the mixture was measured at 412 nm. The
`SH concentration present in samples was then calculated using r: = 1.36 x 104 M-1cm"(18).
`
`To quantity biological activity or rhG-CSF test samples, an assay employing 3H-thymidine uptake in low
`density non-adherent mouse (lemale Balb C) bone marrow cells was used (9). Fully active mo-csF
`usually gives approximately 1.0 x 105 units/mg. Under a wall-calibrated condition. the assay ol various
`analogs was performed in triplicate with dillerent dilutions.
`S
`.
`.
`.
`P