5th International Congress on Nanoscience & Nanotechnology (ICNN2014)

TMU Iranian Nanotechnology Society

138

Highly efficient purification of 6×histidine-tagged recombinant protein-A by MnFe2O4@NH2@2AB-Ni as a novel and highly stable magnetic nanoparticles

H. Naeimia, Z. Rashida, M. Nazarib, M. R. Nejadmoghaddamb, R. Ghahremanzadehb*

aDepartment of Organic Chemistry, Faculty of Chemistry, University of Kashan, Kashan, 87317, I.R. Iran.

bNanobiotechnology Research Center, Avicenna Research Institute, ACECR, 1936773493, Tehran, Iran. r.ghahremanzadeh@yahoo.com.

Abstract: In this study we have developed a simple, cheap, and rapid method for purification of 6×histidine-tagged recombinant proteins with high yields. The new immobilized metal ion affinity adsorbent was synthesized following co-precipitation method in order to synthesis of superparamagnetic manganes ferrit nanoparticles, and subsequently coated with 3-aminopropyltriethoxysilane (APTES) through a silanization reaction. Manganese ferrite nanoparticles afforded bidentate ligands as a result of reaction between isatoic anhydride and amino-functionalized MnFe2O4. The stable surface functionalized nanoparticles were further linked with Ni2+ for purification of 6× His-tagged proteins. The nanoparticles were approximately 75 nm in size and were stable and had negligible non-specific binding for protein. Keywords:Magnetic nanoparticles, Immobilized metal ion affinity, 6×His-tag protein, Proteins purification.

Introduction Along with the revolutionary development of methods for the efficient expression of recombinant proteins, fast and convenient systems to purify target proteins from complex protein mixtures have become increasingly necessary. Affinity chromatography that exploits the unique property of extremely specific biological interactions to achieve separation and purification. Among a variety of affinity chromatograph formats, the 6×His fusion tag technique has gained wide popularity since it was invented [1]. 6×His tag can be easily fused to the proteins of interest without impairing their function due to its small size, and the tagged proteins can be purified with no extra expensive reagents or complicated treatments. Typically, the whole purification can be finished in a single capture step using immobilized metal–ion affinity chromatography (IMAC) [2]. Although IMAC is powerful tool in terms of selectivity and recovery, while it is easy to handle, the column format for the agarose beads is of low throughput [3]. With the demanding of large scale or high throughput purification, magnetic beads support has gained increasing popularity due to their unique advantages in simple handling, fast purification, high throughput, and ease in automation. Currently commercially available agarose-magnetic beads are rather expensive for routine use. Alternatively, there have been reports of preparation of different types of magnetic particles for this aim [4, 5]. This report presents a synthesis of a novel and highly stable magnetic nanoparticles through functionalization of magnetic nanoparticles (MnFe2O4) with 3-aminopropyltriethoxysilane (APTES) followed by the grafting with 2-amino bezamide as a result of the reaction between isatoic anhydride and amino-functionalized nanoparticles afforded (MnFe2O4@NH2@2AB), and

finally reacted with nickel acetate, and a novel magnetic nanoparticles (MnFe2O4@NH2@2AB-Ni) was produced. The product used as magnetic adsorbent for purification of 6×histidine-tagged recombinant proteins directly from bacterial lysates. The result indicated thatMnFe2O4@NH2@2AB-Ni nanoparticles exhibited higher selectivity for the target protein and the separated protein has higher purity from the crude bacterial lysate. Scheme 1 shows the sequence of procedures in the synthesis of MnFe2O4@NH2@2AB-Ni.

Si

NH2

EtOEtO

EtO

OOO

Si NH2

NH

H2N

Si

OO

OO

NSiO

OO Ni

NH

O

H2O

H2O

MeOH, N

i(OAc) 2

reflux

NH

O

O

O

ref lux , -CO2

MeOH

OH

OH

OHHO

HO

HO

OH

OH

MeO

H

Nano MnFe2O4

MnFe2O4@NH2

MnFe2O4@NH2@2AB

MnFe2O4@NH2@2AB-Ni

Scheme 1. Steps for fabricating nickel nanoparticle immobilized

on the surface of superparamagnetic nanoparticles

Materials and method The chemicals used in this work were obtained from Fluka, Merck, and Aldrich and were used without purification. a) Synthesis of MnFe2O4 nanoparticle: MnFe2O4 nanoparticles has been prepared by co- precipitation of MnCl2 and FeCl3 in water in the presence of sodium hydroxide. Briefly, MnCl2.4H2O and

5th International Congress on Nanoscience & Nanotechnology (ICNN2014)

TMU Iranian Nanotechnology Society

139

FeCl3.6H2O were taken in molar ratio of Mn2+: Fe3+ = 1:2 to prepare 0.3 mol.L-1 metal ion solution of 100 ml containing 0.1 mol.L-1 Mn2+ and 0.2 mol.L-1 Fe3+, then was slowly dropped into 100 ml NaOH solution of 3 mol.L-1 at the preheated temperature of 95°C. After aging for 2 h with continuous stirring, the mixture was filtered, washed and dried at 60°C for 12 h. b) Synthesis of amino-functionalized magnetic nanoparticles:

The superparamagnetic nanoparticles (2.2 g) were dispersed in a mixture of ethanol and water (300 ml, 1:1 by volume) and sonicated for 30 min at room temperature. APTES (2.11 ml) was then added, and the solution was heated at 60 ºC with vigorous stirring for 36 h. The final product was washed with deionized water and ethanol by magnetic decantation, and dried under vacuum at room temperature overnight to yield amino functionalized magnetic nanoparticles. c) Grafting of 2-amino benzamide on functionalized magnetic nanoparticles

The amino-functionalized magnetic nanoparticles (0.10 g) were suspended in ethanol (99.5%, 100 ml) with sonication, and then mechanically stirred for 30 min to form a uniform dispersion. To this mixture was added isatoic anhydride (0.016 g, 0.1 mmol) and the resulted mixture was refluxed for 12 h. The prepared functionalized magnetic nanoparticles were separated by magnetic decantation and then washed with ethanol several times to remove the unreacted isatoic anhydride and dried under vacuum at 50 ºC. d) Immobilization of nickel on magnetic nanoparticles

For this aim, 2-amino benzamide immobilized magnetic nanoparticles (0.10 g) were ultrasonically dispersed in 100 ml of ethanol to form a homogeneous dispersion, and then loaded into a round-bottomed flask. Ni(OAc)2.4 H2O (0.012 g, 0.05 mmol) was added to this solution. The mixture was refluxed for 12 h. After stirring, the catalyst was harvested by aid of magnet, washed several times with ethanol to remove unreacted Ni(OAc)2. 4H2O and dried under vacuum at 50 ºC. e) Protein binding, enrichment and separation using MnFe2O4@NH2@2AB-Ni

25 mg of magnetic nanoparticles was taken and washed with buffer (50 mM sodium phosphate, 100 mM NaCl, and pH 7.4) and vortex/sonicated properly for uniform dispersion of the particles. Sonicated cell lysate was then incubated with magnetic particles at 37ºC for 1 h. Magnetic nanoparticles were separated by magnetic concentrator and washed thoroughly with 500 µL wash buffer (50 mM sodium phosphate, 100 mM NaCl, and 50 mM imidazole) to remove all unbound proteins. The recombinant bound protein was then eluted with 250 µL elution buffer (50 mM sodium phosphate, 100 mM NaCl, and various imidazole conc.). The beads were separated

by magnetic separator and the supernatant containing the desired protein was collected boiled with 5x sample buffer and verified in 12% SDS-PAGE gel using Coomassie brilliant blue stain. Results and Discussion Characterization of MnFe2O4@NH2@2AB-Ni FT-IR spectra showed the presence of Fe–O stretching vibration at approximately 580 cm−1, O–H stretching vibration near 3400 cm−1and1630 cm−1 (Fig. 1 a). The significant features observed for the amine-functionalized nanoparticles, were the appearance of the peaks near 1015 cm−1 for Si–O stretching, the peak near 2950 cm−1 due to the –CH2 stretching vibrations and the peak at 3423 cm-1 was probably attributed to the free amino groups, which is overlapped by the O–H stretching vibration (Fig. 1 b). For the immobilized ligand and the catalyst, some weak bands at 1400–1500 cm−1 assigned to stretching vibrations of aromatic rings. In the ligand, the C=O stretch of the amide appeared at 1600 cm-1 which was overlapped with the O–H deformation vibration (Fig. 1 c). While in the complex, this band shifted to higher frequency and appears at 1715 cm−1 because of coordination of the nitrogen with Ni(II) that indicating complexation of amide group of supported ligand with nickel (Fig. 1 d). Fig. 1 The comparative FT-IR spectra for (a) MnFe2O4 (b) MnFe2O4-NH2 (c) MnFe2O4-NH2-2AB (d) MnFe2O4-NH2-2AB-Ni.

Figure 2 (A) presents the XRD-diffraction patterns of the prepared MnFe2O4, MnFe2O4@NH2, MnFe2O4@NH2@2AB, and MnFe2O4@NH2@2AB-Ni. The position and relative intensities of all peaks confirm well with standard XRD pattern of MnFe2O4 indicating retention of the crystalline cubic spinel structure during functionalization of MNPs. It is implied that the resultant nanoparticles are pure MnFe2O4 with a spinel structure and that the grafting process did not induced any phase change of MnFe2O4.

4000 3000 2000 1000

1715

.00

2922

.00

2852

.65

576.

73

1022

.61

1455

.09

1629

.42

3437

.98

Tra

nsm

ittan

ce (%

)

Wavelength (cm-1)

(a)

(b)

(c)

(d)

20 40 60 80

(440

)(5

33)

(333

)

(422

)(3

31)

(400

)

(311

)

(220

)

(d)

(c)

(b)

Position (2Theta)

(a)

5th International Congress on Nanoscience & Nanotechnology (ICNN2014)

TMU Iranian Nanotechnology Society

140

Fig. 2 XRD patterns of (a) MnFe2O4 (b) MnFe2O4-NH2 (c) MnFe2O4-NH2-2AB (d) MnFe2O4-NH2-2AB-Ni.

The size and morphology of MnFe2O4 and the immobilized nickel complex nanoparticle were investigated by scanning electron microscopy (SEM). According to Figure 3 (a), the MnFe2O4 nanoparticles exhibited spherical particles with an average size 30 nm in diameter. SEM image of the particles after functionalization, revealed agglomerates of spherical particles with larger average size 70 nm Figure 3 (b).

Fig. 3The SEM image of (a) MnFe2O4 and (b) MnFe2O4-NH2-2AB-Ni. The successful multi-step modified MnFe2O4 core by APTES and 2AB could also be confirmed by TGA analysis that showed in Figure 4 (A). Vibrating sample magnetometry (VSM) was employed to study the magnetic properties of the synthesized magnetic nanoparticles. The saturation magnetization value of pure MnFe2O4 at room temperature was 54 emu/g. The saturation magnetization of MnFe2O4-NH2-2AB-Ni. was reduced to 38emu/g. Both of them showed superparamagnetic behavior(Figure 5 (B) and (C)).

Fig. 4(A): TGA curves of (a) MnFe2O4@NH2 (b) MnFe2O4@NH2@2AB (c) MnFe2O4@NH2@2AB-Ni. (B): Magnetization curves for the prepared MnFe2O4 (C) MnFe2O4@NH2@2AB-Ni at 40 ºC

To demonstrate that the MnFe2O4-NH2-2AB-Ni selectively bind only the Histag protein, and hence are capable of purifying His-tagged proteins, batch adsorption of His-

tagged Pro A from Escherichia coli extracts was conducted, followed by elution using imidazole and subjecting the eluents to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. As shown in Figure 5, the recombinant His-tagged pro A (68 kDa) has been successfully eluted using 250 mM imidazole, high purity of His-tagged Pro A can be obtained using the MnFe2O4-NH2-2AB-Ni. The desired protein band was seen in the eluted fraction.

Fig. 5. 12% SDS-PAGE analysis of purified recombinant protein A by synthesized magnetic nanoparticle. Lane 1: total soluble protein, Lane 2: Flow through, Lane 3: wash 1, Lane 4: wash 4, Lane 5: elution 1 (E1, 100 mM imidazole), Lane 6: elution 2 (E2, 250 mM imidazole), Lane 7: (E3, 250 mM imidazole), Lane 8: (E4, 500 mM imidazole).

Conclusions In summary, this work provides a facile and efficient synthetic route for the preparation of a magnetic core/functionalized immobilized metal affinity ligand shell structure, as a novel and highly efficient for purification of 6×histidine-tagged recombinant protein A. The MNPs exhibit a high adsorption capacity and excellent specifity towards 6×histidine-tagged recombinant proteins.

References [1] C.F. Ford, I. Suominen, C.E. Glatz, Protein Exp. Purif. 2 (1991) 95. [2] S. Gibert, N. Bakalara, X. Santarelli, J. Chromatogr. B 737 (2000) 143. [3] S. Sahu,A. Chakrabarty, D. Bhattacharya, S.K. Ghosh, P. Pramanik, J Nanopart Res 13 (2011) 2475. [4] M.A. Kadir, S.J. Kim, E. Ha, H.Y. Cho, B.S. Kim, D. Choi, S.G. Lee, B.G. Kim, S.W. Kim, H.j. Paik, Adv. Funct. Mater. 22 (2012) 4032.

a b

1 2 3 4

68kDa

5 6 7 8

5th International Congress on Nanoscience & Nanotechnology (ICNN2014)

TMU Iranian Nanotechnology Society

141

[5] J. Cao, X. Zhang, X. He, L. Chen, Y. Zhang, J. Mater. Chem. B, 1 (2013) 3625.