Development of the Double Cyclic Peptide Ligand for Antibody

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Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection Yiyi Gong, Lin Zhang, Jin Li, Shan Feng, and Haiteng Deng Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00170 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on July 1, 2016

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Bioconjugate Chemistry

Abstract figure 679x350mm (96 x 96 DPI)

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Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein

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Detection

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Yiyi Gong1, Lin Zhang1, 2, Jin Li1, Shan Feng2*, Haiteng Deng1*

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1.

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MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China，， 100084

2.

Tsinghua University-Peking University Joint Center for Life Sciences, Tsinghua University, Beijing, China, 100084

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Author Information

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Corresponding authors

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*E-mail: [email protected];

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[email protected]

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1


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Abstract

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Development of a peptide-based affinity matrix and detection reagent is important for

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biomedical research and the biopharmaceutical industry. In the present work, we designed

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and synthesized an immunoglobin G (IgG)-binding peptide ligand, Fc-III-4C. Fc-III-4C is

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composed of 15 residues and the four cysteine residues form two disulfide bonds to generate

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a double cyclic structure. The binding affinity of the Fc-III-4C peptide towards human IgG

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was determined to be 2.45 nM (Kd), which is higher than that of IgG with Protein A/G

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(Pro-A/G). Importantly, the Fc-III-4C peptide displayed high affinity to various IgGs from

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different species. Fc-III-4C immobilized agarose beads exhibited high stability and

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reusability when compared with that of the Pro-A/G-immobilized beads. The conjugate of

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Fc-III-4C with FITC was demonstrated to be suitable for immuno-fluorescence detection of

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proteins expressed in cells. These results demonstrate that the Fc-III-4C peptide is a useful

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affinity ligand for antibody purification and as a protein detection reagent.

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Phage display and peptidomimetic chemistry are two methods used to find a peptide ligand of

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a targeted protein1-3. Generally, phage display is used for screening a peptide ligand from a

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library composed of a large number of peptides, whereas peptidomimetic chemistry is used to

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further optimize the peptide sequence. The selected peptides with high affinities to a target

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protein can be coupled with agarose beads and fluorescent molecules for enrichment and

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detection of target proteins. These peptides can also be used as affinity tags for expression

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and purification of recombinant proteins4-7.

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The Fc-III peptide that possesses high affinity to an IgG-Fc fragment was originally

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identified by phage display screening with a cyclic peptide library. The Fc-III peptide with

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the sequence DCAWHLGELVWCT adopts a β–hairpin conformation with two Cys residues

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forming a disulfide bond to make a cyclized peptide8. As a mimic of Protein A (Pro-A) or

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Protein G (Pro-G), the Fc-III peptide binds to the hinge region of the IgG-Fc fragment with

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high affinity (Kd = 185 nM)9.

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However, the single disulfide bridge in the Fc-III peptide provides limited

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conformational restraint to the β-hairpin backbone. Dias et al. used D-Pro-L-Pro to link the

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N- and C-termini of Fc-III to form a double cyclic structure denoted as FcBP-2 by using

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peptidomimetic chemistry tool9. The binding affinity of FcBP-2 with IgG is 80-fold higher

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than that of the original Fc-III peptide, indicating that the double ring structure of the peptide

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significantly enhances its affinity towards IgG. However, the D-Pro can not be incorporated

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into Fc-III-tagged recombinant proteins, and N to C cyclization is a challenging for routine

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solid-phase peptide synthesis. In the present work, we modified the Fc-III sequence by

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replacing the D-Pro and L-Pro residues with two cysteine residues at the N- and C-termini of 3


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the peptide to facilitate the formation of a second disulfide bond that mimics the D-Pro-L-Pro

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linkage, which is designated Fc-III-4C. The Fc-III-4C peptide has a double cyclic structure

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with high binding affinity towards human IgG, which is 30-fold higher than that of Fc-III.

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The newly designed Fc-III-4C peptide also showed strong interactions to a variety of IgGs

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from different species. We conjugated the Fc-III-4C peptide to agarose beads and FITC to

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efficient purify IgG molecules from serum and detect expressed proteins in cells, respectively.

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Our results herein demonstrate that the Fc-III-4C peptide is a simple and efficient tool for

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antibody purification and protein detection.

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The Fc-III-4C peptide in the reduced form was synthesized by solid-phase peptide

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synthesis, and the purity was estimated to be 95% by HPLC analysis. Under mild oxidative

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conditions with exposure to air at 37 ºC overnight, the reduced peptide is facile to be

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converted into the fully oxidized peptide with two disulfide bonds. The oxidized Fc-III-4C

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peptide was further purified by HPLC and the purity of the oxidized Fc-III-4C peptide in the

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final product was 98%, as determined by HPLC (Figure S1, S2 and S3). To identify the

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disulfide linkages in the oxidized Fc-III-4C peptide, the peptide was digested with Asp-N

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endopeptidase that hydrolyzes peptide bonds on the N-terminal side of Asp and the resulting

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fragments were analyzed by tandem mass spectrometry (MS). The MS spectrum of the

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oxidized Fc-III-4C peptide showed a doubly charged ion at m/z 867.83, indicating that the

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Fc-III-4C peptide contains two disulfide bonds (Figure 1A). After Asp-N digestion, the mass

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of the product is 18 Da higher than that of the Fc-III-4C and its MS/MS spectrum is presented

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in Figure 1B, in which y1 (241.03 m/z) and y2 (342.08 m/z) ions are the characteristic

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fragments of the disulfide-bond linked peptide. The other peaks in Figure 1B match to 4



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fragments of the Fc-III-4C peptide, as shown in Figure 1C. These results indicate that the

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Fc-III-4C peptide has two disulfide bonds, and its structure is presented in Scheme 1.

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Figure 1. Identification of the disulfide linkages of the Fc-III-4C peptide. (A) The peak at 5


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867.83 Da in the mass spectrum matches to the mass of doubly charged Fc-III-4C peptide

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ions containing two disulfide bonds. After Asp-N digestion, the peak shifts to 876.84 Da

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suggesting one amide bond is hydrolyzed. (B) The MS/MS spectrum of the Asp-N digested

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Fc-III-4C in which y1 and y2 are characteristic peaks indicating that one disulfide linkage is

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between Cys1 and Cys15. (C) Sequence annotations of peaks in the MS/MS spectrum.

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Scheme 1. The proposed structure of the Fc-III-4C double cyclic peptide

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Surface Plasmon Resonance (SPR) was used to determine the binding affinities of

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human IgG with the Fc-III-4C and Fc-III peptides. Human IgG molecules were immobilized

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onto the biacore CM5 chip, and peptides were diluted into five different concentrations and

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interacted with immobilized IgG in the flow cell. The SPR responses were monitored and are

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presented in Figure S4. The data were fit to the two-state kinetic equation to derive the Kd

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values. Previous studies showed that the binding affinity of FcBP-2 with IgG is about 10-fold

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higher than that of Pro-A or the Fc-III peptide9. Consistent with these results, we found that

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the Kd values of the Fc-III-4C, Fc-III and the Z domain (engineered B domain of Pro-A)

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binding to human IgG were 2.45, 70 and 16.8 nM, respectively (Table 1), indicating that 6



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binding affinity of the Fc-III-4C peptide to human IgG is similar to that of the FcBP-2

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peptide and 7 times higher than those of Pro-A/Pro-G. Both Fc-III and Z domain bind to the

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Fc region of IgG. To probe whether the Fc-III-4C peptide binds to the same region in IgG, the

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Fc fragment of IgG (IgG-Fc) was purified and immobilized onto the biacore chip to measure

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the binding affinity of IgG-Fc and the Fc-III-4C peptide. The SPR responses are presented in

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Figure S5, and the calculated Kd value is 8.2 nM, demonstrating that the Fc-III-4C peptide

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binds to IgG-Fc with high affinity.

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We also measured the Kd values of the Fc-III-4C peptide binding to IgGs from rabbit,

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mouse, rat, pig, goat, bovine and horse (Table 1). All Kd values of the Fc-III-4C peptide were

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less than 30 nM, indicating that the Fc-III-4C peptide interacts strongly with different IgGs

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from different species. In comparison, the Kd values of the Fc-III peptide were generally

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lower than those of the Fc-III-4C peptide, and the interactions of the Fc-III peptide with IgGs

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from mouse, rat, goat, bovine and horse were below the detection limit. A similar trend was

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observed for the binding of the Z-domain of Pro-A with IgGs, and the interaction of the

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Pro-A Z-domain with IgGs from goat and horse were below the detection limit.

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Table 1. The dissociation constants of the Fc-III-4C peptide to various IgGs from different

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species

Human

Rabbit

Mouse

Rat

Pig

Goat

Horse

Bovine

Fc-III-4C

2.45 nM

5.67 nM

12.3 nM

18.8 nM

6.97 nM

15.7 nM

17.5 nM

28.6 nM

Fc-III

70 nM

8.9 nM

--

--

23.7 nM

--

--

--

Z-domain

16.8 nM

7.5 nM

33.6 nM

11.5 µM

21.5 nM

--

--

97 nM

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The above data indicate that the Fc-III-4C peptide is a mimic of the structure of the

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FcBP-2 peptide and has higher binding affinity towards IgG when compared with that of the

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Fc-III peptide and Pro-A. The commonly used Pro-A and Pro-G affinity media exhibit a wide

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range of binding affinities towards IgGs from different species, whereas the Fc-III-4C peptide

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possesses high affinities towards all IgGs tested, indicating that the Fc-III-4C peptide has

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advantages over the Pro-A/G affinity matrix for purification of antibodies from different

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species.

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We then used the Fc-III-4C peptide to purify IgGs. The Fc-III-4C peptide was

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immobilized onto NHS-activated Sepharose beads (GE Healthcare) through the primary

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amine at the N-terminus of the peptide with 10-atom spacer arms (Scheme 2), according to

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the manufacturer’s recommended procedures. The Fc-III-4C agarose beads were incubated

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with rabbit serum that consisted of multiple antibodies. After washing, proteins captured by

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the Fc-III-4C peptide immobilized beads were eluted and separated on 1D SDS-PAGE

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(Figure S6). The results showed that Pro-A beads and the Fc-III-4C agarose beads have

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similar enrichment efficiencies for rabbit IgG purification. The dynamic binding capacity at a

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flow rate of 1 ml/min for the Fc-III-4C immobilized agarose beads from 3 different batches

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was measured to be 28.9 ± 3.4 mg rabbit IgG/ml medium, which is slightly lower than that of

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the Pro-A Sepharose FF medium (GE healthcare) and about 20% higher than those of Hitrap

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Pro-A HP and FF column (GE healthcare). The data shown here indicates that the Fc-III-4C

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immobilized agarose beads produced have high binding capacity and low batch-to-batch

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variation.

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Scheme 2. The Fc-III-4C peptide was conjugated to NHS-activated agarose beads

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through the N-terminal amide linkage

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To further test the stability and reusability of the Fc-III-4C immobilized agarose beads,

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we incubated the beads with serum at different pH values (Figure 2A). Both the Fc-III-4C

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and Pro-A beads exhibited the highest binding capacity at pH values between 6.5 and 7. At

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lower pHs, the amount of purified IgGs decreased with the Pro-A beads losing their binding

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capacity more rapidly than the Fc-III-4C beads (Figure 2A).

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The reusability of Fc-III-4C and Pro-A beads was also examined using 6 M GnHCl or 2%

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SDS as the clean-in-place reagent.The affinity of the Fc-III-4C beads towards IgG did not

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change as dramatically as the affinity of Pro-A beads towards IgG. Even after 100

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clean-in-place procedures, the Fc-III-4C beads retained 60% of their affinity to IgG after the

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beads were treated with GnHCl, whereas the Pro-A beads only retained 40% of their affinity

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to IgG Figure 2B & 2C). These results demostrates that the Fc-III-4C peptide immobilized

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beads have higher reusability than the Pro-A beads, and are more stable than the Pro-A beads.

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Figure 2. Purification efficiencies of the Fc-III-4C and ProA immobulized beads after

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treatment with different pH buffers (A), GnHCl (B) and SDS (C). (A) Antibody 10



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purification efficiencies under different pH values. The Fc-III-4C or Protein A beads were

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incubated with serum at different pH values: 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0. Experiments under

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each condition were repeated three times. The relative purification efficiencies were

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normalized by the gray level of purified heavy chain of IgG on SDS PAGE at pH 8.0. (B)

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Purification efficiencies of Fc-III-4C and ProA beads after 6 M GnHCl clean-in-place

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procedures up to 100 times. (C) Purification efficiencies of Fc-III-4C and ProA beads after 2%

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SDS clean-in-place procedures up to 100 times. The relative purification efficiencies for (B)

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and (C) were determined by normalizing against the gray level of purified heavy chain of IgG

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on SDS PAGE before the clean-in-place procedures were performed.

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Immunofluorescence uses a specific antibody to detect protein localization in cells.

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Utilizing the high binding affinity of the Fc-III-4C peptide with the Fc region of IgG, we

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synthesized a fluorescence-labeled Fc-III-4C peptide to detect the primary antibody in

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immunofluorescence experiments. The Fc-III-4C peptides were conjugated with FITC by the

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isothiocyanate-amine reaction through the 6-aminohexanoic acid arm (Scheme 3). The

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products were purified and analyzed by HPLC and detected by both UV absorbance and

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fluorescence (Figure S7, S8 & S9). For a proof-of-principle experiment, we carried out an

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experiment to detect β-tubulin localized in the cytoplasm10, and PCNA (Proliferating cell

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nuclear antigen) localized in nuclei using the FITC-labeled Fc-III-4C peptide as the

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immunofluorescence reagent. PCNA is a nuclear protein that is expressed during the DNA

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synthesis phase of the cell cycle as a cofactor of DNA polymerase11-12. After cell fixation on a

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glass slide, the primary antibodies, the mouse-derived monoclonal anti-β-tubulin antibody 11


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and the rabbit-derived monoclonal anti-human PCNA antibody were respectively incubated

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with the fixed cells, and then the FITC labeled Fc-III-4C peptide was added, followed by

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Hochest 33342 nucleus staining. Fluorescence imaging showed that β-tubulin was present in

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the cytoplasm, whereas PCNA was condensed as small granules that distributed in nuclei

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(Figure 3). The FITC labeled Fc-III-4C peptide exhibited low staining background. Here,

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nuclear staining was not observed when beta-tubulin in the cytosol was stained whereas

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cytosolic staining was not observed when PCNA in the nucleus was stained. This

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demonstrates that the FITC labeled Fc-III-4C peptide is a sensitive and useful detection

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reagent for immunofluorescence experiments.

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Scheme 3. The structure of the FITC-labeled Fc-III-4C peptide

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Figure 3 Immunofluorescence detection of targeted proteins with the FITC-Fc-III-4C

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reagent shown in green. (A) β-tubulin was detected in the cytoplasm. (B) PCNA was

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detected in nuclei. The nuclei were also strained blue by Hochest 33342.

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Conclusions

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The addition of two cysteine residues to the sequence of the Fc-III peptide generates a

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double cyclic structure and increases by 30 folds of the binding affinity of the Fc-III peptide

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to human IgG. The novel Fc-III-4C peptide also exhibits high binding affinities to various

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IgGs from different species. The Fc-III-4C peptide is easy to prepare by routine solid-phase

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synthesis, cyclized under mild oxidation conditions and can be further incorporated into a

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recombinant protein expression system as a fusion tag. Using the primary amine labeling

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approach, the Fc-III-4C peptide was conjugated to agarose beads and FITC through amide

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bond and isothiocyanate-amine reactions, respectively. The Fc-III-4C immobilized agarose

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beads have advantages over the Pro-A/G beads as an affinity matrix for efficient antibody

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purification. Furthermore, the FITC-conjugated Fc-III-4C peptide is a useful detection

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reagent for immunofluorescence analysis.

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Associated Content

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at

17

DOI:

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Figures S1-S9: Oxidized Fc-III-4C peptide purification by HPLC, Fc-III-4C peptide analysis

19

by HPLC, Fc-III-4C peptide detection by MS, SPR analysis of the binding affinities of

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Fc-III-4C, Fc-III and Z-domain to human IgG, SPR analysis of the binding affinity of

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Fc-III-4C to IgG-Fc, the gel image of IgG purified from the serum sample, FITC-labled

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Fc-III-4C peptide purification by HPLC, FITC-labled Fc-III-4C peptide analysis by HPLC 14



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and FITC-labled Fc-III-4C peptide detection by MS.

2 3

Author Information

4

Corresponding Authors

5

*E-mail: [email protected];

6

[email protected]

7

Author Contributions

8

Y.G., S.F. and H.D. designed the experiments. Y.G. and S.F. investigated the biological

9

properties of Fc-III-4C peptide including disulfide bonds sites and binding affinity to IgG.

10

L.Z.and Y.G. applied the Fc-III-4C peptide to antibody affinity and protein detection. J.L. and

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L.Z. mesured the dynamic binding capacity. Y.G., S.F. and J.L. tested the pH stability and

12

reuseability of the Fc-III-4C beads. Y.G., S.F. and H.D. wrote the manuscript.

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Acknowledgments

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We thank the Protein Chemistry Facility at the Center for Biomedical Analysis of

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Tsinghua University for sample analysis. This work was supported by NSFC 31270871

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(H.T.D), NSFC 21502103 (F.S), MOEC 2012Z02293 (H.T.D) and the Chinese Ministry of

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Science and Technology (2014CBA02005 and 2014AA020907) and the Global Science

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Alliance Pro-Gram of Thermo-Fisher Scientific.

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References

22

(1) Sidhu, S. S., Fairbrother, W. J. and Deshayes, K. (2003) Exploring protein-protein

23

interactions with phage display. ChemBioChem 4, 14-25. 15


Page 16 of 24

Page 17 of 24

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


1

(2) Dwyer, M. A., Lu, W., Dwyer, J.J. and Kossiakoff, A.A. (2000) Biosyntheitc phage

2

display: a novel protein engineering tool combining chemical and genetic diversity. Chem.

3

Biol. 4, 263-274.

4

(3) Hruby, V. J. and Cai, M. (2013) Design of peptide and peptidomimetic ligands with novel

5

pharmacological activity profiles. Annu. Rev. Pharmacol. Toxicol. 53, 557-580.

6

(4) Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. and Nygren, P. (1997) Affinity Fusion

7

Strategies for Detection, Purification, and Immobilization of Recombinant Proteins. Protein

8

Expr. Purif. 11, 1-16.

9

(5) Terpe, K. (2003) Overview of tag protein fusions: from molecular and biochemical

10

fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533.

11

(6) Malhotra, A. (2009) Tagging for protein expression. Methods Enzymol. 463, 239-258.

12

(7) Uhlen, M., Forsberg, M. T., Hartmanis, M. and Nilsson, B. (1992) Fusion proteins in

13

biotechnology. Curr. Opin. Biotechnol. 3, 363-369.

14

(8) DeLano, W. L., Ultsch, M. H., de Vos, A. M. and Wells, J. A. (2000) Convergent solution

15

to binding at a protein-protein interface. Science 287, 1279-1283.

16

(9) Dias, R. L., Fasan, R., Moehle, K., Renard, A., Obrecht, D. and Robinson, J. A. (2006)

17

Protein ligand design: from phage display to synthetic protein epitope mimetics in human

18

antibody Fc-binding peptidomimetics. J. Am. Chem. Soc. 128, 2726-2732.

19

(10) Williams, Rc Jr., Shah, C. and Sackett, D. (1999) Separation of tubulin isoforms by

20

isoelectric focusing in immobilized pH gradient gels. Anal. Biochem. 275, 265-267.

21

(11) Cazzalini, O., Sommatis, S., Tillhon, M., Dutto, I., Bachi, A., Rapp, A., Nardo, T.,

22

Scovassi A.I., Necchi, D., Cardoso, M. C., Stivala, L. A. and Prosperi, E. (2014) CBP and

23

p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

24

Nucleic Acids Res. 42, 8433-8448.

25

(12) Leonardi, E., Girlando, S., Serio, G., Mauri, F. A., Perrone, G., Scampini, S., Dalla, P. P. 16



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

1

and Barbareschi, M. (1992) PCNA and Ki67 expression in breast carcinoma: correlations

2

with clinical and biological variables. J. Clin. Pathol. 45, 416-419.

17


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Figure 1 551x1382mm (96 x 96 DPI)



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Figure 2 386x904mm (96 x 96 DPI)


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Figure 3 754x1456mm (96 x 96 DPI)



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

Scheme 1 624x381mm (96 x 96 DPI)


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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


Scheme 2 1928x840mm (96 x 96 DPI)



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

Scheme 3 175x58mm (220 x 220 DPI)


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Development of the Double Cyclic Peptide Ligand for Antibody

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