Makara Journal of Science 19/4 (2015) 137-142

doi: 10.7454/mss.v19i4.5167

Comparison of Immobilized Metal Affinity Chromatography Ni-NTA and

Co-TALON for the Purification of Recombinant Human Erythropoietin

Yana Rubiyana1*, Adi Santoso1, and Irmanida Batubara2

1. Protein Based Terapeutik and Vaccine laboratory, Research Center for Biotechnology,
Indonesian Institute of Science (LIPI), Bogor 16911, Indonesia
2. Department of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor,
Bogor 16680, Indonesia

*E-mail: [email protected]

Abstract
The purification of recombinant proteins is an important stage in biopharmaceutical research. A commonly used
technique is immobilized metal affinity chromatography (IMAC). One of the main advantages of this type of
chromatography is that the column can easily be regenerated for subsequent purification work. The mechanism of
IMAC is based on bonding between metal ions immobilized on a matrix with a specific amino acid. Because of the
strong interactions of the electron donor group on the imidazole ring, histidine is often used in the IMAC purification
system. Two types of commercial IMAC resin use a nitrilotriacetic acid (NTA) matrix: a nickel-based (Ni-NTA) and
cobalt-based (Co-NTA), better known as TALON. This study was aim to investigate the effect of the metal ions Ni2+
and Co2+ to purify recombinant human erythropoietin (rhEPO) expressed in yeast system Pichia pastoris. The results
indicated that both Ni-NTA and Co-TALON gave almost the same level of protein purity; however, Ni-NTA has a
higher binding affinity than Co-TALON might be due to the higher stability complex of Ni+. The average amount of
protein bound by Ni-NTA and Co-TALON was 183.5 and 38.7 g/mL, respectively.

Abstrak
Perbandingan Immobilized Metal Affinity Chromatography Ni-NTA dan Co-TALON untuk Pemurnian
Rekombinan Erythropoietin Manusia. Teknik pemurnian rekombinan protein merupakan tahap yang sangat penting
dalam riset biofarmasetik. Teknik yang umum digunakan adalah kromatografi afinitas logam terimobilisasi (IMAC).
Salah satu kelebihan dari kromatografi jenis ini adalah kolom dapat dengan mudah diregenerasi untuk digunakan pada
proses purifikasi berikutnya. Mekanisme purifikasi IMAC berdasarkan pada ikatan yang sangat spesifik antara metal
ion yang terimobilisasi pada matriks dengan asam amino tertentu. Karena interaksinya yang kuat sebagai kelompok
donor elektron pada cincin imidazol, maka asam amino histidin sering digunakan pada sistem purifikasi IMAC.
Terdapat 2 jenis resin IMAC komersial yang menggunakan matriks asam nitriloasetat (NTA), yaitu resin IMAC
berbasis-nikel (Ni-NTA) dan berbasis-kobalt (Co-NTA) atau lebih dikenal dengan TALON. Penelitian ini bertujuan
untuk mengetahui pengaruh penggunaan ion logam Ni2+ dan Co2+ dalam memurnikan protein rekombinan erithropoietin
manusia (rhEPO) yang diekspesikan dalam sistem ragi Pichia pastoris. Hasil penelitian menunjukkan bahwa baik Ni-
NTA maupun Co-TALON mempunyai tingkat kemurnian yang hampir sama, namun Ni-NTA mempunyai afinitas
pengikatan yang lebih tinggi dibandingkan dengan Co-TALON kemungkinan disebabkan oleh kestabilan komplek
logam Ni yang lebih tinggi. Jumlah rata-rata protein yang diikat oleh Ni-NTA dan Co-TALON masing-masing sebesar
183.5 g/mL dan 38.7 g/mL.

Keywords: Co-TALON, IMAC, Ni-NTA, purification, rhEPO

Introduction hEPO into recombinant human erythropoietin (rhEPO)

as a drug. Since 1980, rhEPO has been a major commodity
Research on human erythropoietin (hEPO) has increased in the biotechnology industry; sales have increased from
as researchers attempt to obtain therapeutic agents. year to year until they reached billions of dollars [1-2].
Cloning and gene expression techniques have developed rhEPO has been used for clinical treatment of patients

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138 Rubiyana, et al.

with anemia caused by cancer, human immunodeficiency polyclonal anti-EPO antibodies. The highest expressing
virus infection (HIV) infection, kidney failure, and bone clone was selected for subsequent study.
marrow transplantation [3].
Recombinant human erythropoietin (rhEPO) pro-
The Research Center for Biotechnology, Indonesian duction. The P. pastoris transformant containing the
Institute of Sciences (LIPI) expressed recombinant -hEPO hEPO gene was grown on the YPD medium (1% Bacto
in the yeast expression system (Pichia pastoris) and yeast, 2% Bacto peptone, 2% glucose 20% and 2% Bacto
showed with the western blotting technique that the agar) at 30 C for 2 days until the clones appeared. The
supernatant contained rhEPO. For the next stage, appro- clones were screened with western blot analysis using
priate purification techniques are required to obtain pure human polyclonal anti-EPO antibodies. The highest-
protein. A purification technique that utilizes specific expressing clone was selected for the study. A single
interactions between the protein and a ligand (metal colony was grown in the BMGY medium (1% yeast, 2%
ions) has been developed. This technique is known as peptone, potassium phosphate 100 mM, 1.34% yeast
immobilized metal affinity chromatography (IMAC) [4-5]. nitrogen base, 0.00004% biotin, and 1% glycerol) at 30 C
Depending on the specific interactions, IMAC can be for 1 day (24 h). Growth of the cells was then resumed
used to purify protein at up to 95% purity [6]. at a concentration of optical density (OD) 1 in the BMMY
medium (the BMGY medium with 1% glycerol was
IMAC utilizes specific interactions between the side replaced with 0.5% methanol) at 30 C for 2 days. After
chains of amino acids with borderline Lewis metal ions 2 days, the cell culture was centrifuged for 10 min at
(such as Cu2+, Co2+, Ni2+, and Zn2+) [7-11]. Metal ions 2,300 g. The supernatant was then analyzed with the
immobilized through a chelating agent attach to the western blot technique [14].
stationary support. The chelating agents most widely
used for this application are iminodiacetic acid (IDA) Ni2+-NTA/Co2+-TALON IMAC purification. A 200 mL
and nitrilotriacetic acid (NTA) [12]. The target protein supernatant was produced from the same culture. Then
is tagged with hexahistidine (6xHis); thus, there is 10 mL of the supernatant was mixed with the IMAC resin
specific binding between the recombinant protein with a (Ni-NTA/Co-TALON), which had been equilibrated with
ligand of the IMAC. Given the specific binding, the phosphate buffer pH 7.4 (20 mM sodium phosphate and
target protein to purify can be separated from other 500 mM NaCl) and then shaken with a shaker (150 rpm)
proteins [4,8]. Two types of commercial IMAC resin for 60 min. The mixture was inserted into the empty
use NTA matrix as a chelating agent, nickel-based (Ni- column, and then the filtrate samples (flow-through) were
NTA) and cobalt-based (Co-NTA) IMAC resin. The accommodated. The column was washed with 10 mL
latter is well known as TALON. In this study, the washing buffer (20 mM sodium phosphate and 500 mM
affinity of metal ions Ni2+ and Co2+ was compared to NaCl) 1 time. Then the column was eluted with the elution
find a suitable IMAC technique for purifying rhEPO buffer containing 20 mM sodium phosphate, 500 mM
protein. NaCl and 500 mM imidazole. The elution results were
fractionated (each 1 mL) and stored in 1.5 mL polypro-
Materials and Methods pylene tubes [15]. This process was repeated four times.
Subsequently, the fraction solutions were analyzed with
Selection of yeast transformant. Escherichia coli DH5 western blotting and measured with a spectrophotometer.
alpha (Invitrogen, San Diego, CA) was used as the host
for the recombinant plasmid cloning [7] experiment and SDS-PAGE and western blot analysis. To confirm the
grown in LB medium (1% tryptone, 0.5% NaCl and protein purification result, the protein was analyzed with
0.5% yeast extract, plus 2% agar in plates). Zeocin was sodium dodecyl sulfate polyacrylamide gel electrophoresis
added at standard concentrations to screen the bacteria (SDS-PAGE) with a separating gel containing 12%
and the selection of the yeast transformant, P. pastoris acrylamide. Western blot analysis was performed to
strain X-33 (Invitrogen). The yeast was cultured in the detect each protein fraction after purification on IMAC.
yeast peptone dextrose (YPD) medium (1% yeast extract, Following SDS-PAGE, the protein inside the gel was
2% peptone and 2% dextrose, plus 2% agar in plates). transferred into Hybond nitrocellulose membrane (GE
The composition of the media for the expression study Healthcare) by using electroblotting. Immunodetection
was buffered glycerol complex medium (BMGY) (1% was achieved by using an anti-hEPO antibody (Cal-
yeast extract, 2% peptone, 1.34% YNB, 4 10-5% biotin, biochem) as the primary antibody and anti-rabbit IgG
1% glycerol and 0.1 M potassium phosphate, pH = 6.0) peroxidase conjugate (Bio-Rad) as the secondary antibody.
and buffered methanol complex medium (BMMY) The band was visualized with the nitro-blue tetrazolium
(BMGY medium containing 0.5% methanol instead of chloride (NBT) and 5-bromo-4-chloro-3'-indolyphosphate
glycerol). The yeast cultures were grown at 30 C. The p-toluidine salt (BCIP) staining reaction, then the
cell-producing strain was selected based on the expression density of the rhEPO bands was calculated based on
level. Several clones were screened for the level of area under the curve (AUC) determined with ImageJ
expression of the EPO protein with western blotting using software (http://imagej.en.softonic.com).

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 Comparison of Immobilized Metal Affinity Chromatography 139

Analysis of total protein by bicinchoninic acid (BCA) To measure how much rhEPO protein was purified
protein assay kit. A series of standard solutions of bovine using these two types of resin, the amount of protein
serum albumin (BSA) with concentrations of 25, 125, eluates was estimated using BCA protein assay. The assay
250, 500, 750, 1000, 1500, and 2000 g/mL was prepared. showed the average amount of protein purified using
The working solution was prepared by mixing 50 mL of Ni-NTA was 183.5 g/mL, while that of Co-TALON was
solution A (sodium carbonate, sodium bicarbonate and 38.7 g/mL (Table 2). Some small variations were
sodium tartrate bicinchoninic acid in 0.1 M sodium observed within the four replications; however, overall,
hydroxide) with solution B containing 4% Cu-sulfate. A these data clearly correlate with those shown in Figure 2
total of 0.1 mL of standard, blank samples was pipetted
into a clean test tube, and then 2 mL of the working
solution was added to each tube and shaken gently until
well mixed. The mixture was incubated in a water bath
with a temperature of 37 C for 30 min. After the solution
had cooled to room temperature, the solution was
immediately measured using a UV-visible spectro-
photometer at a wavelength of 562 nm [16].

Results and Discussion

Identification of the highest-expressing clone. The
expression and subsequent purification of recombinant
proteins are widely performed in molecular and
biochemical studies. A powerful purification method
Figure 1. Western Blot Analysis of rhEPO. Lane 1: Standard
involving the use of peptide affinity tags, which are
rhEPO, Row 2: Protein Marker, Rows 3, 4, and
fused to the protein of interest and used to expedite 5: Supernatant rhEPO Clones 18, 11, and 9
protein purification via affinity chromatography, was
extensively applied. The commonly used commercial
Ni-based IMAC resin (Ni-NTA) and cobalt-based
IMAC resin (Co-NTA) was compared and analyzed. In
the current experiment, purification of rhEPO, which
had been fused with six histidine amino acid residues
at the carboxy terminal, was studied.

The expression level of the rhEPO protein is shown in

Figure 1. In each lane, a band the same size as the rhEPO
standard, which is about 37 kDa, can be observed. These
results indicate that the rhEPO protein was contained in
the supernatant. The result in lane 3 indicates a greater
number of rhEPO in clone 18. The highest-expressing
clone (clone number 18) was selected for the study.

Quantification of rhEPO protein. Clone 18 was then

expressed for further analysis. For a better idea of how Figure 2. Western Blot Analysis of Purified rhEPO. Lane
effective the use of Ni-NTA and Co-TALON resin was 1: Protein Marker, Lanes 25: Ni-NTA Eluate
for purifying the protein, the expression study using Fractions, Lanes 69: Co-TALON Eluate Fractions
clone number 18 was repeated four times from the same
supernatant. The expressed and purified protein was
then analyzed with western blotting. The result is shown Table 1. Area under Curve (ImageJ) of the rhEPO Bands
in Figure 2. The protein eluates using Ni-NTA are Ni-NTA and Co-TALON
shown in lanes 2, 3, 4, and 5. The protein eluates using Area Under Curve (AUC)
Co-TALON resin are in lanes 6, 7, 8, and 9. The density Repetition
Ni-NTA Co-TALON
of the rhEPO bands was analyzed based on the AUC
determined with ImageJ; the results are shown in Table 1 36533.40 6299.229
1 and Figure 3. This analysis clearly shows that the 2 36717.61 10637.96
density of the rhEPO bands purified using Ni-NTA was 3 35299.56 6916.995
higher than that of Co-TALON, indicating that more 4 21125.56 10348.61
rhEPO protein can be captured with Ni-NTA than with
Average 32419.03 8550.69
Co-TALON.

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140 Rubiyana, et al.

used was much higher than when Co-TALON was used.

These data correlate well with the data shown in Figures
2 and 3 and Tables 1 and 2, where the Ni-NTA resin
was more efficient that Co-TALON, indicating that the
Ni-NTA resin has more binding capacity than Co-
TALON.

With the advent of genetic engineering, it has become

easy to design a protein that has a structure for a
simplified purification procedure. One of the most
popular means to do is by adding 610 histidines on the
N-terminus of a protein [18-20]. The protein is then
purified by its ability to bind tightly to a column that
Figure 3. The average AUC of rhEPO Bands for Ni-NTA contains chelated Ni2+ or Co2+ in which it can be washed
and Co-TALON and then eluted with free imidazole or by lowering the
pH to 5.9, where histidine becomes fully protonated and
no longer binds to the chelated metal.
Table 2. Total Protein of the Eluate Purified with Ni-NTA
and Co-TALON However, although IMAC technology is very promising
Concentration (g/mL) and superior to affinity chromatography, the purity level
Repetition of the protein obtained (Figure 4) is not absolute.
Ni-NTA Co-TALON
Protein impurities can still be recognized in this Silver
1 275.6 63.7 stained gel. These impurities might come from the
2 145.2 41.4 media or the host cell proteins containing histidine
3 206.7 25.4 residues. Subsequently, these proteins may bind to the
4 106.4 24.4 IMAC resin.
Average 183.5 38.7
Although histidine occurs infrequently (2% of all
protein residues are histidine), some cellular proteins
may contain two or more adjacent histidine residues.
These proteins have an affinity for the IMAC matrix
and may coelute with the protein of interest, resulting in
significant contamination of the final product. The
problem of this type of impurity is more common in
eukaryotic cells than in prokaryotic cells. This is
because mammalian cells have a higher natural
abundance of proteins containing consecutive histidine
residues. In addition to the presence of natural
consecutive histidine residues, the formation of a
Figure 4. Silver Staining Analysis of purified rhEPO. disulfide bond between the protein of interest and other
Lanes 14: eluate Ni-NTA, lane 5-7: Eluate
Co-TALON
proteins can also lead to contamination [8, 21]. Thus,
although IMAC is a versatile method that can result in
100-fold enrichments in a single purification step [22]
that demonstrated the Ni-NTA resin bound the rhEPO and can achieve at up to 95% purity, the possibility of
protein higher than Co-TALON. The differences of impurities was still present, especially if the protein of
ligand field stabilization energy (LSFE) influenced the interest is eukaryotic in origin.
geometry stability of metal complex [17]. The relative
value of LFSE Ni+ is higher than LFSE Co+, causing Morganti et al. (2002) reported that aromatic nitrogen-
the complex of Ni is more stable. Therefore, the Ni- containing ligands (e.g., histidine and tryptophan) are
NTA resin can capture more proteins. considered borderline Lewis bases. Metal ions at the
boundary of hard-soft acids such as Co, Zn, Cu, and Ni
Purity of the expressed protein rhEPO. To evaluate can coordinate with the aromatic nitrogen atom (the
the rhEPO protein after it was purified using Ni-NTA base border) and with the sulfur atom (the soft base) [9].
and Co-TALON, the eluates were analyzed using SDS- Therefore, Ni and Co can bind in coordination with
PAGE and stained using Silver staining (Fermentas). other proteins from the media or host cells that also
The result (Figure 4) showed that the rhEPO protein have histidine or tryptophan residues. However, Ni
was clearly observed at the size of, approximately, 37 affinity is greater than Co. The Ni addition to the rhEPO
kDa. The amount of rhEPO purified when Ni-NTA was binding protein can also bind to other proteins more.

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 Comparison of Immobilized Metal Affinity Chromatography 141

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