ARTICLES: FORMULATION ANDENGINEERING OF BIOMATERIALS

Different Effects of L-Arginine on Protein Refolding: Suppressing Aggregates of

Hydrophobic Interaction, Not Covalent Binding

Jing Chen, Yongdong Liu, Yinjue Wang, Hong Ding, and Zhiguo SuNational Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences,Beijing 100190, China

DOI 10.1021/bp.93Published online November 24, 2008 in Wiley InterScience (www.interscience.wiley.com).

Arginine is one of the most favorable additives in protein refolding. However, argininedoes not work for certain disulfide-bond-containing proteins, which is not yet well explained.In this work, refolding of three proteins in the presence of 0–2 M arginine was investigatedand compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was success-fully refolded with the help of arginine. The refolding yield could reach almost 100% in thepresence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG-CSF), containing five cysteines, could only achieve 65% refolding yield. The formation ofaggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacet-amide and subsequently refolding of the protein could reduce the aggregate formation sub-stantially. Further investigation on recombinant green fluorescence protein (GFP),containing two cysteines, also revealed the accumulation of oligomers. The content ofoligomers increased with the concentration of arginine, reaching about 30% at 2 M argi-nine. Comparison of reduced and nonreduced SDS-PAGE revealed that the oligomers wereformed through intermolecular disulfide binding. Analysis of the refolding kinetics indicatedthat intermolecular disulfide bonds were probably formed in the intermediate stage wherearginine slowed down the refolding rate and stabilized the intermediates. The accumulatedintermediates with unpaired cysteine possessed more chances to react with each other toform oligomers, whereas arginine failed to inhibit disulfide bond formation.Keywords: refolding, arginine, GFP, CAB, rhG-CSF, aggregation

Introduction

The formation of protein aggregates and misfolded speciescould reduce the recovery of recombinant pharmaceuticalssubstantially. A typical process is the refolding of an inclu-sion body protein where uncontrollable aggregates and unde-sirable misfolded proteins often occur and compete with thecorrect folding structures.1–4 It has been believed that non-specific hydrophobic interaction between folding intermedi-ates plays a crucial role in formation of aggregation. But forthose proteins containing disulfides, formation of intermolec-ular disulfide bonds is another important reason to formaggregates. Because denature-reduced proteins contain freecysteine residues, irreversible mismatching may occur onceproteins are transferred into the oxidative condition. Thus,unproductive aggregates, originated both from nonspecifichydrophobic interaction and incorrect covalent binding oftwo thiol groups, decreased renaturation yields.

How to minimize aggregation and increase refolding yield

is always the chief target of protein refolding research. It has

been found that the balance between aggregates and correct

refolding of proteins could be modulated by various environ-

mental factors, especially by the addition of small molecule

additives. Some of these additives, such as glycerol,5,6 sugar,7

and heparin,8 were called ‘‘folding enhancers’’ because they

may increase the solubility and stability of proteins in the

native states. While some other additives called ‘‘aggregate

inhibitors,’’ such as polyethylene glycol,9 urea,10,11 L-argi-

nine,12 and detergent,13,14 may prevent association of refold-

ing intermediates or unfolded species to suppress aggregation.

Among various folding additives, the amino acid of argi-nine is a popular one because it exhibited excellent effectson protein association, e.g., solubilization of loose inclusionbodies,15 elution of antibodies from protein-A affinity chro-matography,16 and decreasing nonspecific binding of proteinson gel permeation chromatography.17 Because of the effec-tive function of inhibiting protein–protein combination, argi-nine has been universally applied to assist protein refolding,

Correspondence concerning this article should be addressed to Z. Suat zgsu@home.ipe.ac.cn.

Biotechnol. Prog. 2008, 24, 1365�1372 1365

VVC 2008 American Institute of Chemical Engineers

and which is independent on the size and isoelectric point oftarget proteins. Arginine has proved effective in improvingrefolding yields of many proteins, e.g., lysozyme,18,19 caseinkinase II,20 recombinant human neurotrophins,21 interleukin-21,22 immunotoxins,23 recombinant Fab-fragments,24 andhuman plasminogen activator.25 Intriguingly, unlike guani-dine hydrochloride which also contains a guanidine group,arginine shows a more complex pattern of interactions withthe proteins.26 The mechanisms of arginine action on pro-teins, which are highlighted in the last 20 years, are still notunderstood. Three possible mechanisms have been proposed:(1) Arginine may interact favorably with a majority of aminoacid side chains so as to reduce hydrogen bonding, ionicinteractions, and hydrophobic interactions between the pro-teins27; (2) Possibly, arginine selectively increases the freeenergy of protein–protein encounter complexes, therefore,slows down protein association reactions with little effect onthe refolding rate of proteins28,29; and (3) Arginine is likelyto increase the stability and solubility of denatured proteinsand partly folded polypeptides to prevent them from goingdown the path to aggregation.30

However, certain proteins are not fully (or correctly)folded in the presence of arginine even though it appearsthat arginine maintains their solubility. Moreover, Yanceyet al.31 showed that the activity and stability of certainenzymes were perturbed by arginine and concluded that argi-nine is a protein destabilizer which limits the expansion ofits applications. In the previous study of the arginine-assistedrefolding of recombinant consensus interferon (rIFN-con1),we proposed that arginine minimized the formation of pro-tein precipitation, but not the soluble oligomeric species.32

In this work, the mechanisms of arginine action, especiallythe effects of different concentrations of arginine on protein-protein hydrophobic interaction and covalent binding, wereinvestigated using three proteins with different structures. Asresults showed, the concentration of arginine significantlyinfluenced the refolding yield of all the three proteins, butthe effects varied from protein to protein. Our findings mayshed some light on the understanding of arginine-assistedprotein refolding process and provide guidance for thescreening of proper refolding conditions for different pro-teins in vitro.

Materials and Methods

Materials

A recombinant E. coli of green fluorescent protein (GFP)was kindly provided by the Institute of Microbiology, Chi-nese Academy of Sciences. Cloning, expression, and purifi-cation of GFP were carried out as previously described.33

The final preparation was homogeneous on SDS-PAGE. Bo-vine carbonic anhydrase B (CAB) and guanidine chloridewere obtained from Sigma Chemical Co. (St. Louis, MO).Arginine, 2-mercaptoethanol (b-ME), and iodoacetamidewere purchased from Fluka Chemical Corps (Buchs, Switzer-land). All other chemicals were all analytical grades and pur-chased from Shanghai Reagent Company (Shanghai, China).

Expression, isolation, and purification ofinclusion bodies of rhG-CSF

As the source of rhG-CSF, we have used an E. coli strain(DH5a) transformed with an expression plasmid pBV220 forthe human G-CSF gene. In this system, the expression of

rhG-CSF is driven by a strong tightly regulated leftward pro-moter of bacteriophage lambda (PL) which can be thermo-regulated due to the presence of a heat labile cI857repressor. At a low temperature (29–31�C), the cI857repressor maintains the promoter in a repressed state,whereas at 42�C the repressor activity is destroyed, therebyallowing high level expression of the human G-CSF gene.

The cells were grown in 5 L of LB medium containing5 g/L yeast extract, 10 g/L bactopeptone, 5 g/L sodium chlo-ride, and 100 lg/mL ampicillin at 30�C. When the OD600nm

reached 4.0 temperature was increased to 42�C to induceprotein expression. The cells were harvested by centrifuga-tion at 2,800g at 4�C for 15 min after induction for 4 h. Thebacterial pellet was resuspended in 50 mM Tris-HCl (pH8.5) containing 5 mM EDTA and 0.2 mg/mL lysozyme.Cells were then lysed by sonication at 150 kHz, using VC-600-2 sonicator (Sonic & Materials) with a 13 mm probe inan ice-water mixture bath for 5 min (5 s pulse with 5 s inter-vals) which was repeated six times and the inclusion bodieswere collected by centrifugation with 20,000g 20 min at4�C. Then rhG-CSF inclusion bodies were washed by20 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 1 MNaCl, 1% Triton X-100, and 2 M urea for three times. Tengrams inclusion bodies were obtained with a purity of morethan 95% upon SDS-PAGE analysis.

Denaturation/reduction

The purified GFP, CAB, and inclusion bodies of rhG-CSFwere denatured and reduced in 50 mM Tris-HCl (pH 8.5)containing 6 M guanidine chloride, 140 mM b-ME, and1 mM EDTA and incubated for 10 h at room temperature.Insoluble material was removed by centrifugation at 16,000gfor 20 min. The protein concentration was adjusted to20 mg/mL with denaturant.

Renaturation/oxidation

Refolding was initiated by rapid 100-fold dilution of thedenatured proteins into refolding buffer of 50 mM Tris-HCl(pH 8.5) containing 1 mM EDTA and various concentrationof arginine with continuous stirring. And then the solutionwas incubated for 48 h at 4�C without any agitation. Aggre-gation was measured on the turbidity represented by absorb-ance at 340 nm, and then the refolding solution wascentrifuged at 16,000g for 20 min at 4�C and the supernatantwas subjected to the following analysis.

Fluorescence spectra measurement

The formation of fluorophore during the oxidative refold-ing of GFP was monitored by measuring the fluorescencedensity in a Hitachi F-4500 fluorescence spectrophotometer.For the determination of GFP refolding yield, the refoldedsamples were 10-fold diluted before subjected to the analy-sis. The excitation wavelength was 475 nm with emissionwavelength at 509 nm.

For the kinetics analysis of GFP refolding, the denaturedprotein was diluted into different concentration of argininebuffer at the final protein concentration of 10 lg/mL and theexcitation wavelength was 475 nm with emission wavelengthstaying at 509 nm.

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Size-exclusion chromatography analysis

The native and refolded samples of GFP and CAB wereanalyzed by SEC on an AKTA Explorer 10 system (GEHealthcare, Uppsala, Sweden) equipped with a Superdex 75column (300 � 10 mm ID, GE Healthcare). The mobile-phase was 50 mM Tris-HCl (pH 8.5) containing 2 M ureaand 0.15 M NaCl at a flow rate of 0.5 mL/min. One hundredmicrolitre of sample was loaded on the column, and proteinpeaks were detected by UV absorbance at 280 nm.

Aggregated G-CSF was dissolved in 50 mM Tris-HCl (pH8.5) containing 8 M urea with or without 1% (v/v) b-ME.The mobile-phase was 50 mM Tris-HCl (pH 8.5) containing8 M urea with or without 0.1% (v/v) b-ME at a flow rate of0.5 mL/min. The rest experiments were the same as theabove mentioned conditions. All SEC analysis was run atambient temperature.

Iodoacetamide-modified rhG-CSF

Denatured-reduced rhG-CSF was prepared as describedabove at a protein concentration of 20 mg/mL. Iodoacet-amide-modified rhG-CSF was produced by diluting dena-tured-reduced rhG-CSF into 50 mM Tris-HCl (pH 8.5),containing 6 M guanidine chloride, 1 mM EDTA, and200 mM iodoacetamide (presolubilized in 1 M NaOH) andthe reaction mixture was incubated at room temperature for30 min. Then one milliliter samples was subjected to theSephadex G-25 column (130 � 10 mm ID, GE Healthcare)equilibrated with 50 mM Tris-HCl (pH 8.5), containing 6 Mguanidine chloride and 1 mM EDTA at a flow rate of 1 mL/min to recover the indoacetamide- modified rhG-CSF.

CAB activity assay

The esterase activity of the native and refolded enzymewas measured by recording the initial rate of increase in ab-sorbance at 348 nm due to the hydrolysis of p-nitrophenyla-cetate.34 Activity recovery was expressed in percentage oftotal activity of the refolded CAB in the presence of differ-ent concentration of arginine with the same concentration ofnative CAB as the original denatured sample in the presenceof the same buffer.

Protein concentration determination

Protein concentration was determined by Bradford methodusing bovine serum albumin as standard protein.35 Arginineinterfered in the measurement, so the refolded samples mustbe desalted on the Sephadex G-25 column equilibrated with50 mM Tris-HCl, pH 8.5 to remove the arginine beforemeasurement.

SDS-PAGE

SDS-PAGE analysis was performed using 15% gel asdescribed by Laemmli.36 Protein bands were developed bysilver staining. There is no 2-mercaptoethanol in samplebuffer of nonreduced SDS-PAGE.

Kinetic model

The kinetic competition for a monomeric protein can bedescribed with the simplified model depicted by Kiefhaberet al.4 and Hevehan and De Bernardez37:

U�!k1 I�!k2 N

#k3A

This model indicates that unfolded protein (U) undergoes atransient intermediate (I) with native-like secondary structureto form native state protein (N). The process from U to I canbe taken to be an instantaneous reaction because it is a rapidprocess with a time of milliseconds. Thus, this model can befurther simplified to a parallel reaction of the formations ofthe native protein and aggregates (A) from the transient in-termediate (I). Using this model, the yield (y) of native pro-tein with time can be described with the following equation:

y ¼ wftan�1ðð1þ w2Þ expð2k2tÞ � 1Þ1=2 � tan�1 wg (1)

Where

w ¼ ðk2=k3U02Þ1=2 (2)

The final renaturation yield can be obtained from Eq. 1 astime approaches infinity:

y ¼ wfp=2� tan�1 wg (3)

where U0 represents the initial unfolded protein concentra-tion, t the refolding time, k2 and k3 the folding and aggrega-tion kinetic constants, respectively. The larger the k value,the faster the renaturation or aggregation process.

Results and Discussion

Effects of L-arginine on renaturation of carbonicanhydrase B (CAB)

Hydrophobic interaction and covalent binding are twomain reasons to produce aggregates in the process of proteinrefolding. Arakawa and Tsumoto29 reported that the effect ofarginine on refolding was to suppress aggregation, but howdid arginine exert this effect and was this effect fit for bothabovementioned reasons? To address these questions, wefirst choose a model protein CAB, absence of cysteines inthe protein molecule, to investigate arginine’s function ofweakening hydrophobic interaction. Figure 1 showed theeffects of different concentrations of arginine on the mass re-covery and activity recovery for CAB refolding. The massrecovery of soluble protein evidently increased in the pres-ence of arginine, almost reaching 100% in the solution con-taining above 0.75 M arginine. However, the activityrecovery decreased dramatically in high concentrations of ar-ginine, especially above 1 M. To investigate whether thedecrease of activity was produced by formation aggregates,refolded CAB was subjected to SEC analysis which revealedthat the retention time of CAB was shorter when refolded inabove 1 M arginine buffer (Figure 2). An elution peak of bo-vine serum albumin (BSA) was shown for comparison. Themolecular weight of BSA is about two times of CAB. Ifthere was a dimmer of CAB, the peak should be close tothat of BSA. However, the peak distance between CAB and

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BSA is quite large, suggesting that the moving peak was notaggregate, but perhaps a partially unfolded state.

Results mentioned above indicated that ariginine couldeffectively suppress hydrophobic aggregation, both insolubleand soluble states. But when come down to the activityyield, the concentration of arginine became the key factor.Ejima et al.17 reported a requirement for arginine at highconcentrations when arginine was used as an additive in gelpermeation chromatography; however, for aminoacylase, ar-ginine was as good a denaturant as the guanidine chloride orurea.38 Here, we showed that high concentration of argininedose disturb the recovery of proper conformation in therefolding of proteins. So, arginine could be a well aggrega-tion inhibitor to those cysteine-absence proteins. However,

concentration of arginine should be carefully examined toinsure getting the highest activity recovery.

Effects of L-arginine on renaturation of recombinanthuman colony stimulating factor (rhG-CSF)

To examine whether arginine had the same effect on cova-lent binding as on hydrophobic interaction, recombinanthuman G-CSF, which was prone to form disulfide cross-linked aggregates even in native state,39 was selected as amodel protein. RhG-CSF was an important protein medicine,containing two native disulfide bonds at positions 36–42,64–74, and one free cysteine thiol at position 17. As opposedto CAB, the precipitates were visible as soon as denaturedrhG-CSF was diluted into arginine containing buffer, with anonly 65% mass recovery even in 1 M arginine buffer (datanot shown).

Deduced to be produced by disulfide cross-linked process,the precipitates were first resolubilized in denaturant withoutany reductant. As supposed, precipitates could not be totallydissolved. Moreover, the portion dissolved in the denaturantwas also not homogeneous after analyzed by SEC in Figure3. Subsequently, 1% b-ME were added, and after a while theremained precipitates was completely dissolved and only onemonomer peak was observed. All these results clearly indi-cated that the undissolved portion remained after urea treat-ment, as well as oligomers, were the aggregates produced bycovalent binding.

To ensure the inefficient role of arginine in suppressingcovalent binding, the all five cysteines of denature-reducedrhG-CSF were blocked by alkylation with iodoacetamide toavoid disulfide cross-linked action. Both the unmodified andblocked rhG-CSF were diluted into different concentrationsof arginine solution, and then the absorbance at 340 nm wasmeasured to examine the formation of precipitates (Figure4). In the absence of arginine, blocked rhG-CSF producedmore precipitates, which might because the structure of rhG-CSF altered after alkylation to easily associate. However, in0.5 M arginine, the absorbance at 340 nm was about 0.4 forunmodified type rhG-CSF and decreased to 0.1 for iodoacet-amide-modified form. That was, the precipitates of blockedrhG-CSF could be more effectively diminished by arginine

Figure 2. SEC analysis of refolded CAB in different concen-trations of arginine.

One-hundred-microliter sample was injected to a Superdex 75column (300 � 10 mm ID) equilibrated in 50 mM Tris, pH 8.5containing 2 M urea and 0.15 M NaCl and eluted at 0.5 mL/min on AKTA Explorer 10 system. BSA was employed as amolecular weight reference and analyzed at the same condition.

Figure 1. Effect of arginine on the refolding yields of CAB.

Denatured CAB was rapid diluted (1:100) into the refoldingbuffer containing different concentrations of arginine andexamined the activity after 24 h at 4�C as described in the text.The final protein concentration was 0.2 mg/mL.

Figure 3. SEC analysis of precipitates produced in the processof rhG-CSF refolding.

The precipitates were resolubilized in the denaturant of 8 Murea with or without 1% b-ME and centrifuged.

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than unmodified type. This result strengthened the role of ar-ginine as an inhibitor for hydrophobic interaction, and at thesame time revealed arginine was of no effect on covalentbinding.

Effects of L-arginine on renaturation of green fluorescenceprotein (GFP)

Unlike rhG-CSF, most of disulfide-bond-containing pro-teins kept high mass recovery in arginine-assisted refoldingprocess. However, the soluble ingredients were inhomogene-ous for some proteins, such as rIFN-con1,32 which containedquite a few soluble oligomers in refolding supernatant. Greenfluorescent protein was selected as a target protein to investi-gate the effects of arginine on these soluble aggregatesbecause GFP has some outstanding characters, includingrapid refolding process, available activity assay and only one

disulfide bond which could eliminate mismatching of intra-molecular disulfide bonds.

GFP can fluoresce green when exposed to blue light.40 Forwild type GFP, the major excitation wavelength is 475 nmwith the emission peak staying at 509 nm. GFP quenched itsfluorescence when exposed in denaturant. Therefore, the flu-orescence density can be directly used to detect the contentof proper structures in refolded GFP. Figure 5 showed themass recovery and fluorescence density at 509 nm of GFPrefolded in 0–2 M arginine solution. As shown, argininecould increase mass recovery of GFP refolding efficiently. Inthe absence of arginine, the mass recovery was only 28%,which increased to 81% even in 0.25 M arginine buffer; fur-thermore, the mass recovery almost reached 100% in above 0.5M arginine buffer. However, fluorescence density at 509 nm ofGFP dramatically decreased in higher arginine buffer.

To probe the reason why high concentrations of argininedecreased activity yield of GFP, size exclusion chromatogra-phy (SEC) analysis was employed to examine the structureof soluble refolded GFP. As shown in Figure 6, in the ab-sence of arginine the supernatant only presented one peak;on the other hand, diversiform peaks emerged once argininewas added. Comparing the elution volumes of these peakswith that of native GFP, P3 was judged to be the monomerpeak, whereas the other two peaks (P1 and P2) were deducedto be oligomers. Figure 7 depicted the mass recovery of vari-ous refolding species. We found the optimum concentrationof arginine for GFP refolding is 0.5 M at which monomerrecovery reached maximum about 85%. And the content ofprecipitates decreased rapidly until almost disappeared above0.5 M. However the content of the oligomer peaks (P1 andP2) was 10% at 0.25 M arginine and promoted to 30% at2 M arginine. This phenomenon indicated that there are stillself-interactions of protein in the presence of L-arginine,similar as arginine-assisted refolding of rIFN-con1.32 Todeeply explore the interaction of the oligomers formed in therefolding process, oligomer peaks and monomer peaks werecollected respectively, and subsequently analyzed by reducedand nonreduced SDS-PAGE (Figure 8). Although all thesamples showed one band in reduced SDS-PAGE, bands ofP1 and P2 appeared significant mobility retardation on non-reduced SDS-PAGE, confirming that these oligomers wereformed with intermolecular disulfide bonds. The P2 peak,with the 2-fold molecular weight of P3, was judged to be adimmer, while P1 peak was a mixture of several oligomers.Data so far illuminated that soluble aggregation produced by

Figure 5. Influence of 0–2 M arginine on the fluorescence andmass recovery of refolded GFP.

The final protein concentration was 0.2 mg/mL. The excitationwavelength was 475 nm with emission wavelength staying at509 nm.

Figure 4. Effect of arginine on the precipitates of the refoldingof the unmodified rhG-CSF and the iodoacetamide-modified rhG-CSF.

Both denatured forms of rhG-CSF were rapid diluted (1:100)into the refolding buffer containing different concentrations ofarginine as described in the text. The final protein concentra-tion was 0.2 mg/mL.

Figure 6. SEC analysis of refolded GFP in different concentra-tions of arginine.

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covalent binding could not be eliminated, and even accumu-lated in high concentrations of arginine.

Effects of arginine on refolding kinetics constants

To go deep into the reason why soluble oligomers couldaccumulate in high concentrations of arginine in some disul-fide-bond-containing proteins refolding, the kinetics of GFPrefolding process were investigated. Final refolding concen-tration of GFP was restricted to 10 lg/mL to avoid precipi-tates. As observed in Figure 9, the fluorophore of GFP couldbe recovered in a short time. Without arginine added, fluo-rescence density reached to equilibrium within 5 min andlow concentration of arginine (�0.5 M) altered this equilib-rium just a little. However, the higher concentration of argi-nine was added, the longer time was needed to reachbalance.

The renaturation yields of GFP refolded in above-men-tioned conditions were calculated and used as the dynamicexperimental data. The renaturation yield was based on theratio of fluorescence density of refolded GFP to that of

native GFP at the same concentration. Then the model equa-tion (Eq. 1) was fitted to the experimental data by the ‘‘non-linear curve fit’’ imbed in the origin 75 to estimate thevalues of k2 and k3. As shown in Figure 10, the folding ki-netic constant k2 reached the maximum at 0.5 M arginine.Further increase in arginine concentration until 2 M resultedin the rapid decrease of the folding kinetic constant. On thecontrary, a clear minimum value of aggregation kinetic con-stant k3 at 0.5 M arginine concentration was found. W, whichfor a constant protein concentration was proportional to thesquare root of the ratio of k2/k3 (Eq. 2), decreased as the ar-ginine concentration above 0.5 M. The final outcome was adecrease in the final renaturation yield, according to Eq. 3.These results illuminated that high concentrations of argininecould slow down the refolding rate and thereby increase thestability of refolding intermediates. For arginine could notinhibit covalent binding, the refolding intermediates withunpaired cysteines possessed more chances to associate witheach other to form aggregates.

Figure 8. Reduced and nonreduced SDS-PAGE analysis of col-lected samples eluted from Superdex 75 column.

Oligomer peaks (P1 and P2) and monomer peaks (P3) of sam-ple refolded in 1 M arginine were collected from Superdex 75column and analyzed by reduced (A) and nonreduced SDS-PAGE (B).

Figure 7. Effect of arginine on the contents of monomer,oligomer species, and precipitates.

The contents were based on the mass recovery of GFP refoldedin 0–2 M arginine and relative peak area eluted from Superdex75 column.

Figure 9. Kinetics of GFP refolding process measured byfluorescence.

The excitation wavelength was 475 nm with emission wave-length at 509 nm. Arginine concentrations were as follows: 0.5M, 0 M, 1 M, 1.5 M, 2 M (top to bottom).

Figure 10. Effect of arginine concentration on the kinetics con-stants of GFP.

The activity recovery was based on the ratio of fluorescencedensity of refolded GFP to that of native GFP at the sameconcentration. U was defined by Eq. 2.

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Conclusions

To address the mechanism of arginine-assisted proteinrefolding and the reason why arginine does not work for cer-tain disulfide-bond-containing proteins, three proteins, CAB,rhG-CSF, and GFP, were selected as models to investigatethe roles of arginine in suppressing aggregation. We foundthat arginine could suppress aggregates produced by hydro-phobic interaction, not covalent binding, and even indirectlypromoted intermolecular disulfide bonds mismatching.

The refolding yield of CAB reached almost 100% inabove 0.75 M arginine and no oligomers were found in solu-ble proteins. For the aggregates of refolded CAB purelycaused by hydrophobic interaction, the role of arginine inCAB refolding exhibited that arginine could effectively sup-press hydrophobic aggregation. However, arginine was of lit-tle effect on suppressing precipitates of rhG-CSF. Muchcovalent aggregates were found in the precipitates ofrefolded rhG-CSF even in 1 M arginine. But when all cys-teines in rhG-CSF were blocked by iodoacetamide-modifiedprocess, the mass recovery of blocked rhG-CSF was muchincreased by adding arginine. This result powerfully illumi-nated that arginine could not suppress the precipitation pro-duced by covalent binding. Meanwhile, arginine could noteliminate, but to some extent, increased the formation ofsoluble covalent aggregates. Although the precipitation ofGFP could be eliminated by arginine, the contents of cova-lent oligomers were increased to 30% in 2 M arginine. Thiswas because more folding intermediates were left in solutionafter precipitation was inhibited. Furthermore, high concen-trations of arginine could decrease the refolding rate whichprolonged the retention time of free-sulphydryl-containingintermediates. All above-mentioned reasons lead to more op-portunity for the mismatching of disulfide bonds.

These results will be informative for selecting properrefolding conditions when using arginine as a refolding addi-tive. When arginine was used as cosolvent in refolding reac-tions, the concentrations of arginine, combined with themolecular configuration of target protein, turned into the pri-mary factors to investigate. For those proteins without sul-phydryl, arginine is the most favorable cosolvent; however,arginine might abate its effectiveness to some disulfide-bond-containing proteins, and even lose its action to thoseproteins containing free sulphydryl or tending to form disul-fide cross-linked aggregates. But cooperative application ofarginine with other folding additives, such as redox system,folding enhancers and other aggregate inhibitors, could behelpful.

Acknowledgments

The authors are thankful for the financial support from theNational Nature Science Foundation of China (Contract Nos.20536050, 20576136, and 20636010) and National BasicResearch Development Program of China (Contract No.2007CB714305).

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