Appl Biochem Biotechnol (2014) 172:40024012

DOI 10.1007/s12010-014-0818-1

Molecular Modification of Protein A to Improve

the Elution pH and Alkali Resistance in Affinity
Chromatography
Hai-Feng Xia & Zhen-Dong Liang & Sha-Li Wang &
Pu-Qiang Wu & Xiong-Hua Jin

Received: 24 October 2013 / Accepted: 17 February 2014 /

Published online: 6 March 2014
# Springer Science+Business Media New York 2014

Abstract Protein A of Staphylococcus aureus has been widely used as an affinity ligand for
the purification of immunoglobulin. However, the low elution pH and the sensitivity to
alkaline condition restricted the large-scale application of antibody purification. To overcome
these disadvantages, the B domain was selected and mutated to Z domain and the recombinant
Protein A was reconstructed by linking five Z domains. First, a section of six glycines was
inserted into the second loop of Z domain, Z (6G). This increased the elution pH to 4.05.0.
Then, the site-specific mutagenesis was conducted by replacing the 23rd asparagines to
threonine and 30th phenylalanine to alanine, Z (N23T, F30A). These mutations made the
recombinant Protein A shown a higher alkaline resistance than the nature Protein A. The work
confirmed the modification of Protein A and exhibited the characteristics of recombinant
Staphylococcal Protein A for antibody purification.
Keywords Modification . Protein A . Z domain . Affinity chromatography . Elution pH . Alkali
resistance

Introduction
Protein A of Staphylococcus aureus (SPA) is a cell wall protein linking covalently to the
peptidoglycan [1]. SPA is a single polypeptide and has a markedly extended shape [2]. The
conformation of SPA is stable over a wide range of pH (0.9911.8) and partly intact even in
6 M guanidine hydrochloride solution or at the temperature of 80 C [3]. Native Protein A has
H.<F. Xia : Z.<D. Liang : S.<L. Wang : P.<Q. Wu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan
University, Wuxi 214122, China
H.<F. Xia (*)
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu
Avenue, Wuxi 214122, China
e-mail: hfxia@jiangnan.edu.cn
X.<H. Jin
Zhejiang Hisun Pharmaceutical Co., Ltd, Taizhou 318000, China

Appl Biochem Biotechnol (2014) 172:40024012

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a molecular weight of about 42,000 Da and consists of five similar regions (E, D, A, B, and C)
called IgG-binding domains [4]. The SPA-based affinity medium has been widely used in affinity
chromatography systems due to its specific interaction with the Fc portion of IgG [5, 6].
However, the Protein A affinity ligand also needs a further improvement for the application
in the industry. One of which is that the elution of antibodies absorbed by SPA-based affinity
adsorbent is under the condition of low pH, which is generally essential to elute the bonding
IgG from the medium. And because of the strong combination between protein A and
immunoglobulin, the elution solution pH is often as low as 3.0. Such acidic solution might
be harmful to the target antibodies and usually lead to inactivation. Therefore, it is necessary to
increase the elution pH and keep the adsorption capacity at the same time for a healthy
development of Protein A medium. Another problem is the alkaline resistance of Protein A
affinity medium in the chromatography process of regeneration. In order to remove contaminants from the affinity medium such as nucleic acids, lipids, proteins, and microbes, a
cleaning-in-place (CIP) step is often integrated into the purification protocol. Sodium hydroxide solution (0.5 to 1.0 mol/L) probably was the most widely used cleaning agent for this
purpose. However, it was reported that about 1 % of the binding capacity would lose when
SPA is exposed to 0.5 mol/L NaOH for 15 min [7], and the leaked SPA ligand would
contaminate the target antibody products. Thus, it is imperative to improve the stability in
order to enable the SPA-based affinity medium to keep the binding capacity when exposed to
the harsh conditions associated with CIP procedures. According to the two points mentioned
above, it is significant to modify the SPA structure to improve the elution condition and alkali
resistance simultaneously.
Consequently, the work was focused on the B domain of SPA, which is a typical segment in
the five domains. B domain is composed of 58 amino acid residues arranged in an antiparallel
three-helix bundle with two interconnecting loops. The binding function existed in helix 1 and
helix 2 [8, 9], while helix 3 and loop 2 are not involved. The loop sequences not involved in
the function of protein A have been proposed to have some degrees of tolerance in those
protein engineering attempts [1012]. Also, the stability of protein A can be weaken by
lengthening the loop, which resulted in the breakage of the B-IgG interaction at milder
conditions [13, 14]. This property is a breakthrough for the target of easy-elution. Glich
et al. [15] added a section of glycine residues behind loop 2 to lengthen it, and they found most
of the bonding IgG molecules could be eluted at a pH as high as 4.5. Moreover, glycine is the
smallest amino acid and has nearly no structure effect on helix. Thus, it is an effect way to
make the elution condition milder.
In recent years, a kind of commercial Protein A affinity medium named MabSelect SuRe,
which could be tolerant of sodium hydroxide solution as high as 0.5 mol/L, has been
introduced by GE Healthcare. However, less information of the ligand could be found, and
more research work is needed for improving the alkali resistance. In the amino sequence of B
domain, the 30th of phenylalanine (F30) is sandwiched between L44 and L51 of helix 3 in the
hydrophobic core of the domain. The substitution of F30 by alanine (F30A) has a dramatic
effect on the conformational stability. This F30A was located as the scaffold for further
mutation [16] as the bypass mutation method [17, 18]. Besides, Glich et al. [19] reports that
asparagine is one of the most susceptible residue to high pH in the structure of Protein A
resulted from covalent modifications such as deamidation and backbone cleavage. Asparagine
with a succeeding glycine has also been found to be the most sensitive amino acid sequence to
alkaline condition [2022]. These reactions are spontaneous and may also occur in physiological solvent, often resulting in a loss of activity of the protein or peptide [23]. Among the
eight asparagines in the structure of B domain, Linhult et al. [24] found that N23 is the key
residue for the function of IgG binding after alkaline treatment.

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Appl Biochem Biotechnol (2014) 172:40024012

Therefore, depending on the previous researches reported in publications [15, 19, 24],
we integrated the two methods of increasing elution pH and improving alkali resistance
into modifying the B domain. Customarily, the variant of B is named as Z. In the Z
domain, six glycine residues were inserted into loop 2, named Z(6G), to reduce the
interaction between ligand and immunoglobulin. Also, we followed the Linhults work
which two amino acid residues (N23T, F30A) were mutated, named Z(N23T, F30A), to
investigate the stability in alkaline solution. Then, Z domain was obtained. To imitate the
structure and length of nature SPA, five Z gene sequences were linked by isocaudarner to
be the recombinant SPA (rSPA), and the protein was efficiently expressed in Escherichia
coli BL21 (DE3). Furthermore, the recombinant protein was coupled to Sepharose 4 Fast
Flow to be the affinity medium. The chromatographic characteristics of the affinity
medium were carefully evaluated.

Materials and Methods

DNA Constructions and Bacterial Strains
Site-directed mutagenesis was performed using a two-step polymerase chain reaction (PCR)
technique [25]. Isocaudarner Nhe I, Xba I, and restriction enzymes BamH I were used for
linking DNA z. Restriction enzymes BamH I and Noc I were used for ligating rSPA gene with
vector pET-28a. The primers were designed by the software DNAMAN according to the
sequence of SPA gene of S. aureus provided by GenBank. The forward primer of z sequence
F0, CCATGGCTAGCGC GGATAACAAATTCAAC; reverse primer R0, GGATCCTTAACA
TCTAGATTTT GGTGCTTGTGC. The forward primer of Z(6G) F1, ATGACCCAAGCCAA
GGTGGCGGAGGG GGCGGTAGCGCTAACCTTTTA; reverse primer R1, TAAAAGGT
TAGCGCTACCGCCCCCTC CGCCACCTTGGCTTGGGTC. The forward primer of
Z(N23T, F30A) F2, ACTTAACCGAAGAA CAACGCAATGGTGCCATCCAAAGCTTAA;
reverse primer R2 , TTGGATGGCACCATTGCGTT GTTCTTCGGTTA AGT).
Finally, the z sequence was designed as: CCATGGCTAGCGCGGATAACAAATTCAAC
AA AGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAACCGAA
GAACAACGCAATGGTGCCATCCAAAGCTTAAAAGATGACCCAAGCCAAGGTGGC
GGAGGGGGCGGTAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCA
CAAGCACCAAAATCTAGATGTTAAGGATCC. The parts marked by italics are the enzyme
site and the underline are genetically modified part. To obtain a sulfydryl in the Cterminus of SPA, we added a cysteine codon to the end of z. The amino acid sequences
of B and Z were shown in Fig. 1. The helixes were marked with bold type and the
mutations were underlined.
The z gene was cloned into the cloning vector pMD18-T by PCR technique and then
transformed into E. coli JM109 cells. The subscript of zn means the number of z linked. Liner
pMD18-T-z1 was obtained by digesting the recombinant plasmid pMD18-T-z1 with restriction
enzyme Xba I and BamH I. Also, z was obtained by digesting pMD18-T-z1 with restriction
enzyme Nhe I and BamH I. Then, the recombinant plasmid pMD18-T-z2 was obtained by
linking the two digesting productions. The prepared process was shown in Fig. 2 and pMD18T-z5 was obtained with this method repeatedly. Thus, pET-28a-z5 (shown in Fig. 2) was
constructed with plasmid pET-28a and restriction enzyme Noc I and BamH I. At last, the
recombinant plasmid pET-28a-z5 was transformed into competent E. coli BL21 (DE3) cells
chemically. The clones were analyzed by a restriction digestion for an insert release of the
recombinant plasmid isolated from the confirmed clones.

Appl Biochem Biotechnol (2014) 172:40024012

(a)

Helix 1

Loop1

4005

Helix 2

Loop 2

Helix 3

B: ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK
Z(N23T)

Z(F30A)

Z(6G)

Cys

Z: ADNKFNKEQQNAFYEILHLPNLTEEQRNGAIQSLKDDPSQGGGGGGSANLLAEAKKLNDAQAPKC

(b)

Helix 3

Helix 1

C-terminal

Z(F30A)

Loop 2

Loop 1

N-terminal
Z(6G)

Z(N23T)

Helix 2

Fig. 1 a The amino acid residues of B and Z domains. b The three-dimensional structure of B domain

Expression and Purification of the Recombinant SPA

E. coli BL21 (DE3) with the expression plasmid pET-28a-z5 was cultured in LuriaBertani broth medium containing 50 g/mL of kanamycinat at the temperature of
37 C. Then, the culture was induced by isopropyl -D-1-thiogalactopyranoside
(IPTG) when the A600 is at the range of 0.6 to 0.8 and was cultured at 30 C
overnight. After that, the cells were harvested by centrifugating at 8,000 rpm 4 C for
10 min, then suspended in 50 mmol/L TrisHCl buffer (pH 7.5, including 150 mmol/
L NaCl). The rSPA was released by ultrasonication, and the crude rSPA solution was
obtained by centrifugating at 8,000 rpm.
The recombinant SPA was purified by affinity adsorbent of IgG Sepharose 6 Fast Flow. The
chromatographic process was conducted with KTA purifier system. Ten milliliters of
adsorbents was packed in a 1.6-20-cm column. TrisHCl buffer (50 mmol/L, pH 7.5,
including 150 mmol/L NaCl) was selected as the binding buffer, and acetic acid-ammonium
acetate buffer (0.5 mol/L, pH 3.4) as the elution buffer. The rSPA was lyophilized after
desalting with dialysis bag. The protein was analyzed by SDS-PAGE following the method
of Laemmli [26].

Digested with
Amp

BamH I
Xb a I
p MD1 8 T-z1

o ri

Eco R

Nh e

Nhe I and

BamH

z5

BamH I
Xb a I

z1

BamH I

z1
Nh e I
No c I

Amp

p MD18 T-z2

Kan

Nh e
T7 promoter
p ET2 8a-z5

z2

Digested with
Nhe I and
BamH I

BamH

Nhe
Nco

Xba
o ri

plasmid

z1

Fig. 2 The schematic diagram of preparation process of recombinant plasmid pET28a-z5

o ri

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Appl Biochem Biotechnol (2014) 172:40024012

Preparation of rSPA Sepharose 4 Fast Flow

The Sepharose 4 FF was modified to NHS Sepharose 4 FF through three steps. Firstly,
Sepharose 4 FF was activated with allyl glycidyl ether according to the method described
by Johansson et al. [27] with some modifications. Ten milliliters of sepharose 4 FF was
mixed with 10 mL of dioxane, 300 L of boron trifluoride ether, and 0.1 g of allyl
glycidyl ether in a 100-mL conical flask at 140 rpm 35 C for 45 min. After the
reaction, the agarose gel was washed with dioxane, 70 % of acetone, 30 % of
acetone, and deionized water, respectively. Then, the gel was coupled with
thioglycollic acid by adding 1.20 mL of thioglycollic acid, 10 mL of deionized water,
and 0.25 g of ammonium persulfate at 140 rpm 60 C for 8 h. After the reaction, the
agarose gel was washed with deionized water, 30, 70, and 100 % of acetone,
respectively. Then, the gel was modified with NHS by being mixed with 10 mL of
dioxane, 1 g of NHS, and 1 g of DCC at 140 rpm 25 C for 8 h. Finally, the gel was
washed with dioxane, 70 % of acetone, 30 % of acetone, and deionized water,
respectively.
To couple the rSPA onto Sepharose 4 Fast Flow, the purified rSPA was dissolved in
phosphate buffer (50 mM, pH 7.6). Then, the solution was mixed with the NHS agarose gel
and 20 mg of sodium borohydride at 150 rpm 25 C for 6 h. Then, the gel was separated by
filtration and suspended in phosphate buffer containing 0.5 mol/L of mercaptoglycerol at
150 rpm 25 C for 3 h. The adsorbents were washed with excessive distilled water and stored
at the temperature of 4 C in 20 % of ethanol. The schematic diagram of preparation process
was shown in Fig. 3.
Preparation of Crude IgG Sample
The IgG from bovine colostrum was selected as the target protein. The lipids in fresh bovine
colostrums were removed by centrifugating at 8,000 rpm 4 C for 10 min. Then, the bovine
colostrum was adjusted to be in a condition of pH 4.6 with 0.01 mol/L of hydrochloric acid
and was centrifugated at 8,000 rpm 4 C for 10 min to remove the casein. After that, the crude
IgG was obtained by salting out with 35 % of ammonium sulfate and dialyzing overnight with
phosphate buffer (20 mmol/L, pH 7.6).
Chromatographic Properties in Packed Bed
The chromatographic properties of rSPA Sepharose 4 FF and GE rProtein A Sepharose 4 FF
were measured in a column (110 cm) with KTA purifier system from GE Healthcares
(Uppsala, Sweden). An amount of 3.9 mL of adsorbents was packed to the height of 5 cm. The

OH
OH

OH

allyl glycidyl ether

CH2

OH

S
OH

NHS

thioglycollic acid

DCC

O
O

OH
S

rSPA

rSPA

O
O

N
O
O

Fig. 3 The schematic diagram of preparation process of rSPA Sepharose 4 Fast Flow

Appl Biochem Biotechnol (2014) 172:40024012

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phosphate buffer (50 mmol/L, pH 7.6) was selected as the equilibrium buffer and the citric
acid-disodium hydrogen phosphate buffer (0.1 mol/L, pH 3.06.0) was selected as the elution
buffer. The flow rate was 1 mL/min, and the injection volume of crude IgG sample was 2 mL.
Finally, the protein collected was analyzed by SDS-PAGE following the method of Laemmli
[26].
Evaluation of Alkali Resistance
To simulate the CIP process, the column of self-made adsorbents and GE rProtein A Sepharose
4 FF were both washed with different concentrations of sodium hydroxide solution at flow rate
of 1 mL/min and kept in the alkaline condition for certain time. Then, the adsorbents were
washed with equilibrium buffer until the pH reached 7.6. After that, the chromatographic
properties of the column were evaluated as stated in the Chromatographic Properties in
Packed Bed section. Here, the elution pH was set at 3.0.

30
25

Detected Signal, mV

breakthrough

20
15
10

elution

5
0
0

20

40

60

80

100

Volume, mL

97.2kD
66.4kD
44.3kD

35.5kD rSPA
29.0kD

20.1kD
14.3kD

Fig. 4 Purification and analysis of rSPA. a Process of purification; b SDS-PAGE profiles: 1 elution of rSPA, 2
marker, 3 penetration of rSPA sample, 4 rSPA sample from E. coli BL21(DE3)/pET28a-z5

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Appl Biochem Biotechnol (2014) 172:40024012

Results and Discussion

Preparation of rSPA and rSPA Affinity Adsorbents
Within the plasmid pMD18-T, five z sequences were connected in series by Nhe I, Xba I, and
BamH I. After transforming the recombination plasmid pET-28a-z5 into competent E. coli
BL21 (DE3), the engineered E. coli BL21 (DE3)/pET-28a-z5 was obtained. The rSPA was
over-expressed in the bacterium, and the results were analyzed by SDS-PAGE (Fig. 4b, lane
4). It distinguished the recombinant protein in the induced samples as 35.5 kDa band, which
corresponds to the expected molecular weight.
There was only one elution peak and one breakthrough peak (Fig. 4a) in the purification process
of rSPA with IgG Sepharose 6 FF. A thick band which indicated rSPA eluted from the elution peak
appeared in the SDS-PAGE profiles (Fig. 4b). Analyzed by software ImageJ, the purity of the crude
sample and the purified rSPA was recognized as 44.1 % and almost 100 %, respectively.

30

Detected Signal, mV

25
20
15
rProtein A Sepharose 4 FF

10

rSPA Sepharose 4 FF

5
0
0

20

40

60

80

Volumn, mL

1
200.0kD

4
IgG

116.0kD
97.2kD
66.4kD

44.3kD

Fig. 5 Purification of IgG from bovine colostrum with rSPA and rProtein A Sepharose 4 FF. a Process of
purification; b SDS-PAGE profiles for purification by rSPA Sepharose 4 FF: 1 marker, 2 elution, 3 breakthrough,
4 sample of IgG

Appl Biochem Biotechnol (2014) 172:40024012

4009

Activation with NHS is one of the first choices on the preparation of affinity media [28].
Also, the coupled ligand rarely leaked in the process of storage and chromatography [29].
According to the different concentrations of rSPA, before and after the coupling, the density of
rSPA onto agarose was calculated as 6.64 mg/mL, which is similar to the commercial
adsorbents rProtein A Sepharose 4 Fast Flow.
Purification of Bovine IgG by rSPA Sepharose 4 Fast Flow
The IgG from bovine colostrum was purified in one affinity step to measure the capacity of
self-made and commercial adsorbents. The chromatographic process was shown in Fig. 5a.
From the SDS-PAGE profiles (Fig. 5b), the purity of crude IgG in sample was recognized as
54.4 %, and that of the eluted IgG was almost 100 %, respectively. The capacity of adsorbing
IgG was only 0.701 mg for rSPA Sepharose 4 Fast Flow and 0.765 mg for GE adsorbents.
Thus, the recovery of IgG was 11.3 % for self-made adsorbents, lower than 12.3 % for GE
adsorbents. It is known that the IgG from bovine colostrum include bovine IgG1 and IgG2,
and protein A binds to bovine IgG1 weakly while it binds to bovine IgG2 strongly. The band
of IgG for breakthrough fraction shown in Fig. 5b should be mainly pointed to bovine IgG1.
Nevertheless, for the adsorption capacity of rSPA Sepharose 4 Fast Flow and GE adsorbents,
the decline in capacity of adsorbing IgG could be attributed to the comparatively weak
interaction between rSPA and IgG. However, the tiny distinction would not influence the
application of rSPA Sepharose 4 Fast Flow.
Evaluation of Elution pH
Generally, pH 3.0 is used as the elution pH for purification of immunoglobulin by Protein A
affinity. Hence, this pH was assumed as the condition that can elute 100 % bonding IgG from
columns. As seen in Fig. 6, at the pH 4.0, 97.7 % of IgG can be eluted from the self-made
column, compared with 79.5 % from GE column. Then, at the pH 5.0, 89.4 % of IgG was
eluted from the self-made column, whereas only 66.3 % was eluted from the GE column. The
results show that a higher proportion of bonding IgG can be eluted from the self-made column
than the GE column at the same pH. This might be attributed to the mutant in the second loop
100
rSPA Sepharose 4 FF
rProtein A Sepharose 4 FF

90
80

Elution rate, %

70
60
50
40
30
20
10
0
4

pH
Fig. 6 Effect of pH on IgG elution rate by rSPA and rProtein A Sepharose 4 FF

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Appl Biochem Biotechnol (2014) 172:40024012

100

0.203
80

0.196

60

0.189
0.182

40

0.175
20

Adsorption capacity of bovine IgG on rSPA Sepharose 4 FF

0.168

Binding rate of bovine IgG on rSPA Sepharose 4 FF

0.161

Binding rate of bovine IgG on rProtein A Sepharose 4 FF

Binding rate, %

Adsorption capacity, mg/mL

0.210

Adsorption capacity of bovine IgG on rProtein A Sepharose 4 FF

0.0

0.5

1.0

1.5

2.0

Concentration of NaOH, mol/L

Fig. 7 Adsorption analysis of bovine IgG on two kinds of adsorbents as the function of concentration of sodium
hydroxide

of B domain. The interaction between IgG and rSPA is much weaker than that between IgG
and natural SPA. The elution pH (4.05.0) for rSPA Sepharose 4 FF is significantly milder
than the pH (3.0) commonly used today. The results are similar to the published report [19].
The engineered ligands described here might be attractive in affinity chromatography for IgGpurification, especially for the product that is susceptible to extreme pH values.
Stability Analysis in Alkaline Environment
In the chromatographic process, cleaning-in-place (CIP) using high concentration of sodium
hydroxide is an important step for regeneration of medium. Generally, Protein A ligand has
poor ability to resist such high alkaline condition. Here, the key sites in the B domain were
modified to improve alkali resistance. IgG in excess was loaded, and the amount of eluted IgG
was measured after each separation process to determine the total adsorption capacity of the
column. As shown in Fig. 7, the IgG-binding capacity of rSPA Sepharose 4 FF was stable after
having been exposed in sodium hydroxide solution (02.0 mol/L) for 1 h, while that of GE
rProtein A Sepharose 4 FF decreased gradually. The results could also be found in the
100
90

0.225

80
0.200

70
60

0.175

50
0.150
0.125

40
30
Adsorption capacity of bovine IgG on rSPA Sepharose 4 FF

20

Binding rate of bovine IgG on rSPA Sepharose 4 FF

0.100

Binding rate, %

Adsorption capacity, mg/mL

0.250

Adsorption capacity of bovine IgG on rProtein A Sepharose 4 FF

10

Binding rate of bovine IgG on rProtein A Sepharose 4 FF

0
0

10

12

14

Time of exposure, h

Fig. 8 Adsorption analysis of bovine IgG on two kinds of adsorbents as the function of exposed time to
2.0 mol/L sodium hydroxide

Appl Biochem Biotechnol (2014) 172:40024012

4011

calculation of binding rate: the data of rSPA Sepharose 4 FF was almost 100 % at the sodium
hydroxide concentration of 2.0 mol/L, while that of rProtein A Sepharose 4 FF decreased to
94.4 %.
To test the alkali resistance further, we extended the time of our adsorbents being exposed
in as high as 2.0 mol/L of sodium hydroxide (Fig. 8), although this concentration was hardly
used in the regenerating process. After 7 h, the binding capacity of IgG on rSPA Sepharose 4
FF remained about 86.1 %, while that of GE adsorbents was 72.8 %. When the exposure time
increased to 14 h, the capacity of the former was still 79.1 %, while the capacity of the latter
decreased to 59.7 %.
Comparing with the report [24], we used a higher concentration of sodium hydroxide to
treat the SPA and proved the stability of the ligand. The unmodified rProtein A Sepharose 4 FF
also showed the similar high affinity capacity after treated by high concentration of sodium
hydroxide. However, another important thing was whether the Protein A or Protein A fragment
would leak and contaminate the immunoglobulin. This problem would be studied in the future
work.

Conclusions
A recombinant SPA (rSPA) was obtained by linking five Z domains, which was modified as
six glycine residues (Z6G) inserted behind loop 2 and two amino acid residues of (N23T,
F30A) mutated in the B domain. The extended residues in loop 2 of Z domain reduced the
interaction between ligand and protein and kept the adsorption capacity at the same time,
resulting in the elution pH increased from 3.0 to 4.05.0. Moreover, the recombinant ligand
became more stable in alkaline condition and could even resist as high as 2 mol/L of sodium
hydroxide for 1 h due to the mutation of key residues of (N23T, F30A). The work confirmed
the effect of two aspects of modification and the rSPA showed potential for large-scale
antibodies purification.
Acknowledgments This work was supported by the National High Technology Research and Development
Program of China (863 Program, 2012AA021201), the National Natural Science Foundation of China
(21206054), the Research Fund for the Doctoral Program of Higher Education of China (20110093120001),
the 111 Project (No. 111-2-06), and the Project Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions.

References
1. Sjquist, J., Movitz, J., Johansson, I. B., & Hjelm, H. (1972). Localization of protein A in the bacteria.
European Journal of Biochemistry, 30, 190194.
2. Bjrk, I., Petersson, B. ., & Sjquist, J. (1972). Some physicochemical properties of protein A from
Staphylococcus aureus. European Journal of Biochemistry, 29, 579584.
3. Sjholm, I. (1975). Protein A from Staphylococcus aureus. Spectropolarmetric and spectrophotometric
studies. European Journal of Biochemistry, 51, 5561.
4. Moks, T., Abrahmsn, L., Nilsson, B., Hellman, U., Sjquist, J., & Uhln, M. (1986). Staphylococcal protein
A cosists of five IgG-binding domains. European Journal of Biochemistry, 156, 637643.
5. Lindmark, R., Thorn-Tolling, K., & Sjquist, J. (1983). Binding of immunoglobulins to protein A and
immunoglobulin levels in mammalian sera. Journal of Immunological Methods, 62, 113.
6. Goding, J. W. (1978). Use of staphylococcal protein A as an immunological reagent. Journal of
Immunological Methods, 20, 241253.

4012

Appl Biochem Biotechnol (2014) 172:40024012

7. Hale, G., Drumm, A., Harrison, P., & Phillips, J. (1994). Repeated cleaning of protein A affinity column with
sodium hydroxide. Journal of Immunological Methods, 171, 1521.
8. Deinenhofer, J. (1981). Crystallographic refinement and atomic models of a human Fc fragment and its
complex with fragment B of Protein A from Staphylococcus aureus at 2.9- and 2.8- resolution.
Biochemistry, 20, 23612370.
9. Tashiro, M., Tejero, R., Zimmerman, D. E., Celda, B., Nilsson, B., & Montelione, G. T. (1997). Highresolution solution NMR structure of the Z domain of Staphylococcal Protein A. Journal of Molecular
Biology, 272, 573590.
10. Brunet, A. P., Huang, E. S., Huffine, M. E., Loeb, J. E., Weltman, R. J., & Hecht, M. H. (1993). The role of
turns in the structure of an -helical protein. Nature, 364, 355358.
11. Castagnoli, L., Vetriani, C., & Cesareni, G. (1994). Linking an easily detectable phenotype to the folding of a
common structural motif. Selection of rare turn mutations that prevent the folding of Rop. Journal of
Molecular Biology, 237, 378387.
12. Predki, P. L., & Regan, L. (1995). Redesigning the topology of a four-helix-bundle protein: monomeric Rop.
Biochemistry, 34, 98349839.
13. Nagi, A. D., & Regan, L. (1997). An inverse correlation between loop length and stability in a four-helixbundle protein. Folding and Design, 2, 6775.
14. Nagi, A. D., Anderson, K. S., & Regan, L. (1999). Using loop length variants to dissect the folding pathway
of a four-helix-bundle protein. Journal of Molecular Biology, 286, 257265.
15. Glich, S., Uhln, M., & Hober, S. (2000). Protein engineering of an IgG-binding domain allows milder
elution conditions during affinity chromatography. Journal of Biotechnology, 76, 233244.
16. Cedergren, L., Andersson, R., Jansson, B., Uhln, M., & Nilsson, B. (1993). Mutational analysis of the
interaction between staphylococcal protein A and human IgG1. Protein Engineering, Design & Selection, 6,
441448.
17. Kotsuka, T., Akanuma, S., Tomuro, M., Yamagishi, A., & Oshima, T. (1996). Further stabilization of 3isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation
method. Journal of Bacteriology, 178, 723727.
18. Sieber, V., Plckthun, A., & Schmid, F. X. (1998). Selecting proteins with improved stability by a phagebased method. Nature Biotechnology, 16, 955960.
19. Glich, S., Linhult, M., Uhln, M., Nygren, P. ., & Hober, S. (2000). Stability towards alkaline conditions
can be engineered into a protein ligand. Journal of Biotechnology, 80, 169178.
20. Kossiakoff, A. A. (1988). Tertiary structure is a principal determinant to protein deamidation. Science, 240,
191194.
21. Lura, R., & Schirch, V. (1988). Role of peptide conformation in the rate and mechanism of deamidation of
asparaginyl residues. Biochemistry, 27, 76717677.
22. Kosky, A. A., Razzaq, U. O., Treuheit, M. J., & Brems, D. N. (1999). The effects of alpha-helix on the
stebility of Asn residues: deamidation rates in peptides of varying helicity. Protein Science, 8, 25192523.
23. Geiger, T., & Clarke, S. (1987). Deamidation, isomerization, and racemization at asparaginyl and aspartyl
residues in peptides. Succinimide-linker reactions that contribute to protein degradation. Journal of
Biological Chemistry, 262, 785794.
24. Linhult, M., Glich, S., Grslund, T., Simon, A., Karlsson, M., Sjberg, A., Nord, K., & Hobe, S. (2004).
Improving the tolerance of a Protein A analogue to repeated alkaline exposures using a bypass mutagenesis
approach. Proteins, 55, 407416.
25. Higuchi, R., Krummel, B., & Saiki, R. K. (1988). A general method of in vitro preparation and specific
mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Research, 16, 351
7367.
26. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, 227, 680685.
27. Johansson, B. L., Belew, M., Eriksson, S., Glad, G., Lind, O., Maloisel, J. L., & Norrman, N. (2003).
Preparation and characterization of prototypes for multi-modal separation media aimed for capture of
negatively charged biomolecules at high salt conditions. Journal of Chromatography. A, 1016, 2133.
28. Cuatrecasas, P., & Parikh, I. (1972). Adsorbents for affinity chromatography. Use of N-hydrox ysuccinimide
esters of agarose. Biochemistry, 11, 22912299.
29. Van Sommeren, A. P. G., Machielsen, P. A. G. M., & Gribnau, T. C. J. (1993). Comparison of three activated
agaroses for use in affinity chromatography: effects on coupling performance and ligand leakage. Journal of
Chromatography. A, 639, 2331.