RESEARCH

Studies on the Refolding Process of Recombinant HorseradishPeroxidase

Sedigheh Asad • Bahareh Dabirmanesh •

Nasser Ghaemi • Seyed Masoud Etezad •

Khosro Khajeh

� Springer Science+Business Media, LLC 2012

Abstract Horseradish peroxidase (HRP) is an important

heme-containing glyco-enzyme that has been used in many

biotechnological fields. Valuable proteins like HRP can be

obtained in sufficient amounts using Escherichia coli as an

expression system. However, frequently, the expression of

recombinant enzyme results in inclusion bodies, and the

refolding yield is generally low for proteins such as plant

peroxidases. In this study, a recombinant HRP was cloned

and expressed in the form of inclusion bodies. Initially, the

influence of few additives on HRP refolding was assessed

by the one factor at a time method. Subsequently, factors

with significant effects including glycerol, GSSG/DTT, and

the enzyme concentration were selected for further opti-

mization by means of the central composite design of

response surface methodology (RSM). Under the obtained

optimal condition, refolding increased about twofold. The

refolding process was then monitored by the intrinsic

fluorescence intensity under optimal conditions (0.35 mM

GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and

2 mM CaCl2 in 20 mM Tris, pH 8.5) and the reconstitution

of heme to the refolded peroxidase was detected by the

Soret absorbance. Additionally, samples under unfolding

and refolding conditions were analyzed by Zetasizer to

determine size distribution in different media.

Keywords Horseradish peroxidase � Inclusion body �Intrinsic fluorescence � Soret band � Zetasizer

Introduction

Horseradish peroxidase (HRP, EC 1.11.1.7) with 308

amino acids is a heme-containing glyco-enzyme that uti-

lizes hydrogen peroxide to oxidize a wide variety of

organic and inorganic compounds [1]. Peroxidase from

horseradish root has been extensively used as a component

of clinical diagnostic kits and for immunoassays. Although

the term horseradish peroxidase is used somewhat gener-

ally, the root of the plant contains a number of distinctive

peroxidase isoenzymes of which the C isoenzyme (HRP C)

is the classic one. HRP C has been commonly used for the

biochemical studies of peroxidases and is probably the

most extensively studied member of the plant peroxidase

superfamily [2–5]. Production of recombinant proteins is

necessary for structure and function studies. Moreover,

heterologous expression by means of optimized host sys-

tems is the best approach for the production of those

enzymes with an industrial interest. Bacterial cultivation

processes are based on low-cost media in which fast

growth and high cell concentrations can be obtained. These

high cell concentrations combined with higher production

rates of the bacterial expression system result in higher

volumetric productivities. High-level expression of plant

and fungal peroxidase genes has been achieved in Esche-

richia coli; however, the recombinant protein usually

accumulates in a non-glycosylated and insoluble form in

cytoplasmic inclusion bodies [6]. Although producing an

inactive target protein in the form of inclusion bodies is an

important drawback, it also has several advantages such as

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12033-012-9588-6) contains supplementarymaterial, which is available to authorized users.

S. Asad � N. Ghaemi

Department of Biotechnology, College of Science,

University of Tehran, Tehran, Iran

B. Dabirmanesh � S. M. Etezad � K. Khajeh (&)

Department of Biochemistry, Faculty of Biological Science,

Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

e-mail: khajeh@modares.ac.ir

Mol Biotechnol

DOI 10.1007/s12033-012-9588-6

the high degree of purity of the target protein in the

aggregate fraction and the increased protection from pro-

teolytic degradation compared to the soluble form [7]. The

level of active protein is limited by the low yield obtained

from in vitro refolding procedures, which involves the

formation of native disulfide bridges and incorporation of

heme and Ca2? ions. Refolding conditions were first

optimized for horseradish peroxidase with a 2–3 % active

enzyme yield after E. coli expression of a synthetic gene

[4]. In comparison, manganese peroxidase and lignin per-

oxidase from the white-rot fungus Phanerochaete chry-

sosporium were expressed in E. coli with lower yields of

active enzyme than that obtained for HRP [8, 9]. The aim

of this study was to optimize the conditions for higher

refolding yield and to monitor the refolding process and

size distribution in different media. After the preliminary

screening tests, a central composite design and response

surface methodology (RSM) was applied to optimize the

conditions for maximal refolding and recovery of HRP.

Intrinsic fluorescence intensity and Soret band absorbance

were then monitored to investigate the conformational

changes during the process of folding in an optimized

condition. Furthermore, Zetasizer was used as an appro-

priate instrument for analyzing the size measurement under

refolding conditions.

Materials and Methods

Materials

Oxidized glutathione (GSSG), crystallized phenol, H2O2,

4-aminoantipyrine, isopropyl-b-D-thiogalactopyranoside

(IPTG), and dithiothreitol (DTT) were purchased from

Sigma Aldrich (St. Louis, MO, USA). Thermostable DNA-

polymerase from Pyrococcus furiosus (Pfu) was from

Stratagene (USA), Restriction endonucleases and T4 DNA

ligase were obtained from Fermentas (Germany). Molec-

ular biology kits were from Bioneer (Korea). All other

reagents were purchased from Merck (Darmstadt, Ger-

many) and were of analytical grade. Escherichia coli XL1-

Blue cells and E. coli BL21 (DE3) cells, pET26b(?) vector

were purchased from Novagen (USA). The recombinant

hrp gene was a generous gift from Prof. Frances H. Arnold

(Caltech, CA, USA).

Cloning

All DNA manipulations were carried out by standard

techniques. The DNA fragment encoding hrp gene was

cloned into the pET26b(?) vector via a BamHI–HindIII

double restriction. Sequence analysis confirmed the cloning

procedure (Macrogen, Korea).

Protein Expression

The recombinant plasmid pET26b(?) containing hrp gene

was transformed into E. coli BL21(DE3). Transformants

were grown in Luria–Bertani (LB) medium supplemented

with 50 lg/ml kanamycin at 37 �C. When the optical

density (OD) at 600 nm reached about 0.5, IPTG was

added to a final concentration of 0.5 mM. After incubation

for further 3 h at 30 �C, cells were harvested by centrifu-

gation (8,0009g, 15 min, 4 �C).

Isolation of Inclusion Bodies

Bacterial pellets were resuspended in lysis buffer A

(50 mM Tris–HCl pH 8, 1 mM EDTA, 10 mM DTT).

After 1 h incubation on ice, the solution was sonicated for

five 30 s pulses. Centrifugation at 8,0009g for 40 min

allowed isolation of inclusion bodies. Other proteins were

removed by washing with buffer B (buffer A amended with

2 M urea) as confirmed by SDS-PAGE. Subsequently, the

precipitate was solubilized in 5 ml of buffer C (50 mM

Tris–HCl pH 8, 1 mM DTT, and 6 M urea) and centrifuged

at 8000 9 g for 40 min. The supernatant was used for

refolding [10].

Purification

Unfolded protein was loaded on Ni–NTA agarose column

(Amersham Biosciences) that had been equilibrated with

buffer C. Bound proteins were eluted with buffer C con-

taining 200 mM imidazole and the purity was checked by

SDS-PAGE according to the Laemmli method [11]. The

gel was stained by coomassie brilliant blue R-250. Protein

concentration was determined by the Bradford method

[12, 13].

Optimization of In Vitro HRP Refolding

Protein refolding was studied in dilute aqueous solutions

with controlled pH. The solubilized inclusion bodies were

added drop by drop to the refolding medium and incubated

at 4 �C for 24 h.

One Factor at a Time Method

This step served as a screening test to identify which fac-

tors had a significant effect on the refolding. In this set of

experiments, the level of each factor was changed, while all

other experimental factors remained constant. In this case,

different refolding media were prepared by varying the

concentration of urea, oxidized glutathione, and hemin

with a fixed concentration of CaCl2 (2 mM) in 20 mM

Tris–HCl, pH 8.5. Various enhancers including ionic

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liquids (ILs), glycerol, sodium chloride, glucose, lactose,

sorbitol, trehalose, immidazole, glycine, L-arginine, DTT,

PEG, a-cyclodextrin, and Triton X100 were also added to

the blank refolding buffer (Table 1) and the refolding yield

was studied after 24 h. To determine the optimal pH,

denatured HRP was diluted in the refolding buffer with

different pH values ranging from 7 to 10.

Response Surface Methodology (RSM)

Based on the results obtained at one factor at a time

experiments, RSM was applied to optimize the refolding

condition with evaluating the influence of the selected

variables. The experiments were conducted in triplicate

and the results were analyzed by the Design Expert soft-

ware (version 7.0, Stat-Ease, Inc., Minneapolis, MN).

A central composite design (CCD) has been applied to

determine the optimal conditions for the refolding. The

levels of experimental variables investigated in this study

are presented in Table 2. The CCD permits the response

surface to be modeled by fitting a second-order polynomial

with a number of experiments equal to 2f ? 2f ? n, where

f and n are the number of factors and center runs, respec-

tively (f = 3, n = 6). The repetition of the central runs was

carried out to provide information on the variation of the

responses about the average, the residual variance, and

eventually estimate the pure experimental uncertainty. A

three factor-three coded level (Table 2) CCD, 20 runs, was

carried out to obtain the optimal refolding conditions of

HRP.

Data in Table 3 represent the means of three indepen-

dent experiments. The average values of these three sets

were used as final values for developing the model. The

quality of the fit of the polynomial model equation was

expressed by different criteria. Furthermore, the accuracy

of the model was verified by comparing the model pre-

dictions with the experimental data which were not inclu-

ded in the model estimation. At last, the optimum values of

the selected variables were obtained by calculating the

regression equation and also by analyzing the response

surface contour plots [14].

Table 1 Effect of various additives on HRP refolding

Additives Activity (U)

Blank (without additives) 0.037

Sorbitol (0.1 M) 0.033

NaCl (150 mM) 0.024

Arginine (0.4 M) 0.022

Glucose (0.1 M) 0.034

Lactose (0.1 M) 0.034

Glycine (0.1 M) 0.025

Imidazole (0.1 M) 0.004

[BMIm] [Cl] (2 % v/v) 0.02

[HMIm] [Cl] (2 % v/v) 0.003

Trehalose (0.2 M) 0.03

TFE (5 % v/v) 0.001

Cyclodextrin a (4 mM) 0.04

PEG (2 mg/ml) 0.025

Triton X100 (1 % v/v) 0.015

Glycerol (4 % v/v) 0.12

Refolding mixture used contained 2 mM CaCl2, 1.7 M urea, and

0.7 mM GSSG in 20 mM Tris–HCl (pH 8.5). Experiments were

performed at least in triplicate and the standard deviations were ±5 %

Table 2 Coded levels and range of independent variables for

experimental design

Level Coded

level

Uncoded level

Glycerol

(v/v %)

GSSG/

DTT

Enzyme

(mg/ml)

High ?1 7 8.0 0.20

Mid 0 5 5.5 0.15

Low -1 3 3.0 0.10

Table 3 Experimental design and results of central composite design

Run x1 x2 x3 Peroxidase activity

Predicted Experimental

1 -1 ?1 ?1 0.0699 0.085

2 ?1 0 0 0.1389 0.14

3 0 0 0 0.1341 0.12

4 0 0 -1 0.0923 0.099

5 0 ?1 0 0.1427 0.136

6 0 0 0 0.1126 0.12

7 -1 0 0 0.0996 0.098

8 0 0 ?1 0.1136 0.106

9 ?1 -1 ?1 0.0386 0.05

10 -1 ?1 -1 0.0927 0.082

11 0 0 0 0.1165 0.126

12 ?1 -1 -1 0.0529 0.039

13 0 0 0 0.1071 0.12

14 -1 -1 ?1 0.0207 0.011

15 0 0 0 0.1275 0.12

16 0 -1 0 0.0494 0.055

17 ?1 ?1 -1 0.1161 0.126

18 -1 -1 -1 0.0387 0.047

19 0 0 0 0.1196 0.12

20 ?1 ?1 ?1 0.1837 0.176

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Enzymatic Activity

Peroxidase activity was measured by the method of Wagner

and Nicell [15]. The reaction was carried out in the mixture

containing 200 mM potassium phosphate buffer (pH 7.0),

phenol (42.5 mM), 4-aminoantipyrine (0.625 mM), and

H2O2 (0.75 mM). The rate of H2O2 consumption was fol-

lowed colorimetrically at 510 nm with a molar extinction

coefficient 7,100 M-1 cm-1 at 25 �C. One unit of enzymatic

activity was defined as the quantity of enzyme necessary to

produce 1 lmol product per minute under the assay condi-

tion. Results presented in this paper are the mean values of at

least three repeated experiments in a typical run to confirm

reproducibility.

Spectroscopic Studies

Refolding of apoHRP was studied using Perkin Elmer

LS55 fluorescence spectrometer. Samples were excited at

280 nm and emission was monitored from 300 to 400 nm.

The excitation and emission slit were both set to 5 nm. The

UV spectra were also recorded with Scinco S 2100

spectrophotometer.

Circular dichroism (CD) measurements were conducted

using a JASCO (Tokyo, Japan) J-715 spectropolarimeter.

The protein concentration used for far-UV CD

(195–245 nm) was 0.25 mg ml-1 in 200 mM potassium

phosphate buffer. Results are expressed as molar ellipticity,

[H] (deg cm2 dmol-1), based on a mean amino acid resi-

due weight (MWR) assuming average weights of 110. The

molar ellipticity was calculated from the formula

[H]k = (h 9 100 MRW)/(cl), where c is the protein con-

centration in mg/ml, l is the light path length in centime-

ters, and h is the measured ellipticity in degrees at

wavelength k.

Size Measurements Analysis

Measurements were performed on a Zetasizer, Malvern

Nano-ZS spectrometer. The wavelength of the laser was

632.8 nm, and the scattering angle was 90 and 173.

Results and Discussion

The heterologous expression of foreign genes in E. coli

often leads to production of the expressed proteins in

insoluble inclusion bodies which must then be solubilized

and refolded into active conformation. The lack of post-

translational modifications in prokaryotic hosts such as

E. coli is also another problem in the production of

eukaryotic proteins like HRP which is glycosylated at eight

asparagines’ sites in the native form. Although several

guidelines have been proposed, the search for proper

refolding conditions can be very laborious and time

consuming.

Optimization of Refolding Conditions

One Factor at a Time Experiments

The primary centrifugation of disrupted cells removed sol-

uble E. coli proteins. Treatment of the precipitate with

buffer containing 2 M urea provided further purification of

the inclusion bodies (supplementary data, Fig. 1S). Rena-

turation was attempted by diluting the denatured HRP

solution with various solvents at 4 �C. The expressed pro-

tein in 6 M urea was added into small-scale folding mixtures

with varying concentrations of urea and GSSG. As the

apoHRP captures heme after refolding [16], hemin was

added at the end of the refolding step to the apoenzyme. The

maximum recovery was obtained at 0.35 mM and 1.7 M of

GSSG and urea, respectively (supplementary data Fig. 2S).

2 and 1.8 M urea were previously reported for refolding of

HRP and tobacco anionic peroxidase, respectively [4, 17].

The effect of pH on HRP refolding was not significant over

the range of 7–10 (data not shown).

As no universal refolding buffer can be used to refold an

enzyme, it was necessary to screen a limited set of condi-

tions. The formation of incorrectly folded species, and in

particular aggregates, is usually the cause of decreased

renaturation yields. A very efficient approach to suppress

aggregation is the inhibition of the intermolecular interac-

tions leading to aggregation by the use of low molecular

weight additives. Based on previous studies, several com-

monly used additives were chosen to examine their effects on

the refolding [18]. Among these additives, only glycerol had

significant effect on this process (Table 1). Therefore, dif-

ferent concentrations of glycerol were examined. The high-

est recovered activity was obtained at 5 % glycerol (Fig. 1a).

Other additives like glycine, Triton X-100, and L-arginine,

which were previously shown to suppress aggregation or to

enhance the solubility of refolding intermediates, showed no

significant effect. Since HRP has four disulfide bonds formed

between non-consecutive cysteine residues [3], we assumed

that the oxidation rate might be accelerated by means of an

oxido-shuffling system. Because of the low efficacy of

disulfide bond formation by oxidation with molecular oxy-

gen, exchange reactions with low molecular weight thiols in

reduced and oxidized form are generally used for protein

disulfide bond formation [18]. Herein, the influence of the

redox conditions was investigated by changing the GSSG/

DTT ratio in the folding reaction, and the maximal yield was

found at a ratio of 5:1 (Fig. 1b).

The folding process is a first-order reaction and its

conversion is independent of protein concentration.

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Nevertheless, the competing aggregation pathway is an

intermolecular reaction and its rate increases, accordingly,

to some power of the protein concentration. Previous

studies have also shown that protein concentration has a

strong effect on aggregation [19, 20]. Here, refolding was

increased by raising the enzyme concentration up to

0.15 mg/ml (Fig. 2). The optimal heme concentration was

6 lM and the time required for the activity to reach the

plateau was 1 h after the heme addition (data not shown).

Experimental Design

Results obtained by one factor at a time method revealed

the prominent effect of three factors (glycerol, GSSG/DTT,

and the enzyme concentration) on the yield of refolding.

The approximated values of the key factors were set at a

middle level in the CCD for further optimization. A total of

20 experimental runs with different combinations of the

three factors were performed (Table 3).

In order to analyze and determine the effect of factors,

the regression equation was obtained to check all the

polynomial models to fit the CCD data. The quadratic

model was selected as the most appropriate. The statistical

significance of the model was then determined by the

analysis of variance (ANOVA).The larger the F value, the

more significant the corresponding terms (Table 4).

The F value of 21.06 implies that the model is significant,

which means that there is only 0.01 % chance that a model

with a large F value could occur due to noise. Moreover, the

plot of experimental response (HRP activity) versus the

predicted ones was made. Since the point’s cluster is around

the diagonal line, it can be concluded that there is no sig-

nificant difference between the predicted and experimental

values (Fig. 3). Empirical models are only valid in the ran-

ges defined by the model variables and any extrapolation

may lead to significant errors. Among the interacting effects,

glycerol-GSSG/DTT and glycerol-enzyme were the signif-

icant ones in the variable ranges investigated. In order to

understand these effects, the 3D contour plots of these terms

were also generated, which delineate predicted response

over a range in the design surface. In 3D contours, the

responses were studied taking two factors at a time, while

keeping the other at a fixed level.

A significant interaction (P value: 0.0273) between the

concentration of glycerol and enzyme was seen. At low

concentrations of glycerol, the highest enzyme recovery

was observed at 0.15 mg/ml of denatured enzyme. As the

concentration of glycerol increased, the optimum refolding

yield was obtained at 0.18 mg/ml of unfolded enzyme

(Fig. 4a). Glycerol is known as a stabilizer of the native

state proteins [21]. The liquid-phase viscosity increases

with increasing glycerol concentration; so, the intermo-

lecular interactions are reduced by the lowered diffusivity

of the protein, and aggregation will decrease. Protein

concentrations in a range of 0.1–0.5 mg/ml have been typ-

ically used for refolding of peroxidases. 0.2 and 0.1 mg/ml

Fig. 1 Effect of glycerol, glutathione, and DTT on refolding. a Effect

of different concentrations of glycerol on the refolding recovery yield.

Glycerol was added to the refolding buffer containing 0.35 mM

GSSG, 1.7 M urea, and 2 mM CaCl2 in 20 mM Tris–HCl (pH 8.5).

b The ratio between oxidized glutathione and DTT concentrations on

the yield of active enzyme in the course of refolding. Refolding

carried out in 20 mM Tris–HCl (pH 8.5) containing 0.35 mM GSSG,

1.7 M urea, 5 % glycerol, and 2 mM CaCl2. Refolding mixtures were

incubated at 4 �C for 24 h

Fig. 2 Effect of protein concentration on refolding. Refolding was

carried out in 20 mM Tris–HCl (pH 8.5) containing 0.35 mM GSSG,

1.7 M urea, 2 mM CaCl2, 0.07 mM DTT, and 5 % glycerol

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were previously reported for HRP and Pleurotus eryngii

versatile peroxidase, respectively [4, 6].

The calculated P value (0.0163) also showed a signifi-

cant interaction between glycerol and GSSG/DTT. It is

shown that increasing both glycerol (up to 7 %) and GSSG/

DTT (up to 8) leads to an increase in the refolding yield

(Fig. 4b). At higher values, refolding improvement was

negligible. A previously ratio of 6:1 was reported for lignin

peroxidase and manganese peroxidase [8, 9].

Spectroscopic Studies

In order to monitor structural changes during refolding,

further purification of the solubilized inclusion bodies was

required. So the soluble inclusion bodies in buffer C were

loaded onto Ni-column equilibrated with the same buffer

(Table 5).

The purity of the eluted protein was confirmed by SDS-

PAGE. Having optimized the folding condition, the puri-

fied HRP in 6 M urea was added to the optimized refolding

buffer (20 mM Tris, 0.35 mM GSSG, 7 % glycerol, and

2 mM CaCl2 and 0.044 mM DTT) for spectroscopy stud-

ies. Fluorescence emission has long been established to

study denaturation/renaturation of proteins. The intensity

of fluorescence emission is related to the protein confor-

mation which may expose or bury the internal Trp and Tyr

residues [22, 23]. Samples were taken every 4 h for 48 h.

Each sample was split with a portion being used for fluo-

rescence study and the remaining being used to determine

the activity and changes in the heme environment by

studying Soret absorbance measurements. HoloHRP has

absorption bands in the visible and near ultraviolet region

due to either p ? p* or charge-transfer transition as a

result of interaction between the porphyrin and iron. The

Soret band has the highest extinction with a maximum at

403 nm [24, 25]. As shown in Fig. 5 (inset), in the absence

of heme, apoproteins have no absorption in the visible

region, but in the presence of heme, the polypeptide con-

formation affects the spectroscopic properties of the heme

group. Therefore, changes in the absorbance within the

Soret band correlate with the conformation and function of

the enzyme. During the process of refolding, an increase in

both fluorescence intensity and Soret absorbance was

observed. Results exhibited that the fluorescence intensity,

Table 4 Analysis of variance

(ANOVA) for response surface

model

Source Sum of Squares df Mean square F value P value

Prob [ F

Model 0.031 9 0.003497 21.06 \0.0001

X1 0.004 1 0.004351 26.20 0.0005

X2 0.016 1 0.016 98.67 \0.0001

X3 0.0001 1 0.000114 0.69 0.4262

X1X2 0.0014 1 0.001381 8.31 0.0163

X1X3 0.0010 1 0.001107 6.66 0.0273

X2X3 0.0007 1 0.00074 4.47 0.0605

X12 0.000004 1 0.000004 0.024 0.8795

X22 0.001 1 0.00163 9.87 0.0105

X32 0.000843 1 0.00084 5.08 0.0479

Residual 0.001661 10 0.00016

Lack of Fit 0.001173 5 0.00023 2.41 0.1786

Pure error 0.00487 5 0.000097

Core total 0.033 19

SD 0.013 R2 0.9499

Mean 0.098 Adj R2 0.9048

C.V. % 13.10 Pred R2 0.2636

PRESS 0.024 Adeq precision 18.160

Fig. 3 Relationship between the observed and predicted values of

activity recovery

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Table 5 Steps of recovery of the purified recombinant HRP in different refolding conditions through optimization

Volume (ml) Total protein (mg) Total activity (U) Specific activity (U/mg)

Purification steps

Solubilized in 2 M urea 10 15 – –

Solubilized in 6 M urea 5 12 – –

Ni–NTA agarose column 4 10 – –

Different refolding conditions

Basic refolding conditiona 20 2.2 11.5 5.22

One factor at a time techniqueb 20 3.0 24.3 8.09

Response surface methodc 20 3.6 36.0 10

a 20 mM Tris–HCl (pH 8.5) containing 1.7 M urea, 2 mM CaCl2, 0.35 mM GSSG, and 0.11 mg/ml enzymeb 20 mM Tris–HCl (pH 8.5) containing 1.7 M urea, 2 mM CaCl2, 5 % glycerol, 0.35 mM GSSG, 0.07 mM DTT, and 0.15 mg/ml enzymec 20 mM Tris–HCl (pH 8.5) containing 1.7 M urea, 2 mM CaCl2, 7 % glycerol, 0.35 mM GSSG, 0.044 mM DTT, and 0.18 mg/ml enzyme

Experiments were performed at least in triplicate and the standard deviations were ±5 %

Fig. 4 Response surface plot showing the effect of the three variables/experimental factors on refolding yield; a Effect of glycerol and enzyme,

b effect of GSSG/DTT and glycerol

Fig. 5 Changing in

recombinant HRP apoenzyme

reactivity (dotted line) and

fluorescence intensity (dashedline) upon excitation at 280 nm

and observation of emission at

340 nm during refolding at 4 �C

for various times. The insetshows the changes in

absorbance of the Soret band at

403 nm. Standard deviations

were within 6 % of the

experimental values

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activity and Soret absorbance reached a plateau at around

13 h indicating that complete folding might have appeared.

In order to evaluate the secondary structural content of

commercial and freshly refolded recombinant HRP, far-UV

CD measurements were carried out (Fig. 6). Minor dif-

ferences in the secondary structure might be for the lack of

glycosylation in the recombinant enzyme.

Size Measurement Analysis

The folded state of most proteins is a compact and ordered

structure, whereas the unfolded state (random coil) is usu-

ally significantly larger and substantially less ordered. In

order to confirm refolding, the size of HRP under unfolded

and folded conditions was investigated. As expected, the

unfolded state showed larger size distribution than the fol-

ded state. As the refolding condition improved, the peak

area increased (Fig. 7) indicating the presence of more

folded protein species in the solution which was also

accompanied by a higher specific activity (Table 5).

In summary, the objective of this study was to obtain

renatured HRP from inclusion bodies by means of a simple

and efficient procedure. In recent years, the use of RSM in

the evaluation of biological processes has gained impor-

tance and is becoming an innovative approach in many

research studies. Previously, the refolding condition of

lipase from Pseudomonas sp. and xylanase from Bacillus

halodurans S7 was reported by means of RSM [26, 27].

Response surface method proved to be a generalized and

reliable method for developing the model, optimizing

factors, and analyzing interaction effects. To improve the

refolding yield, in the first step, the one factor at a time

method was used to select the most significant factors.

Subsequently, the refolding process was evaluated in dif-

ferent conditions through statistical methods. The response

surface methodology (RSM) made it possible to investigate

successfully the optimal conditions of in vitro refolding

and to elucidate interactions between refolding factors with

a minimum number of experiments. Incubation in 20 mM

Tris buffer (pH 8.5) containing 0.35 mM GSSG, 0.044 mM

DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl2 for about

13 h at 4 �C provided the best results, which was con-

firmed by fluorescence and size determining Nano-ZS

spectrometer (Figs. 5, 7).

Acknowledgments We would like to thank the research council of

Tarbiat Modares University and the University of Tehran for the

financial support of this investigation.

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