THE JOURNAL OF BIGLOGICAL CHEMISTRY
`© 1992 by The American Society for Biochemistry and Molecular Biology,Inc.
`
`Vol. 267, No. 13, Issue of May 5, pp. 8770-8777, 1992
`Printed in U.S.A.
`
`Folding and Oxidation of Recombinant Human Granulocyte Colony
`Stimulating Factor Produced in Escherichia coli
`CHARACTERIZATION OF THE DISULFIDE-REDUCED INTERMEDIATES AND CYSTEINE — SERINE
`ANALOGS*
`
`Hsieng S. Lut, Christi L. Clogston, Linda O. Narhi, Lee Anne Merewether, WayneR. 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, I, and
`I,, were detected by kinetic studies using high perform-
`ance liquid chromatography.I, exists transiently and
`disappears quickly with the concomitant formation of
`I,. In contrast, I, requires a longer time to fold into the
`final oxidized form, N. CuSQ, catalysis increases the
`folding rate of I, from I,, while CuSO, and elevated
`temperature (37 °C) have a dramatic effect on the fold-
`ing rate of N from I,. These observations suggest the
`following sequential oxidative folding pathway.
`I, —Pk
`fast
`
`rate-limiting
`
`species, since they are usually short-lived. As described by
`Creighton and others (8-7), 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 bondis 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 duringfolding.
`Humangranulocyte 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 humanclinical studies to treat
`cancer patients suffering from chemotherapy-induced neutro-
`penia (13-15). E. coli-produced rhG-CSFis a 175-aminoacid
`polypeptide chain containing an extra Met (at position —1)
`at its NH, 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 aminoacids.
`Like other bacteria-derived recombinant proteins, rhG-CSF
`produced in E. coli requires an oxidative folding procedure in
`order to recoverits biological activity (17). In this paper, we
`describe the kinetic study of a folding and oxidation procedure
`for the reduced rhG-CSFsolubilized 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
`The mechanistic study of protein folding is important in
`also describe the biological and physicochemical characteriza-
`understanding the structure and function of proteins (1, 2).
`tion of intermediates and analogs made bysite-directed mu-
`Thedifficulty in elucidating a protein folding pathwaylies in
`tagenesis at the Cys residues.
`measuring the structural properties of intermediate protein
`
`
`Peptide map analysis of the iodoacetate-labeled in-
`termediates revealed that I, represents the fully re-
`duced granulocyte colony-stimulating factor contain-
`ing 5 free cysteines; I. 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
`I,, Iz, N, and several Cys > Ser analogs made bysite-
`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 Ser. Recombinant human granulocytecol-
`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
`lowerbiological activity. These studies show that di-
`sulfide bond formation is crucial for maintaining the
`molecule in a properly folded and biologically active
`form.
`
`* The costs of publication of this article were defrayed in part by
`the payment of page charges. This article must therefore be hereby
`marked “advertisement” in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`+ 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; GdnHCl, guanidine HCl; RP-HPLC, reverse-
`phase high performanceliquid chromatography; DTT,dithiothreitol;
`TFA,trifluoroacetic acid; DTNB,5,5’-dithiobis(nitrobenzoic acid).
`
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`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
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` Absorbanceat215nm(1.2AUFS)—»
`
`Retention Time (Minutes)
`
`Fic. 2. Folding of rhG-CSFat 25 °C in CuSO, monitored by
`RP-HPLCatdifferent times. Chromatograms 1-6, 20 min, 1, 2, 4,
`8, and 12 h, respectively. Approximately 50 ug of rhG-CSFin folding
`mixture was injected. Intermediates I; and I, 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 METHODS?
`
`RESULTS
`
`Folding Intermediates and Folding Kinetics of rhG-CSF—
`Fig. 1 shows the RP-HPLCelution 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-CSFin
`6 M GdnHClis 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 thefully
`denatured and reduced rhG-CSF. The difference in the chro-
`matographic elution times among these rhG-CSF forms has
`allowedus to detect folding intermediates and to study folding
`kinetics by RP-HPLC.
`Shownin 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 CuSQ,.
`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 I, as depicted
`in Fig. 2 (chromatogram 1) is the starting reduced rhG-CSF.
`At the 20-min incubation time,I, has already accumulated to
`a level of 33% of the total rhG-CSF. At 1, 2, and 4 h, the
`generation of I, has proceeded further with the concomitant
`disappearance of I, and appearanceofthe final oxidized form
`N (Fig. 2, chromatograms 2-4). The folding into form N
`reaches its maximum in 12h andis greater than 95% complete
`(Fig. 2, chromatogram 6).
`Theinitial first order folding rate for conversion of I, into
`I, was estimated to be 1.9 X 10°? s" andtheinitial rate for
`conversion of I; into the final oxidized rhG-CSF (form N) to
`be approximately 3.1 x 107? s“! (Table 1). The half-maximal
`conversion of I, to I, takes approximately 30 min, while the
`
`? 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.
`
`Oxidative folding of hG-CSF
`
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`half-maximal conversion of I, to N takes almost 4.5 h. Kinetic
`studies also indicate that the oxidative folding of form N from
`I, is biphasic. The folding rate from I, to N duringthefirst
`phaseis faster. The second phase of folding starts at approx-
`imately 8 h with a muchslowerrate.
`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 I, formation at both 25 and 37 °C
`are similar (1.0 X 10°? s“') andslightly slower than the folding
`of rhG-CSF in the presence of coppersulfate (1.9 x 107? s“').
`However, at 25 °C the generation of completely oxidized rhG-
`CSF(form N) is relatively slow (rate = 6.6 x 107* s~'). In this
`case,
`Ip persists much longer and approximately 20 h are
`required to reach half-maximal folding of form N from lL
`versus 4.5 h in the presence of copper sulfate at 25°C. At
`37 °C the folding of form N from I, without CuSO,is faster,
`but the biphasic kinetics becomes apparent. Thefirst 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 usinga sulfoethyl
`polyaspartamidesilica-based column (data not shown).
`To estimate the stoichiometry of labeling, the modification
`was also performed using [*H]C.-iodoacetic acid. Table 2 lists
`the labeling results for rhG-CSF andthe purified intermedi-
`ates. In the absence and presence of 6 M GdnHC]l,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 I, gives 3.74 mol of label and I, 1.9 mol of label/mol
`of protein. These results indicate that 4 and 2 free cysteinyl
`residues are available for the labeling in I, and I, respectively.
`To further characterize the structure of intermediates, the
`°H-labeled I, and I, 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 I, derivative. Peptide fractions
`were pooled and aliquots were analyzed for determination of
`peptide concentration, radioactivity counting, and NH.,-ter-
`minal sequence analysis. Table 3 summarizes the isolation
`and characterization of the labeled and unlabeled peptides
`derived from I, and I;. For form Iz, only peptides 7 and 8
`(Leu*’ 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
`I, (Table 2). For form I, Cys and Cys”, found in peptides 7
`and8, as well as Cys** and Cys* in peptide 2 (Lys* to Glu**)
`were radioactively labeled. This confirms that the 4 cysteines
`at positions 36, 42, 64, and 74 in IJ, are not involvedin disulfide
`bonding.
`As indicated in Table 3, peptide 4 is the NH.-terminal
`peptide of rhG-CSF containing Cys at position 17. Analysis
`of peptide 4 derived from both intermediatesI, and I,indicated
`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 bysite-directed mutagenesis of
`
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`Oxidative folding of hG-CSF
`
`the rhG-CSFgene 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-CSFand could be isolated
`with equivalent recovery. In contrast, rhG-CSF[Cys***? >
`Ser*®?] exhibited very slow folding in the absence of CuSO,
`(greater than 4 days) and was recovered in low yield. The
`[Cys"* —» Ser™] analog lacking the Cys®*-Cys” disulfide bond
`also folded moderately slowly but correctly in the absence of
`CuSO,
`(more rapidly but
`incorrectly in the presence of
`CuSO,), but recoveredin low yield.
`Fig. 4A showsthe elution of purified rhG-SCF[Cys” —>
`Ser™] from a C-4 reverse-phase HPLC column.It elutes 2.20
`min (chromatograms2 and 3 at pH 3 and 7, respectively) later
`than the wild type rhG-CSF (chromatogram 1). I, lacking the
`Cys®-Cys” 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***? — Ser***?} (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 hasanactivity
`of approximately 1.0 x 10° units/mg. Carboxymethylated
`intermediatesI, and I, have only approximately 3-5% activity
`of the wild type rhG-CSF. Both rhG-CSF[Cys**? — Ser**?}
`and rhG-CSF[Cys”™ > Ser”] 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.
`Conformationalstabilities of rhG-CSF intermediates and
`analogs were compared by denaturation with GdnHCl at pH
`7.2. Fig. 54 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 aminoacids 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 appearsto stay in the native state below
`2 M GdnHCl concentration, denatures quickly above 2.5 M
`denaturant, and is completely unfolded at 3.6 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 DTNBreaction. The increased acces-
`sibility of Cys’’ is coincident with the denaturation of rhG-
`CSF detected by the change in absorbance at 290 nm.
`As shownin Fig. 6, similar GdnHCl denaturation studies
`were also performed on L, I, rhG-CSF[Cys™ — Ser], and
`rhG-CSF[Cys*** — Ser****], 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 Keal/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[Cys™ — Ser] analog, the rhG-CSF[Cys**” >
`Ser***?] analog, and the folding intermediates at pH 7.5. rhG-
`CSFis rich in a-helix, as evidenced by the minima at 222 and
`
`208 nm.All of the molecules tested exhibit a-helical structure,
`but the native molecule and the rhG-CSF[Cys**** > Ser*®*?]
`molecule contain substantially higher helical content than the
`other species examined,
`i.e. 64% helix versus 38% helix at
`neutral pH, calculated using the Greenfield-Fasman equation
`(19). The native and rhG-CSF[Cys** — Ser**’] are the only
`species that show an increasein helicity (up to 75%) at pH
`3.5 (Fig. 7B). The far UV CD spectra of J, and I, with or
`without iodoacetate modification are identical.
`Asseen 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,althoughthe intensities
`are greater, indicating that the Trp fluorescence might be
`somewhat less quenched by the surrounding environment.
`The spectra of I, and I, differ slightly from those of the
`respective labeled derivatives; the peaks are broader, again
`reflecting a difference in the environmentof 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 changein the Tyr fluorescence. The spectrum of the rhG-
`CSF[Cys*°*? — 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” —> Ser“analog, and Cys***? — Ser®*?
`analog as determinedby gelfiltration. 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 increasein flexibility.
`
`DISCUSSION
`
`Thepresent study demonstrates that the folding of reduced
`rhG-CSF proceeds through identifiable intermediates I, and
`I, (Fig. 2). The originally fully reduced (I,), partially oxidized
`(Iz), and the final oxidized (N) forms can be separated and
`quantitated by HPLCfor 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 uM (17). The kinetic data
`show that copperion increasesthe folding rate of intermediate
`I, from J, approximately 2-fold and increases the folding rate
`of N from I, 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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`
` 320
` 320
`
`
`
`FluorescenceIntensity(ArbitraryUnits)
`
`FluorescenceIntensity(ArbitraryUnits)
`
`Fic. 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[Cys™ — Ser”); spectrum 3, rhG-
`CSF[Cys**? — Ser®*“?]; spectrum 4, in-
`termediate I, derivative; spectrum 5, re-
`duced rhG-CSF (or intermediate I, de-
`rivative).
`
`B
`
`300
`
`340
`
`360
`
`380
`
`400
`
`420>
`
`300
`
`340
`
`am
`
`360
`
`380
`
`400
`
`420>
`
`Emission Wavelength
`
`suggest that the mechanistic pathway for rhG-CSF folding
`under the described conditions is sequential: I, > I, — N.
`Thefolding from I; to I, is fast, while the folding from I, to
`N is rate-limiting (Table 1).
`I, and I, were the two predominant intermediates observed
`during folding and no otherspecies containing intermolecular
`disulfide forms were detected. The absence of such species
`was supported by other experimental evidence. For example,
`asmall amount (<5%) of interdisulfide-linked rhG-CSF dimer
`or aggregate formsis 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.
`
`Forstructural characterization, I, and I, 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[Cys*" —> Ser®**?] and rhG-
`CSF[Cys"* —» 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-CSFpurification procedures.
`In the folding studies, the Cys***? — Ser***? analog under-
`goes a sequential oxidation and folding pathway similar to
`that observed for the wild type rhG-CSF going from I, (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 Ser***? analogis
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`Oxidative folding of hG-CSF
`
`slower than the wild type G-CSF, the formation of Cys**-
`Cys‘ disulfide in the folding of wild type G-CSF seemsto be
`important for providing the initial stabilization force for the
`conformational integrity of the molecule, and for allowing
`moderately rapid formation of the Cys®-Cys™ bond. More-
`over, it is interesting to note that the far UV CD spectra of
`rhG-CSF[Cys***” — Ser®®?] at neutral and acidic pH values
`resemble those obtained for native rhG-CSF (>60% helix),
`suggesting that formation of the rhG-CSFsecondary structure
`is independent of formation of the Cys**-Cys*? disulfide bond.
`The rhG-CSF(Cys™% — Ser™] analog and I, that do not
`contain the Cys“-Cys™ bond have some commonproperties.
`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[Cys™ — Ser] is only half that of the native rhG-
`CSF,andis similar to that of the isolated I, (~35% a-helix),
`it appears that both I, and rhG-CSF[Cys™ — Ser™] contain
`partial secondary (or helical) structure. The low a-helical
`content observed for I, and rhG-CSF[Cys” — Ser”) 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 absenceofdisulfide bonds on the
`tertiary structural folding and conformational stability of
`rhG-CSF were studied by fluorescence spectrometry and
`GdnHC!idenaturation. Under acidic pH the wild type rhG-
`CSFexhibits 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” 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[Cys**”? =>
`Ser***?], rhG-CSF[Cys’ — Ser], and the trapped I, and I,
`are conformationally less stable than the wild type rhG-CSF
`or rhG-CSF[Cys’’ — Ser’’]. Interestingly, AGE2° determined
`for both Cys**“? — Ser*** and Cys” — Ser” 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 I, and I,. The inaccessibility of Cys’?
`by iodoacetate modification suggests that both intermediates
`maybepartially 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 a-helical structure
`(16). The formation of this helical structure suggests that
`Cys*’ may belocated 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 DTNBreactivity 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 madeto 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 retainsfull 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.° 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.° 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.
`
`Acknowledgmenis—Weare indebted to Dr. Keith Langley, Depart-
`ment of Protein Chemistry, Amgen Inc., for his critical reading of the
`manuscript and to Joan Bennett for her help in typing the manu-
`script.
`
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`Page 5
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`

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`
`MATERIALS ANO METHOOS:
` Materiais
`E. coli-produced rhG-CSF was purified according to methods described previously (11). Measurement
`of biciagical activity was periormed by a standardized jn yitro mouse bone marrow assay(9) (see
`“G-CSF biological assay”). Purity was assessed by sodium dodecyl sulfate-polyacrylamide gal electro-
`phoresis (SDS-PAGE)and reverse-phase HPLC. The protein is monomeric as juciged by getfiltration.
`The extinction coefficiant at 280 nm in 20 MM sodium acetate (pH 4.0 - 7.0) is 16,400 M-1 cmr1.
`Sarkosyl (laurylsarkosine) was purchased from J.T. Baker and DTT from Calbiochem. GdnHC!in
`solution (8 M) was obtained trom Pierce Chemical Co.
`lodoacetate was trom Sigma Chemical Co. and
`was recrystallized twice in petroleum etherpriorto use. [t]-C2-iodaacetate was obtained from New
`England Nuclear. HPLC solvents and water were purchased from Burdick and Jackson.
`aureus protease (V-8 strain} was from Miles. All of the sequencing reagents and
`solvents were fram Applied Biosystems (Foster City, CA).
`
`Supplementary Material to: FOLDING AND OXIDATION OF RECOMBINANT RUMAN
`GRANULOCYTE COLONY STIMULATING FACTOR PRODUCED IN ESCHERICHIA COLI:
`CHARACTERIZATION OF THE DISULFIDE-REDUCED INTERMEDIATES AND CYS~4SER
`ANALOGS. Hsieng S. Lu*, Christi L. Clogston, Linda O. Narhi, Lee Ann Merewather, Wayne Pearl
`and Thomas C. Boone, AmgenInc., Amgen Center, Thousand Oaks, CA 91320
`To determine the thermodynamic constants for denaturation, a two-state transition approximation was
`used and the free energy change. 4Gp, was calculated for the reaction, F (folded) — U (unfolded), at a
`givan GdnHCl concentration using the equation,
`Ag
`-A
`(1)
`AG, = -ATIng . Ay
`where A is the observed absorbanceat 290 nm, and AF and AU are the absorbancesof the tolded and
`unfolded states, respectively. aap? was the free energy for denaturation in the absence of the
`denaturant determined bya linear relation method (see equation 2).
`(2)
`AGp = AGH?- miGdnHi