β-Cyclodextrin-Modified Magnetic Nanoparticles Immobilized on

Oct 9, 2018 - Department of Animal Nutrition, Poznan University of Life Sciences, 60-637 ... Institute of Biochemistry and Biophysics, University of T...
0 downloads 0 Views 1MB Size
Subscriber access provided by UNIV OF CAMBRIDGE

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

#-Cyclodextrin-Modified Magnetic Nanoparticles Immobilized on Sepharose Surface Provide an Effective Matrix for Protein Refolding Marziyeh Ghaeidamini, Ali Nemati Kharat, Thomas Haertlé, Faizan Ahmad, and Ali Akbar Saboury J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07226 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

TOC 266x241mm (96 x 96 DPI)

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 41

β-Cyclodextrin-Modified Magnetic Nanoparticles Immobilized on Sepharose Surface, Provide an Effective Matrix for Protein Refolding Marziyeh Ghaeidamini†, Ali N. Kharat†, Thomas Haertlé ‡# §, Faizan Ahmad⊥ and Ali A. Saboury*§ †

School of Chemistry, University Collage of Science, University of Tehran, Tehran, Iran.



Poznan University of Life Sciences, Department of Animal Nutrition, Poznan, Poland.

#

Biopolymers, Interactions, Assemblies, UR 1268, Institute National de la Recherche

Agronomique, Nantes, France. §

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran,

[email protected]. ⊥

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-

110025, India.

ACS Paragon Plus Environment

1

Page 3 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

ACS Paragon Plus Environment

2

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 41

ABSTRACT

In this manuscript, we propose an impressive and facile strategy to improve protein refolding using solid phase artificial molecular chaperones consisting of the surface-functionalized magnetic nanoparticles. Specifically, mono-tosyl-β-cyclodextrin (Ts-β-CD) connected to the surface of 3-aminopropyltriethoxysilane (APES) - modified magnetic nanoparticles (APESMNPs), is immobilized on the Sepharose surface to promote interaction with exposed hydrophobic surfaces of partially folded (intermediates) and unfolded states of proteins. Their efficiencies were investigated by circular dichroism (CD) spectroscopy and photoluminescence spectroscopy (PL) of the protein. Although the mechanism of this method is based on principles of hydrophobic chromatography, this system is not only purging the native protein from inactive inclusion bodies but also improving the protein refolding process. We chose β-cyclodextrin considering multiple reports in the literature about its efficiency in protein refolding and its biocompatibility. To increasing surface area/volume ratio of the Sepharose surface by nanoparticles, more β-cyclodextrin (β-CD) molecules are connected to the Sepharose surface to make better interaction with proteins. We suppose that proteins are isolated in the nanospace created by bound cyclodextrins on the resin surface so intermolecular interactions are reduced. The architecture of nanoparticles was characterized by Fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), scanning electron microscopy images (FESEM), energy dispersive X-ray spectroscopy (EDX), nuclear magnetic resonance (H1NR, C13NMR) and dynamic light scattering (DLS).

Keywords: Protein refolding; β-cyclodextrin; Sepharose; Magnetic nanoparticles

ACS Paragon Plus Environment

3

Page 5 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

1. INTRODUCTION Heterologous recombinant proteins that are expressed in various host cells usually form biologically inactive and insoluble aggregates known as inclusion bodies 1-2. There is a kinetically competitive process between protein folding and protein aggregation, which can significantly lead to low yields of biologically active forms of proteins through direct contact between exposed hydrophobic patches and by erroneous folding. Since, protein aggregation is an unwanted process in in vitro biotechnology and pharmaceutical studies, protein refolding should be useful in a range of biomedical applications 3. The common procedure used to recover the active form of the protein from inactive inclusion bodies, involves several stages including isolation and purification of inactive inclusion bodies, solubilization, renaturation to active form and final purification. Renaturation of solubilized inactive proteins is the most important step 46. To convert inclusion bodies into biologically active proteins, the inclusion bodies must be dissolved in a buffer containing a denaturant such as guanidinium chloride (GdmCl) or 6-8 M urea. The efficacy of this step can be relatively high and then the solubilized inactive protein is refolded into its active form with use of diverse approaches 6-9. One of refolding strategies is the dilution, which reduces denaturant concentration 10-11. The second one is free solution in the presence of several species (e.g., additives 12, micelles 13, nanoparticles 14-18, nanotubes 19). Another method is solid state or on-column refolding 20-22 (e.g., size-exclusion chromatography (SEC) 23-26, ion exchange chromatography (IEC) 27-28, immobilized metal affinity chromatography (IMAC) 29-30 and hydrophobic interaction chromatography (HIC) 31-32), and the most well-known and advanced method, which is an imitation of the in vivo folding called

ACS Paragon Plus Environment

4

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 41

“artificial chaperone-assisted refolding” 29, 33-34. Rozema and Gellman developed this method, which has two steps capturing and stripping 35. First, hydrophobic patches of denatured protein are shielded by the detergent and a protein-detergent complex is formed. In fact the protein is captured by a detergent. Second, β-cyclodextrin is added 32, 36 to the mixture striping the detergent from the protein. Other studies introduced some molecules with small cavities like nanogels 36-37 and nanotubes 19, cycloamyloses 38 as artificial molecular chaperones. The implementation of this strategy leads to protein refolding but its efficacy is limited by the purification step. Depending on the protocol used, extent of the protein refolding can be improved by applying relevant chaperones39. In some investigations aiming amplifying of refolding efficiency, natural or artificial chaperones were immobilized on resin and a hydrophobic interaction chromatography (HIC)40 was used. A successful immobilization of foldases catalysts like GroEL 41

on resin to help protein refolding was discussed by Dong et al

42.

Also some studies were

carried out on the preparation of solid phase assisted refolding like β-CD-acrylamide copolymer beads 43, β-CD-polyurethane polymer 44, β-CD-epichlorohydrin copolymer 45 as solid adsorbents. However, to the best of our knowledge there is no studies of modifications of resin surface with functionalized nanoparticles. We have applied a combination of methods for the protein refolding for the first time. In this strategy, an artificial molecular chaperone was connected to the surface of magnetic nanoparticles, which were immobilized on the resin surfaces. We chose β-cyclodextrin considering multiple reports in the literature about its efficiency in the protein refolding and its biocompatibility. β-Cyclodextrin was used in order to manage chromatography column pores and interparticle spacing. Immobilized β-cyclodextrin molecules increase optimal interactions with

ACS Paragon Plus Environment

5

Page 7 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

α-amylase because they have specific site(s) to bind to critical site(s) of denatured proteins. Thus they prevent protein aggregation and stimulate return protein to its original fold. Because of the preparation and surface modification methods of nontoxic and cheap MNPs are very simple, they were used to extend Sepharose surface for grafting much more chaperones. The development of refolding chromatography columns based on nanoparticles for protein refolding could become highly promising for industrial use in the future.

2. EXPERIMENT SECTION

2-1. Material Iron (III) nitrate nonahydate, sodium azide, p-toluenesulfonyl chloride (98%), bacterial αamylase, Sepharose 6B, guanidine hydrochloride, triphenyl phosphine, 1,1-carbonyldiimidazole (CDI), PEG-300, 1,1,2,2 tetrachloroethane and Nile red were purchased from Sigma-Aldrich. 3aminopropyltriethoxysilane (APES, 98%) was obtained from Alfa Aesar. Sodium hydroxide and 3, 5 dinitrosalicylic acid (DNS) were purchased from Merck. β-Cyclodextrin (99%) was purchased from Across-organic. Pyridine [Sigma] and N, N-dimethylformamide (DMF) [Fluka] were distilled before use and other chemical reagents were of analytical reagent grade and used without further purification.

2-2. Preparation of APES-Fe3O4 magnetic nanoparticles Fe3O4 magnetic nanoparticles were synthesized with the assistance of the microwave irradiations 46. In this procedure, 2 mmol of Fe(NO3)3.9H2O was dissolved in polyethylene glycol 300 (PEG-300) and then gently stirred and heated until 70 ◦C with a mechanical stirrer. A solution of 5 mmol of NaOH in PEG was added to the above mixture and continuously stirred for 10 min. Then a final solution was placed in a domestic microwave oven and reaction was

ACS Paragon Plus Environment

6

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 41

carried out for 8 min (30 s on for every 60 s interval). After cooling down the container to the room temperature the magnetic precipitates were collected and washed with ethanol and hot distilled water. Obtained product dried in a drying oven at 50 ◦C under vacuum and then annealed under argon atmosphere at 200 ◦C for 3 hours. To synthesize APES-coated Fe3O4 nanoparticles

47,

170 mg of Fe3O4 nanoparticles were dissolved in 100 ml ethanol and 2 ml

water. The mixture was placed in the ultrasonic bath for 30 min to completely disperse it. 70 µL 3-aminopropyltriethoxysilane (APES) was added to the above solution and was shaken for 7 hours at the room temperature. The product was collected by using a magnet, washed with ethanol for several times and then dried at room temperature under vacuum 48-49.

2-3. Activation of resin (Sepharose) surface In a typical activation method, 3 g of Sepharose 6B-CL was washed with 20 ml of each of water, dioxane/water and dioxane. The moist gel was suspended in 5 ml dioxane, and to this was added a solution containing 0.12 g of 1, 1-carbonyldiimidazole (CDI) in 5 ml dioxane. This mixture was shaken for 3-4 hours at room temperature followed by washing with dioxane, and this activated Sepharose gel was used immediately. It should be noted that in the whole washing process a Buchner funnel for separating Sepharose from the solvent was used 50-51.

2-4. Conjugation of Fe3O4-APES on the resin surface (Resin-MNPs-APES) To immobilize APES-Fe3O4, the activated Sepharose gel was suspended in a sodium carbonate buffer (pH 9.7), and 50 mg of APES-Fe3O4 was added to this solution, followed by incubation at 25 ◦C for l6 hours 51.

2-5. Synthesis of mono-6-(p-toluenesulfonyl)-deoxy-6-cyclodextrin (Ts-β-CD) 6-Ts-β-CD was obtained according to the Nature protocol described by Tang and Ng 52. First, 5 g β-CD and 100 ml dried pyridine were added into three-necked flask, and the flask was kept

ACS Paragon Plus Environment

7

Page 9 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

on the magnetic stirrer. A solution containing 0.8 g p-toluenesulfonyl chloride in pyridine was added gently to the flask with glass syringe over one hour at 0 ◦C, then the reaction was allowed to continue overnight at room temperature. 200 ml acetone was added into the flask while the solution was stirred violently, then separated the solid by Buchner funnel and washed with acetone several times.

2-6. Preparation of stationary phase by grafting of β-cyclodextrin on the Resin-MNPsAPES surface (Resin-MNPs-APES-CD) To graft β-cyclodextrin on the APES-Fe3O4-resin surface, the activated gel connected to APES-Fe3O4 nanoparticles was suspended in sodium carbonate buffer (pH 9.7) and mixed with β-cyclodextrin in a 1-5:1 stoichiometry ratio of β-cyclodextrin to the APES-Fe3O4-Sepharose gel. To this was added 50-250 mg of mono-6-(p-toluenesulfonyl)-deoxy-6-cyclodextrin (Ts-βCD)/ml of Sepharose, and this mixture was kept at 25 ◦C for 6-8 hours 51.

2-7. Synthesis of mono-6-amino-deoxy-6-cyclodextrin (NH2-β-CD) At first we should synthesize mono-6-azide-deoxy-6-cyclodextrin (CD-N3). Following the protocol

52,

2.5 g Ts-β-CD and 2.5 g sodium azide in 250 ml deionized water were refluxed

overnight. After cooling the reaction to the room temperature the mixture was filtered to remove any insoluble compounds. The filtered solution was concentrated to reduce its volume to 30 ml. To this was added 2.5 ml 1,1,2,2 tetrachloroethane dropwise and stirred for 30 min. Collected product was dried at 60 ◦C under vacuum. NH2-β-CD was obtained by adding 2.9 g N3-β-CD, 1.5 g triphenylphosphine and 10 ml DMF in one-neck flask equipped with condenser and magnetic stirrer. Reaction was followed for 2 hours at room temperature. Then 1 ml deionized water was added into the flask, and let the reaction continue at 90 ◦C for 3 hours. After this solution was

ACS Paragon Plus Environment

8

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 41

cooled down to the room temperature followed by the addition of 50 ml acetone. The product was collected and washed with acetone several times and dried at 60 ◦C under vacuum 52.

2-8. Preparation of modified Sepharose gel with β-CD (β-CD-Resin) as a blank stationary phase This step is the same as the coupling of Fe3O4-APES on resin. So, initially, the activated gel was suspended in sodium carbonate buffer (pH 9.7) and to this was added 50 mg of NH2-βCD/ml of Sepharose. And the mixture was incubated at 25 ◦C for 16 hours 51.

2-9. Characterization of resin-MNPs-APES-CD, resin –CD Fourier transform infrared (FT-IR) spectra of samples prepared in KBr were recorded in Shimadzu FTIR1650 Spectrometer (Japan) in the range of 400–4000 cm-1. X-ray diffraction patterns were examined in X’PertPro Philips diffractometer using Cu Kα radiation (λ=1.5406 Å) in the 2θ range 20-70 ◦C. FESEM (MIRA3TESCAN-XMU) was used to determine the size and morphology of MNPs and MNPs-APES. Also purity and chemical analysis was studied by FESEM, which was equipped with an energy dispersive X-ray spectroscopy (EDX). 1H and 13Cspectra, respectively in DMSO and CDCL3 were measured in Bruker ECX 300 (300.132 MHz) at 298 ◦K.

2-10. Preparation of chromatography column 30 ml modified Sepharose 6B gel, which was prepared in previous (1-6, 1-8) steps were packed in a cylindrical column with sectorial area of 1 cm2 for fast protein liquid chromatography (FPLC). The solution containing denatured α-amylase and denaturant was loaded onto the column with nanosphere pores and thoroughly washed with a refolding buffer 40, 53-55.

Protein was eluted from the column in correct form, what was confirmed by various

measurements. Prior to on-column refolding, the column was pre-equilibrated with phosphate

ACS Paragon Plus Environment

9

Page 11 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

buffer, then α-amylase was applied to the column at a relatively high flow rate of 0.5 ml/min at 4 ◦C.

The fractions contain protein were collected, freeze-dried and their enzymatic activity was

checked.

2-11. Denaturation and renaturation of α-amylase. The native α-amylase was added to 50 mM phosphate buffer solution (pH 7.5) containing 6 M GdmCl and left for 16 h at room temperature with rolling at 150 rpm. The formation of the denatured protein was confirmed by circular dichroism (CD) spectroscopy and fluorescence analysis as well as enzyme activity determination

56.

The refolding of α-amylase (2 mg/ml, 0.5

mg/ml) was performed in renaturation buffer containing 50 mM phosphate buffer (pH 7.5) using a hydrophobic affinity chromatography column. β-CD (50 mg/ml of Sepharose) was grafted onto the Sepharose surface in the absence and presence of the MNPs. Sepharose 6B with 10-1000 kDa pore size is not suitable to trap α-amylase (55.4 kDa) individually. Bonding β-cyclodextrin to the Sepharose surface can manage the pore size poorly, but in the presence of MNPs can be controlled, particularly in the result of increasing ratio surface/volume.

2-12. Structure analyses of α-amylase by fluorescence spectra and far-UV circular dichroism To observe tertiary structural changes of refolded α-amylase, fluorescence experiments were performed using a Varian Cary Eclipse Fluorescence Spectrophotometer at 25 ◦C, with a 1 cm path length sample cuvette. For Nile red fluorescence studies, to the refolded α-amylase solution was added required amount of the stock solution of Nile red (2 mM), to obtain a final concentration of 20 µM. Fluorescence of Nile red (dye 9-diethylamino-5-benzo phenoxazinone) increases on transferring it from polar environment to hydrophobic environments, hence it is used as a probe to confirm the presence of hydrophobic clusters on protein surface exposed to

ACS Paragon Plus Environment

10

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 41

solvent water, to which the dye binds. 5 µl of this probe (2 mM) was added to all samples (495 µl) which were prepared in water pH 1.6 and were incubated for 30 min at 25 ◦C. Then the change in fluorescence spectrum of Nile red in each sample was recorded in the range 600-700 nm after exciting the sample at 553 nm (excitation wavelength). Intrinsic fluorescence spectra of the native, unfolded and refolded α-amylase samples were recorded in the wavelength range 300400 nm; the excitation wavelength was 280 nm. The secondary structural variations of α-amylase samples were evaluated by the far-UV CD measurements. CD spectra were recorded in the wavelength range of 195-260 nm using a 0.1 cm path length cell. The concentration of the native, unfolded and refolded samples was 0.2 mg/ml and all of them were filtered by an Amicon filter.

2-13. Determination of biological activity and protein concentration The enzyme activity of the denatured, native and refolded α-amylase (2 mg/ml) was determined by incubating starch with enzyme in a buffer at pH 7.4 and 37 ◦C 57. The activity was ceased by adding DNS to the samples in 0-120 minutes at intervals of 30 minutes. The tubes were heated for 5 minutes in boiling water bath then cooled. The optical density of the solution containing the reduction product was measured using Varian (Carry 100 Bio) UV-vis spectrophotometer at 540 nm. Glucose was used as standard then unit activity was calculated. One enzyme unit is defined as the amount of the enzyme that catalyzes the conversion of 1 µmole of substrate per minute. The specific activity was calculated by dividing the unit activity by the amount of protein (mg). It is interesting that the enzyme activity spontaneously recovered up to 77% after loading on the hydrophobic chromatography column with an appropriate concentration of β-CD. β-CD illustrates especially higher chaperone-like activity in this solidstate method. The protein concentration of the refolded and native α-amylase was determined by

ACS Paragon Plus Environment

11

Page 13 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

reading the absorbance at 280 nm using a value of 2.6 M-1cm-1 for an extinction coefficient at 280 nm (ε280).

3. RESULT AND DISCUSSION Scheme1 illustrates the activation of Sepharose surface with 1, 1-carbonyldiimidazole (CDI) and the preparation strategy of ß-CD grafted onto the MNPs-APES-Sepharose surface. We coupled MNPs-APES to the activated Sepharose surface applying substitution reaction at the carbonyl position by NH2 groups of MNPs-APES. Consequently, we grafted the CD moiety onto the MNPs-APES, immobilized on the Sepharose surface through replacement reaction at C-6 position of ß-CD by the rest of NH2 groups of APES. Moreover, for preparation of blank column mono-6-amino-deoxy-6-cyclodextrin (NH2-β-CD) molecules were connected to the activated Sepharose surface through NH2 groups of NH2-CD. These two modified Sepharose 6B gels were used as the stationary phase in the chromatography column. FT-IR, XRD, H1NMR, EDX, SEM and DLS were used to characterize participants in all processes.

ACS Paragon Plus Environment

12

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 41

Scheme 1. Mechanism of grafting of β-cyclodextrin to APES-MNPs is immobilized on Sepharose surface (Resin-MNPs-APES CD).

3-1. Characterization of magnetic nanoparticles X-ray diffraction (XRD) pattern was used to investigate crystalinity, phase structure and purity of MNPs and APES-MNPs through six strong Bragg diffraction peaks at 2Ɵ = 30.2, 35.6, 43.2,

ACS Paragon Plus Environment

13

Page 15 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

53.7, 57.2 and 62.9 which matched well with the standard XRD data (JCPDS card, file No. 750033) (Fig. 1). Standard XRD data with a cubic spinal structure illustrate characteristic peaks, including (220), (311), (400), (422), (511) and (440). And by using the Debye-Scherrer’s formula (d = kλ/βcosƟ), one can calculate MNPs average crystalline diameter from the XRD pattern regarding the line width of half maximum intensity. According to XRD pattern (Fig. 1 (a, b)) Fe3O4 and APES-MNPs nanoparticles were of average size of 14 and 23 nm, respectively. However APES coating did not change the result phase, but corresponded to the formation of amorphous structure what can be seen as a weak broadband in 2Ɵ =18-29 (Fig. 1 (b)) 49, 58.

Figure 1. (a) XRD pattern of magnetic nanoparticles (MNPs), (b) XRD pattern of APESmodified magnetic nanoparticles (APES-MNPs). Energy-dispersive X-ray spectroscopy (EDX) spectrum presented in Fig. 2 (a) indicates that there are only Fe and O as elements and confirmed purity of MNPs. Also the presence of Si and

ACS Paragon Plus Environment

14

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 41

N elements shown in Fig. 2 (b) revealed that MNPs were coated with APES as well (Fig. 2 (a, b)).

Figure 2. (a) Energy-dispersive X-ray spectroscopy (EDX) of magnetic nanoparticles (MNPs), (b) EDX of APES-modified magnetic nanoparticles (APES-MNPs).

FT-IR spectrum (Fig. 3 (a)) of MNPs, which exhibit Fe-O band in the octahedral and tetrahedral sits appear at 492 and 549 cm-1, respectively. In addition, the stretching vibrations of OH absorbed on the surface of Fe3O4 nanoparticles are observed at 3230 cm-1. FT-IR spectrum (Fig. 3 (b)) of APES-MNPs confirms APES coated surface of MNPs because OH peak at 3200 is absent and a weak broadband at 3400 cm-1 replaces also a weak band, which appears at 1630 cm1.

They both belong to N-H stretching vibrations of free NH2 groups. Bands at 981 and 1008 cm-1

are due to Si-O-H and Si-O-Si groups of silan polymer. Also is observed a little shift at Fe-O bands after coating with APES 48-49, 58.

ACS Paragon Plus Environment

15

Page 17 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 3. (a) FT-IR spectrum of magnetic nanoparticles (MNPs), (b) FT-IR spectrum of APESmodified magnetic nanoparticles (APES-MNPs).

Scanning electron microscopy (SEM) was used to determine the size distribution and morphology of MNPs and APES-MNPs. SEM images of these two well shaped spherical compounds are presented in Fig. 4 (a, b) and Fig. 4 (c, d), respectively. It is evident that the size of MNPs changed after coating from 10 nm to 16 nm, as expected. Although the size distribution for MNPs from SEM, XRD and DLS techniques was coherent, in case APES-MNPs the size, obtained by DLS method was larger than other methods. DLS method was applied for measurements in the solution, so that presence of silan polymer on the surface of MNPs may lead to the binding of additional H2O molecules on its surface and to the increase in its hydrodynamic diameter (Fig. 5 ) 49, 58.

ACS Paragon Plus Environment

16

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 41

Figure 4. (a, b) Scanning Electron Microscopy (SEM) of magnetic nanoparticles (MNPs), (c, d) SEM of APES-modified magnetic nanoparticles (APES-MNPs).

ACS Paragon Plus Environment

17

Page 19 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 5. (a) Dynamic light scattering (DLS) spectrum shows Size distribution of MNPs in water, (b) DLS spectrum shows Size distribution of MNPs-APES in water.

3-2. Characterization of grafted β-CD on the MNPs surfaces immobilized on the Sepharose surfaces The percent of β-CD on the surfaces of magnetic nanoparticles immobilized on the Sepharose surface, was confirmed by FT-IR spectra. Fig. 6 (a) illustrates spectrum of the inert Sepharose having characteristic bands at 1042 and 1115 cm-1 assigned to C-O-C and C-O groups, respectively, band at 3353 cm-1 and 2912 cm-1 related to O-H stretching and to C-H vibration, respectively. Fig. 6 (b) shows a peak at 1370-1410 cm-1 due to C-N stretching and to C-H bond bending, the absorption band near 1670 cm-1 refers to C=O functional group of the activating agent connected to the Sepharose surface (1,1-carbonyldiimidazole). After surface activation, the absorption band of OH was weaker than that of the inert Sapharose. Fig. 6 (c) confirms substitution of APES-MNPs instead of the activation groups and the presence of Fe having bands at 446 cm-1 and 506 cm-1. Furthermore, NH2 groups of APES emerge at 3342 cm-1, C-N stretching and C-H bond bending are documented by peak at 1405 cm-1(small red shift), and C=O group with a slightly blue shift appears at 1645 cm-1. Results presented in Fig. 6 (d), confirmed the grafting of β-CD on the surfaces of magnetic nanoparticles which were immobilized on the Sepharose surface. All the significant bands of β-CD emerge in the range of 900-1200. Available OH groups of β-CD lead to enhanced peak intensity that shows that β-CD is connected to NH2 groups of APES-MNPs successfully.

ACS Paragon Plus Environment

18

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 41

ACS Paragon Plus Environment

19

Page 21 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 6. (a) FT-IR spectrum of Sepharose, (b) FT-IR spectrum of activated Sepharose gel with CDI, (c) FT-IR spectrum of grafted APES-MNPs on activated Sepharose gel surface, (d) FT-IR spectrum of coupled β-CD-NH2 on the surface of modified nanoparticles, (e) FT-IR spectrum of conjugated β-CD on the resin surface (blank stationary phase).

3-3. Characterization of grafting of β-CD on the resin surfaces To confirm coupling of β-CD on the resin surfaces, FT-IR spectra of β-CD were recorded at different steps of the preparation. As shown in Fig. 7 (a), FT-IR spectrum of β-CD with peaks at 1019 and 1115 cm-1 can be attributed to stretching vibrations of C-O-C and C-O groups, band at 3294 cm-1 can be assigned to O-H groups, and peak at 2926 cm-1 originates from C-H bending. Fig. 7 (b) shows FT-IR spectrum of TS-β-CD that did not have any functional groups thus, this compound was characterized by 1H NMR and 13C NMR. FT-IR spectrum of CD-N3 is shown in Fig. 7 (c). Peak at 2042 cm-1 belongs to stretching band of N3. Fig. 7 (d) relates to CD-NH2 that illustrates N-H bend at 1654 cm-1. Fig. 6 (e) from conjugated β-CD on the resin surface shows all the bonds of β-CD in the range of 900-1200 cm-1 with O-H, N-H, C-H bands at 2700-300 cm-1 and C=O group emerging at 1670 cm-1.

ACS Paragon Plus Environment

20

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 41

Figure 7. (a) FT-IR spectrum of β-CD, (b) FT-IR spectrum of β-CD-OTS, (c) FT-IR spectrum of β-CD-N3, (d) FT-IR spectrum of β-CD-NH2.

TS-β-CD was characterized by 1H NMR (300 MHz, DMSO): δ 7.75 (d, J= 8.4 Hz, 2H), 7.44 (d, J= 8.4 Hz, 2H), 5.5-5.8 (M, 13H), 4.19-4.9 (M, 16H) 3.45-3.72 (m, 28H), 3.15-3.47 (m, overlapping with HDO, 14H), 2.41 (s, 3H) (Fig. 8), and by 13C NMR (75 MHz, DMSO): δ 142.9, 128.5, 127.4, 125.9, 102.4, 82, 73.5, 72.9, 72.5, 60.4 (Fig. 9).

ACS Paragon Plus Environment

21

Page 23 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 8. H1 NMR spectrum of Ts-β-CD.

Figure 9. C13 NMR spectrum of Ts-β-CD.

3-4. CD-APES-MNPs-Sepharose chaperone α-amylase refolding Artificial chaperone immobilized on the resin surface remarkably stimulates protein refolding without use of any additional agent and any need of repurification

43, 59.

Although artificial

chaperone-assisted refolding have been successfully used in a free solution earlier as well, the protein purification was a painstaking job. For, in addition to the protein of interest, several

ACS Paragon Plus Environment

22

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 41

compounds are present in the solution such as denaturing agents, buffer, detergent and stripping agent which prevents protein aggregation by covering the hydrophobic region of the unfolded protein. These additional compounds should be removed during protein purification

13.

On

contrary, in solid-state method described in this study, β-CD is used as an artificial chaperone and is fixed on the modified Sepharose (APES-MNPs-Sepharose) surfaces so two processes, namely protein refolding and purification happen simultaneously. Thus the production costs will be decreased 34, 55.

3-5. Structural analyses of the refolded α-amylase by far-UV circular dichroism and fluorescence To evaluate the efficiency of the prepared column, the protein folding was confirmed by circular dichroism spectroscopic study [36, 53]. Far-UV circular dichroism spectra of the refolded α-amylase using different concentrations of β-CD immobilized on modified Sepharose surface were recorded and compared with CD spectrum of the native protein. The CD spectra of the refolded α-amylase at its two different concentrations, namely 0.5 and 2 mg/ml are shown in Figs. 10 (a) and 10 (b), respectively. The column comprising of CD-APES-MNPs-Sepharose suppressed α-amylase aggregation to some extent, for it is expected that intermolecular interactions will be diminished and intramolecular interactions will be increased. In fact, unfolded α-amylase is adsorbed through flexible spacer arms (β-CD) that are hanging on the modified Sepharose surface34. The optimal amount of β-CD was measured by changing the stoichiometric ratio of 1-5:1 β-CD to the APES-Fe3O4. Fig. 10 (a, b) shows CD spectra of 0.5 and 2 mg/ml α-amylase in the native, refolded and unfolded states in 195-260 nm range. We

ACS Paragon Plus Environment

23

Page 25 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

used 2 concentrations of α-amylase to show that the column can be used for renaturation of high as well as low concentrations of the protein. Unfolded α-amylase is devoid of characteristic CD of secondary structures. At three highest concentration of β-CD (150-250 mg/ml of Sepharose) attached to the modified Sepharose surface (β-CD-APES-MNPs-Sepharose), the far-UV spectra of the refolded α-amylase display negative ellipticity bands at 208 and 222 nm which are characteristics of α-helix and resemble the spectrum of the native protein. Estimate of contents of secondary structure (helix, strand, turn) and unordered structure including ±0.05 error was obtained by DICHROWEB, an online server for protein secondary structure analyses from CD spectra data 60-61. Analysis of these results are given in Tables 1 and 2. It is seen in these tables that the secondary structure of the native αamylase consists of 7 % α-helix 1 and 11 % helix 2, and the denatured protein has almost no helix. As it can be seen in Fig. 10 (a and b) and Tables 1 and 2, an increase in the amount of βCD attached to the modified Sepharose surface (β-CD-APES-MNPs-Sepharose) leads to increase the secondary structure, i.e., the artificial chaperone refolds the denatured α-amylase. It is also seen these figures that the CD spectra of the refolded protein from 150-250 mg/ml β-CD-APESMNPs-Sepharose resemble to the native form. It is also seen in these figures that the CD spectrum of the refolded protein from the 250 mg/ml β-CD-APES-MNPs-Sepharose column has less negative ellipticity than that of the refolded protein from 200 mg/ml β-CD-APES-MNPsSepharose column in the wavelength region 240-205 nm. This observation is discussed below. Intrinsic fluorescence spectroscopy is a sensitive probe to measure change in the environment of Trp residue in the protein 44. It is known that transfer of Trp from polar to non-polar environment leads to an increase in fluorescence intensity. We have measured fluorescence spectra of αamylase in the native and denatured states and those of the refolded proteins eluted from β-CD-

ACS Paragon Plus Environment

24

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 41

APES-MNPs-Sepharose columns having different amounts of β-CD (50-250 mg/ml of Sepharose). The results of these measurements are shown in Fig. 11 (a). This figure also shows emission spectrum of the denatured protein eluted from the β-CD-Sepharose column. Fig. 11 (b) shows change in emission intensity at λmax of refolded proteins with increasing concentration of β-CD attached to APES-MNPs-Sepharose. These results show that more and more Trp residues get buried in the refolded protein on increasing the concentration of β-CD. The fluorescence intensity at λmax for the native α-amylase is close to 1000 while that for refolded α-amylase is about 800. Bound β-CD on the Sepharose surface without using MNPs is not as much effective as other columns. Another interesting observation is that at the high concentrations of β-CD (250 mg/ml of Sepharose) fluorescence intensity of the refolded protein is less that of the refolded protein at 200 mg β-CD/ml of Sepharose, suggesting less refolding at the higher concentration. This observation is similar to that from the far-UV measurements.

Table 1. Estimation of secondary structure (helix, strand and turns) and unordered structure of αamylase (0.5 mg/ml) using DICHROWEB Result

Helix1

Helix2

Strand1

Strand2

Turns

Unordered

Unfolded protein

0

0.027

0.293

0.195

0.228

0.252

Blank column

-0.002

0.030

0.262

0.169

0.233

0.291

Column contain 50 mg β-CD/ml

-0.001

0.018

0.292

0.138

0.219

0.33

Column contain 100 mg β-CD/ml

0.007

0.001

0.004

0.087

0.277

0.593

Column contain 150 mg β-CD/ml

0.053

0.06

0.43

0.149

0.15

0.15

Column contain 200 mg β-CD/ml

0.062

0.097

0.188

0.123

0.232

0.306

Column contain 250 mg β-CD/ml

0.071

0.11

0.25

0.109

0.215

0.222

ACS Paragon Plus Environment

25

Page 27 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Native protein

0.078

0.114

0.24

0.105

0.214

0.225

Table 2. Estimation of secondary structure (helix, strand and turns) and unordered structure of αamylase (2 mg/ml) using DICHROWEB

Result

Helix1

Helix2

Strand1

Strand2

Turns

Unordered

Unfolded protein

0

0.027

0.293

0.195

0.228

0.252

Blank column

-0.003

0.037

0.273

0.176

0.236

0.283

Column contain 50 mg β-CD/ml

-0.005

0.031

0.304

0.141

0.205

0.317

Column contain 100 mg β-CD/ml

0

0.018

0.292

0.139

0.219

0.33

Column contain 150 mg β-CD/ml

0.052

0.153

0.233

0.149

0.163

0.242

Column contain 200 mg β-CD/ml

0.063

0.113

0.269

0.114

0.212

0.209

Column contain 250 mg β-CD/ml

0.063

0.109

0.262

0.113

0.214

0.216

Native protein

0.078

0.114

0.24

0.105

0.214

0.225

ACS Paragon Plus Environment

26

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 41

Figure 10. (a) Far-UV circular dichroism spectra of native, refolded, unfolded α-amylase (0.5 mg/ml) samples, (b) Far-UV circular dichroism spectra of native, refolded, unfolded α-amylase (2 mg/ml) samples.

Figure 11. (a) Intrinsic fluorescence spectra of native, refolded, unfolded α-amylase (2 mg/ml) samples, (b) Emission intensity at λmax refolded α-amylase (2 mg/ml) samples in the present of different columns.

Hydrophobic dyes such as Nile red are used to identify the presence of hydrophobic exposed patched on proteins, for the transfer of such dyes from polar to non-polar mediums leads to an increase in the fluorescence intensity with a blue shift. To follow refolding of α-amylase, we measured fluorescence spectra of Nile red in the presence of α-amylase in the native and

ACS Paragon Plus Environment

27

Page 29 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

denatured states and in the presence the refolded proteins eluted from β-CD-APES-MNPsSepharose columns having different amounts of β-CD (50-250 mg/ml of Sepharose). Fig. 12 (a) shows Nile red fluorescence spectra recorded in the wavelength range of 600-700 nm

62.

All

refolded samples were incubated in the presence of Nile red (20 µl) in distilled water pH 1.6 at room temperature. The following observations can be made from measurements of fluorescence spectra of Nile red shown in Fig. 12 (a). (i) Fluorescence intensity is almost zero in the presence of the native protein, suggesting an absence of exposed hydrophobic patches in the native protein. (ii) Fluorescence intensity in the presence of denatured protein is very high, suggesting presence of exposed hydrophobic patches on the protein. (iii) Fluorescence intensity decreases in the presence of refolded proteins suggesting burial of exposed hydrophobic patched due protein refolding. (iv) Refolding monitored by fluorescence measurements increases with an increase in the number of β-CD attached to APES-MNPs-Sepharose. (v) Refolding of the denatured protein at 250 mg β-CD/ml of Sepharose is the same as 200 mg β-CD/ml of Sepharose. Fig. 12 (b) shows functional activity measurements on the native, denatured and refolded proteins proportion to time. Fig. 12 (c) illustrates that specific enzyme activity of α-amylase increase with an increase in the concentration of β-CD attached to the modified Sepharose (APES-MNPs-Sepharose) from 50200 mg β-CD/ml of Sepharose in the optimal codition (37 ºC and 90 min, pH 7.4). Above this concentration refolding decreased. The far-UV CD, Trp fluorescence, Nile red fluorescence and enzyme activity measurements suggest that refolding ability of β-CD attached to the modified Sepharose (APES-MNPsSepharose) (i) increases with an increase in the concentration of β-CD from 50 to 200 mg/ml of Sepharose, and (ii) decrease at β-CD concentration above 200 mg/ml. A most probable

ACS Paragon Plus Environment

28

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 41

explanation of the latter observation is that the access to the cyclodextrin hydrophobic pockets was hindered at the highest concentration of β-CD.

ACS Paragon Plus Environment

29

Page 31 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 12. (a) Nile red fluorescence spectra of native, refolded, unfolded α-amylase (2 mg/ml), (b) Enzyme activity assay of refolded α-amylase (2 mg/ml) with different columns versus time, (c) Specific enzyme activity of refolded α-amylase after 90 minutes incubation at 37 ºC in buffer pH 7.4.

4. CONCLUSION A novel hydrophobic chromatography column was prepared by grafting artificial chaperones on the MNPs which immobilized on the Sepharose surface. β-cyclodextrin showed high molecular chaperone activity in the solid-state method. Our results demonstrate that the maxim refolding yield observed reaching 77% in case of chemically denatured α-amylase. The far-UV CD spectra, intrinsic fluorescence, Nile red fluorescence and enzyme assay confirmed that the structure of the refolded α-amylase eluted from the column with attached β-CD at a concentration of 200 mg/ml of Sepharose, is the same as the native form. Hence β-CD -APESMNPs immobilized on the Sepharose (resin) surface could be used as a suitable stationary phase in a chromatography column assisting protein renaturation.

AUTHOR INFORMATION Corresponding Author *Email: [email protected].

Notes

ACS Paragon Plus Environment

30

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 41

The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to acknowledge the financial support of the University of Tehran.

ACS Paragon Plus Environment

31

Page 33 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

REFERENCES 1. Jungbauer, A.; Kaar, W., Current Status of Technical Protein Refolding. Journal of Biotechnology 2007, 128, 587-596. 2. Mitraki, A.; King, J., Protein Folding Intermediates and Inclusion Body Formation. Nature Biotechnology 1989, 7, 690-697. 3. Clark, E. D. B., Refolding of Recombinant Proteins. Current Opinion in Biotechnology 1998, 9, 157-163. 4. Middelberg, A. P., Preparative Protein Refolding. TRENDS in Biotechnology 2002, 20, 437-443. 5. Walther, C.; Mayer, S.; Sekot, G.; Antos, D.; Hahn, R.; Jungbauer, A.; Dürauer, A., Mechanism and Model for Solubilization of Inclusion Bodies. Chemical Engineering Science 2013, 101, 631-641. 6. Eiberle, M. K.; Jungbauer, A., Technical Refolding of Proteins: Do We Have Freedom to Operate? Biotechnology Journal 2010, 5, 547-559. 7. Clark, E. D. B., Protein Refolding for Industrial Processes. Current Opinion in Biotechnology 2001, 12, 202-207. 8. Swietnicki, W., Folding Aggregated Proteins into Functionally Active Forms. Current Opinion in Biotechnology 2006, 17, 367-372.

ACS Paragon Plus Environment

32

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 41

9. Singh, S. M.; Sharma, A.; Upadhyay, A. K.; Singh, A.; Garg, L. C.; Panda, A. K., Solubilization of Inclusion Body Proteins Using N-Propanol and Its Refolding into Bioactive Form. Protein Expression and Purification 2012, 81, 75-82. 10. Pan, S.; Zelger, M.; Hahn, R.; Jungbauer, A., Continuous Protein Refolding in a Tubular Reactor. Chemical Engineering Science 2014, 116, 763-772. 11. Bao, X.; Xu, L.; Lu, X.; Jia, L., Optimization of Dilution Refolding Conditions for a Camelid Single Domain Antibody against Human Beta-2-Microglobulin. Protein expression and Purification 2016, 117, 59-66. 12. Yamaguchi, S.; Yamamoto, E.; Mannen, T.; Nagamune, T., Protein Refolding Using Chemical Refolding Additives. Biotechnology Journal 2013, 8, 17-31. 13. Yang, L.; Xiaoyan, D.; Yan, S., New Development of Reverse Micelles and Applications in Protein Separation and Refolding. Chinese Journal of Chemical Engineering 2008, 16, 949955. 14. Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S., Protein− Nanoparticle Interactions: Opportunities and Challenges. Chemical Reviews 2011, 111, 5610-5637. 15. Xu, C.; Qu, X., Cerium Oxide Nanoparticle: A Remarkably Versatile Rare Earth Nanomaterial for Biological Applications. NPG Asia Materials 2014, 6, e90. 16. Lynch, I.; Dawson, K. A., Protein-Nanoparticle Interactions. Nano Today 2008, 3, 40-47. 17. De, M.; Rotello, V. M., Synthetic “Chaperones”: Nanoparticle-Mediated Refolding of Thermally Denatured Proteins. Chemical Communications 2008, 3504-3506.

ACS Paragon Plus Environment

33

Page 35 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

18. Filipe, V.; Hawe, A.; Jiskoot, W., Critical Evaluation of Nanoparticle Tracking Analysis (Nta) by Nanosight for the Measurement of Nanoparticles and Protein Aggregates. Pharmaceutical Research 2010, 27, 796-810. 19. Kameta, N.; Masuda, M.; Shimizu, T., Soft Nanotube Hydrogels Functioning as Artificial Chaperones. ACS Nano 2012, 6, 5249-5258. 20. Talaga, D. S.; Li, J., Single-Molecule Protein Unfolding in Solid State Nanopores. Journal of the American Chemical Society 2009, 131, 9287-9297. 21. Oukhaled, A.; Cressiot, B.; Bacri, L.; Pastoriza-Gallego, M.; Betton, J.-M.; Bourhis, E.; Jede, R.; Gierak, J.; Auvray, L. c.; Pelta, J., Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force. ACS Nano 2011, 5, 3628-3638. 22. Kumada, Y.; Hamasaki, K.; Nakagawa, A.; Sasaki, E.; Shirai, T.; Okumura, M.; Inoue, M.; Kishimoto, M., Immobilization and Functional Reconstitution of Antibody Fab Fragment by Solid-Phase Refolding. Journal of Immunological Methods 2013, 400, 70-77. 23. Lambrecht, M. A.; Rombouts, I.; Van Kelst, L.; Delcour, J. A., Impact of Extraction and Elution Media on Non-Size Effects in Size Exclusion Chromatography of Proteins. Journal of Chromatography A 2015, 1415, 100-107. 24. Saremirad, P.; Wood, J. A.; Zhang, Y.; Ray, A. K., Oxidative Protein Refolding on Size Exclusion Chromatography at High Loading Concentrations: Fundamental Studies and Mathematical Modeling. Journal of Chromatography A 2014, 1370, 147-155.

ACS Paragon Plus Environment

34

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 41

25. Saremirad, P.; Wood, J. A.; Zhang, Y.; Ray, A. K., Oxidative Protein Refolding on Size Exclusion Chromatography: From Batch Single-Column to Multi-Column Counter-Current Continuous Processing. Chemical Engineering Science 2015, 138, 375-384. 26. Saremirad, P.; Wood, J. A.; Zhang, Y.; Ray, A. K., Multi-Variable Operational Characteristic Studies of on-Column Oxidative Protein Refolding at High Loading Concentrations. Journal of Chromatography A 2014, 1359, 70-75. 27. Freydell, E. J.; van der Wielen, L.; Eppink, M.; Ottens, M., Ion-Exchange Chromatographic Protein Refolding. Journal of Chromatography A 2010, 1217, 7265-7274. 28. Chen, Y.; Leong, S. S. J., Adsorptive Refolding of a Highly Disulfide-Bonded Inclusion Body Protein Using Anion-Exchange Chromatography. Journal of Chromatography A 2009, 1216, 4877-4886. 29. Nian, R.; Tan, L.; Yoo, I.-K.; Choe, W.-S., Chaperone-Assisted Column Refolding of Gloshedobin with the Use of Refolding Cocktail. Journal of Chromatography A 2008, 1214, 4758. 30. Mondal, K.; Bohidar, H. B.; Roy, R. P.; Gupta, M. N., Alginate-Chaperoned Facile Refolding of Chromobacterium Viscosum Lipase. Biochimica et Biophysica Acta (BBA)Proteins and Proteomics 2006, 1764, 877-886. 31. Wang, F.; Liu, Y.; Li, J.; Ma, G.; Su, Z., On-Column Refolding of Consensus Interferon at High Concentration with Guanidine-Hydrochloride and Polyethylene Glycol Gradients. Journal of Chromatography A 2006, 1115, 72-80.

ACS Paragon Plus Environment

35

Page 37 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

32. Li, J.-J.; Venkataramana, M.; Sanyal, S.; Janson, J.-C.; Su, Z.-G., Immobilized ΒCyclodextrin Polymer Coupled to Agarose Gel Properly Refolding Recombinant Staphylococcus Aureus Elongation Factor-G in Combination with Detergent Micelle. Protein Expression and Purification 2006, 45, 72-79. 33. Lu, D.; Liu, Z.; Zhang, M.; Wang, X.; Liu, Z., Dextran-Grafted-Pnipaam as an Artificial Chaperone for Protein Refolding. Biochemical Engineering Journal 2006, 27, 336-343. 34. Jhamb, K.; Jawed, A.; Sahoo, D. K., Immobilized Chaperones: A Productive Alternative to Refolding of Bacterial Inclusion Body Proteins. Process Biochemistry 2008, 43, 587-597. 35. Rozema, D.; Gellman, S. H., Artificial Chaperone-Assisted Refolding of Carbonic Anhydrase B. Journal of Biological Chemistry 1996, 271, 3478-3487. 36. Sawada, S.-i.; Sasaki, Y.; Nomura, Y.; Akiyoshi, K., Cyclodextrin-Responsive Nanogel as an Artificial Chaperone for Horseradish Peroxidase. Colloid and Polymer Science 2011, 289, 685-691. 37. Sasaki, Y.; Akiyoshi, K., Nanogel Engineering for New Nanobiomaterials: From Chaperoning Engineering to Biomedical Applications. The Chemical Record 2010, 10, 366-376. 38. Machida, S.; Ogawa, S.; Xiaohua, S.; Takaha, T.; Fujii, K.; Hayashi, K., Cycloamylose as an Efficient Artificial Chaperone for Protein Refolding. FEBS Letters 2000, 486, 131-135. 39. Takeda, S.; Takahashi, H.; Sawada, S.-i.; Sasaki, Y.; Akiyoshi, K., Amphiphilic Nanogel of Enzymatically Synthesized Glycogen as an Artificial Molecular Chaperone for Effective Protein Refolding. RSC Advances 2013, 3, 25716-25718.

ACS Paragon Plus Environment

36

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 41

40. Wang, F.; Liu, Y.; Chen, J.; Su, Z., Chromatographic Refolding of Proteins: Molecular Action and Column Control. China Particuology 2005, 3, 337-342. 41. Kim, Y. E.; Hipp, M. S.; Bracher, A.; Hayer-Hartl, M.; Ulrich Hartl, F., Molecular Chaperone Functions in Protein Folding and Proteostasis. Annual Review of Biochemistry 2013, 82, 323-355. 42. Wang, G. Z.; Dong, X. Y.; Sun, Y., Acyl Cystamine: Small‐Molecular Foldase Mimics Accelerating Oxidative Refolding of Disulfide‐Containing Proteins. Biotechnology Progress 2011, 27, 377-385. 43. Yamaguchi, S.; Hong, C.; Mannen, T.; Tsukiji, S.; Nagamune, T., Solid-Phase Artificial Chaperone-Assisted Refolding Using Insoluble Β-Cyclodextrin–Acrylamide Copolymer Beads. Biotechnology Letters 2004, 26, 1787-1791. 44. Esmaeili, M. A.; Yazdanparast, R., Solid-Phase Assisted Refolding of Carbonic Anhydrase Using Β-Cyclodextrin-Polyurethane Polymer. The Protein Journal 2008, 27, 334. 45. Jimmy, C. Y.; Jiang, Z.-T.; Liu, H.-Y.; Yu, J.; Zhang, L., Β-Cyclodextrin Epichlorohydrin Copolymer as a Solid-Phase Extraction Adsorbent for Aromatic Compounds in Water Samples. Analytica Chimica Acta 2003, 477, 93-101. 46. Emadi, H.; Kharat, A. N., Single Source Preparation of Superparamagnetic Fe 3 O 4 Nanoparticles by Simple Cyclic Microwave Approach. Materials Research Bulletin 2013, 48, 3994-4001.

ACS Paragon Plus Environment

37

Page 39 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

47. Liu, X.; Ma, Z.; Xing, J.; Liu, H., Preparation and Characterization of Amino–Silane Modified Superparamagnetic Silica Nanospheres. Journal of Magnetism and Magnetic Materials 2004, 270, 1-6. 48. Zhu, J.; He, J.; Du, X.; Lu, R.; Huang, L.; Ge, X., A Facile and Flexible Process of ΒCyclodextrin Grafted on Fe 3 O 4 Magnetic Nanoparticles and Host–Guest Inclusion Studies. Applied Surface Science 2011, 257, 9056-9062. 49. Wang, H.; Zhou, Y.; Guo, Y.; Liu, W.; Dong, C.; Wu, Y.; Li, S.; Shuang, S., ΒCyclodextrin/Fe 3 O 4 Hybrid Magnetic Nano-Composite Modified Glassy Carbon Electrode for Tryptophan Sensing. Sensors and Actuators B: Chemical 2012, 163, 171-178. 50. Bethell, G.; Ayers, J.; Hearn, M.; Hancock, W., Investigation of the Activation of Various Insoluble Polysaccharides with 1, 1′-Carbonyldiimidazole and of the Properties of the Activated Matrices. Journal of Chromatography A 1987, 219, 361-371. 51. Besselink, G.; Beugeling, T.; Bantjes, A., N-Hydroxysuccinimide-Activated GlycineSepharose. Applied Biochemistry and Biotechnology 1993, 43, 227. 52. Tang, W.; Ng, S.-C., Facile Synthesis of Mono-6-Amino-6-Deoxy-Α-, Β-, Γ-Cyclodextrin Hydrochlorides for Molecular Recognition, Chiral Separation and Drug Delivery. Nature Protocols 2008, 3, 691-697. 53. Vohra, R. S.; Murphy, J. E.; Walker, J. H.; Homer-Vanniasinkam, S.; Ponnambalam, S., Functional Refolding of a Recombinant C-Type Lectin-Like Domain Containing Intramolecular Disulfide Bonds. Protein Eexpression and Purification 2007, 52, 415-421.

ACS Paragon Plus Environment

38

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 41

54. Altamirano, M. M.; Golbik, R.; Zahn, R.; Buckle, A. M.; Fersht, A. R., Refolding Chromatography with Immobilized Mini-Chaperones. Proceedings of the National Academy of Sciences 1997, 94, 3576-3578. 55. Luo, M.; Guan, Y.-X.; Yao, S.-J., On-Column Refolding of Denatured Lysozyme by the Conjoint Chromatography Composed of Sec and Immobilized Recombinant Dsba. Journal of Chromatography B 2011, 879, 2971-2977. 56. Gao, Y. G.; Guan, Y. X.; Yao, S. J.; Cho, M. G., On‐Column Refolding of Recombinant Human Interferon‐Γ with an Immobilized Chaperone Fragment. Biotechnology Progress 2003, 19, 915-920. 57. Bernfeld, P., Enzymes of Starch Degradation and Synthesis. Advances in Enzymology and Related Areas of Molecular Biology 1951, 12, 379-428. 58. Feng, B.; Hong, R.; Wang, L.; Guo, L.; Li, H.; Ding, J.; Zheng, Y.; Wei, D., Synthesis of Fe 3 O 4/Aptes/Peg Diacid Functionalized Magnetic Nanoparticles for Mr Imaging. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2008, 328, 52-59. 59. Liu, H.; Dong, X.; Sun, Y., Grafting Iminodiacetic Acid on Silica Nanoparticles for Facilitated Refolding of Like-Charged Protein and Its Metal-Chelate Affinity Purification. Journal of Chromatography A 2016, 1429, 277-283. 60. Whitmore, L.; Wallace, B., Dichroweb, an Online Server for Protein Secondary Structure Analyses from Circular Dichroism Spectroscopic Data. Nucleic Acids Research 2004, 32, W668W673.

ACS Paragon Plus Environment

39

Page 41 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

61. Whitmore, L.; Wallace, B. A., Protein Secondary Structure Analyses from Circular Dichroism Spectroscopy: Methods and Reference Databases. Biopolymers 2008, 89, 392-400. 62. Hawe, A.; Sutter, M.; Jiskoot, W., Extrinsic Fluorescent Dyes as Tools for Protein Characterization. Pharmaceutical Research 2008, 25, 1487-1499.

TOC Graphic

ACS Paragon Plus Environment

40