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An easy and simple separation method for Fc and Fab fragments from chicken immunoglobulin Y (IgY)
Journal Pre-proofs An Easy and Simple Separation Method for Fc and Fab Fragments from Chicken Immunoglobulin Y (IgY) Xin Zhou, Yanru Wang, Dong Uk Ahn, Zhaoxia Cai PII: DOI: Reference:
S1570-0232(19)31654-X https://doi.org/10.1016/j.jchromb.2020.122011 CHROMB 122011
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
9 November 2019 25 January 2020 30 January 2020
Please cite this article as: X. Zhou, Y. Wang, D. Uk Ahn, Z. Cai, An Easy and Simple Separation Method for Fc and Fab Fragments from Chicken Immunoglobulin Y (IgY), Journal of Chromatography B (2020), doi: https:// doi.org/10.1016/j.jchromb.2020.122011
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© 2020 Published by Elsevier B.V.
An Easy and Simple Separation Method for Fc and Fab Fragments from Chicken Immunoglobulin Y (IgY)
Xin Zhou1, Yanru Wang1, Dong Uk Ahn2, Zhaoxia Cai1*
1. College of Food Science and Technology, Huazhong Agricultural University, National Research and Development Center for Egg Processing, Wuhan, Hubei, 430070, PR China 2. Department of Animal Science, Iowa State University, Ames, IA 50011, USA
Abstract Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.
Keywords:
IgY;
Fab
fragment;
Anion-exchange chromatography;
Fc
fragment;
(NH4)2SO4
precipitation;
1 Introduction Chicken IgY, a type of immunoglobulin mainly existing in egg yolk, is composed of two identical Fab segments and one Fc segment [1, 2]. IgY has many advantages over mammalian IgG, 1) it can be obtained easily from egg (100-150 mg IgY/each egg yolk)[3]; 2) has high avidity to mammalian antigens; (3) has less cross-reactivity with mammalian IgG; 4) does not activate or recognize the mammalian complements, and 5) does not react with rheumatoid factors (RF) [4]. Recent studies indicated that the fragments, as well as intact immunoglobulins, have significant effects on immune responses [5]. Fab fragment is composed of partial H-chain (VH+CH1) and entire L-chain (VL+CL1), and has the antigen-binding sites. Due to the cleavage of Fc fragment (antigen portion), Fab fragment has been developed to an antimicrobial agent that performs better than an intact antibody [6], and is widely used in diagnostics and biopharmaceutical research [7-9]. Besides, Fab fragment can be used as a ligand in purification study due to its affinity-binding ability [10]. Crystallizable fragment (Fc) is vital to trigger immune defense through the interactions with specific Fc receptors, which contains two or three domains of the entire heavy chain, CH2, CH3 and CH4. Nhg et al. (2018) and Smith et al. (2014) reported that the glycosylation of Fc can provide a strategy to engineer antibodies with high therapeutic efficacy and pharmacokinetics [11, 12]. Many researchers also used the Fc region to study the mechanism of IgY transport into egg yolk [13, 14]. Vermeer, Norde, & Van (2000) showed that stable Fab and Fc fragments could behave as independent subunits within an intact immunoglobulin and suggested that the functions of Fab and Fc could be studied independently using the isolated Fab and Fc fragments [15]. Thus, it is meaningful to collect Fab and Fc fragments separately. Due to the advanced recombinant DNA technologies, Fab and Fc fragments can be produced through the transient expression of appropriate constructs in mammalian cells or Escherichia coli cells [16-18]. However, it is very expensive and sometimes the
synthesized Fc could be inactive [14]. Some data shows that the Fc fragment of IgY obtained by papain digest mainly refers to Fc3-4 (residues 346-566), and its size is 53 kDa while the value of Fab is 44 kDa [19, 20]. Pepsin is typically used to produce active fragments, but it hydrolyzes Fc fragment into pieces [21]. Protein A and G have been commonly used as immunoglobulin-binding proteins (IBPS) to purify antibodies like IgG and IgA through binding to the Fc domain. However, due to the differences in amino acid sequence and conformation, IBPS has no affinity to IgY. Recently, Jiang, Diraviyam, & Zhang (2016) developed a new ligand, human mycoplasma protein (protein M), as an IBPS to purify IgY through the interactions with the Fab domain. However, the pH of the elution buffer used in the affinity purification was about 3 that reduced the activity of fragments. Also, the affinity column used was not stable for the purification and had a low capacity [22]. Due to similar molecular sizes, the Fab and Fc fractions collected from the size exclusion chromatography contained contaminants [17]. Gradiflow is a preparative instrument to purify Fab from IgY digest. However, the purified Fab was still contaminated with low-molecular-weight fragments [23]. Ion exchange chromatography (IEC) has been broadly used to purify Fab fragment from IgG [24, 25], but the purification of high-purity immunoglobulin fragments could be achieved only when a subsequent multi-column chromatography was used [19]. The objectives of this study were 1) to establish the optimal conditions for purifying Fab and Fc fragments from the papain hydrolysate, and 2) to identify separated Fab and Fc fractions using Western blot and MALDI-TOF-MS.
2 Materials and Methods 2.1 Materials Fresh eggs were purchased from Wuhan Jiufeng farm (Jiufeng Farm, Wuhan, China). Anti-chicken IgG (Fc) (rabbit) antibody and anti-chicken IgG (Fab) (rabbit) antibody were purchased from Rockland immunochemical (Gilbertsville, PA).
Chicken IgY standard was purchased from Bio Unit (Shanghai, China). Papain was purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents used in SDS-PAGE were purchased from Guge Biotechnology (Wuhan, China). The Bradford kit was purchased from Bio Time Biotechnology (Shanghai, China). The deionized distilled water was produced by a water purification system from Bio-Legend Bio-Tech Co., Ltd. (Shanghai China). Other chemical reagents were all analytical grade.
2.2 Papain digestion of IgY IgY was purified using the salting-out method of previous works [26, 27]. The purified IgY was dissolved in 0.1 M PBS (pH 7.0) at a concentration of 1.25 mg/mL and then the IgY content was determined using the Bradford method. Papain solution (0.2 mg/mL) was prepared by dissolving papain powder in 0.1 M, pH 7.0 phosphate-buffered saline (PBS) containing 20 mM ethylenediaminetetraacetic acid disodium salt (EDTA-Na) activated by adding 100 mM (final concentration) cysteine and then incubated at 37 oC for 3 h. The digestion of IgY was performed by combining 4 mL of IgY solution with 1 mL of activated papain (0.2 mg/mL) and incubating at 37 oC for 5h. The reactions were stopped by adding 5 mL of 60 mM iodoacetamide and placing it on ice for 1h. After concentrating hydrolysate using an ultrafiltration unit (50 kDa and 30 kDa cut-off sizes), 100% saturated ammonium sulfate (NH4)2SO4 was added to the final concentrations of 75%, 70%, 65%, 60%, 55%, 50% and 45% saturation to precipitate Fc and Fab fragments. The solution was centrifuged at 10,000 × g for 15 min at 4 oC, the precipitant collected, redissolved with distilled water, desalted using an ultrafiltration unit, and then freeze-dried (Alpha2-4LD, Christ, Germany).
2.3 Separation of IgY fragments using DEAE sepharose ion exchange chromatography The freeze-dried IgY digest was dissolved in 0.1M Tris-HCl (pH 8.0) at 6 mg/mL and then loaded on a DEAE-Sepharose Fast Flow (HiTrap, 5 mL) column (GE Healthcare Bio-Sciences AB, Björkgatan 30, SE75184 Uppsala, Sweden). The chromatographic elution was carried on a liquid chromatography system AKTATM pure instrument (GE Healthcare Bio-Sciences AB, Björkgatan 30, SE75184 Uppsala, Sweden) at a flow rate of 1 mg/mL, and the effluent was monitored at 280 nm. The pH and ionic strength are two important factors that influence the separation efficiency of ion-exchange chromatography [28]. Thus, the pH effect of the equilibrating solution and the ionic strength of the eluting solution on the separation IgY fragments were studied. The loaded column was washed with 10 mM Tris-HCl buffer (pH 8.5, 8.0 and 7.6) for 25 min, and then eluted with a linear gradient of 0 to 0.21 M, 0.4 M and 0.7 M NaCl in 10 mM Tris-HCl buffer, during 120 min. All eluents were collected, desalted, and then freeze-dried. All samples were then analyzed by SDS-PAGE. All experiments were conducted in triplicate. Later, the mixture of Fab and Fc obtained in step 2.2 was separated by DEAE chromatography under the optimal conditions. Each peak was collected, desalted by dialysis, and then freeze-dried.
2.5 Yield and Purity of Fab and Fc Fragments The yield of Fab and Fc fragments was calculated according to the formula given below: 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑜𝑤𝑑𝑒𝑟 × 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 × 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑝𝑢𝑟𝑖𝑡𝑦
Yield (100%)=
𝑤𝑒𝑖𝑔ht 𝑜𝑓 𝐼𝑔𝑌 × 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑜𝑓 𝐼𝑔𝑌
∗ 100%
Protein concentration was calculated by measuring absorbance at 595 nm using the Bradford kit following the manufacture`s instruction. The purity of Fab and Fc fragments was checked by non-reducing SDS-PAGE. Western blot was used to identify the target fragments. The separation process was triplicated.
2.6 SDS-PAGE and Western Blotting All samples were analyzed using 4-12% SDS-PAGE according to the previous work with some modifications [29]. The SDS-PAGEs were run both under reducing and non-reducing conditions. For the natural gel electrophoresis, a non-reducing protein loading buffer was added to the sample directly at a ratio of 3:1. The image of the stained protein was taken using the Gel DOC 2000 imaging system (Bio-Rad, Hercules. CA, USA). Western-blotting analysis of Fab and Fc fragments were conducted as described by Bozena Kubickova [30]. The proteins in the gel were transferred to polyvinylidene fluoride membranes (PVDF). The transfer was done for 20 min at 200 mA. Subsequently, the transferred membrane was blocked in 5% non-fat milk powder solution (dissolved in phosphate-buffered saline with Tween-20, PBST) for 1 h at 37 oC. The primary antibodies used were anti-chicken IgG (Fc) rabbit IgG (1:1000 diluted in blocking buffer) and anti-chicken IgG (Fab) rabbit IgG (1:1000 diluted in blocking buffer). The immune reaction was performed in an incubation solution that contained specific primary antibodies and kept shaking overnight at 4 oC. After washing 5-6 times with PBST, the membrane was reacted with the secondary rabbit anti-chicken IgY antibody conjugated to horseradish peroxidase (HRP) for 2 h at 37 oC. After the completion of the secondary antibody incubation, the membrane was washed 5-6 times with PBST to remove excessive secondary antibody and then exposed to ECL reagent for 5 min.
2.7 Mass spectrometry The target bands in the SDS-gel were cut and then reacted with trypsin for 20 h at
37
oC.
One
μL
enzymatic
hydrolysate
was
mixed
with
0.6
μL
saturatedα-Cyano-4-hydroxyzine amic acid (CHCA) matrix solution (diluted in 50% acetonitrile, ACN 0.1% trifluoroacetic acid, TFA), deposited on a sample plate, and then allowed to air-dry. The mass spectrometric analysis was performed using a 5800 MALDI-TOF/TOF MS (AB SCIEX, USA). The mass spectrometer was operated in the positive ion mode and pulsed nitrogen laser was operated at 349 nm with Nd: YAG duration pulses. The acceleration voltage was set as 2 kV. The resolution of the mass spectrometer was 100 ppm at m/z 800-4000 Da. The result was analyzed by the Mascot database (http://www.matrixscience.com).
3 Results and Discussion 3.1 Papain digest of IgY Pepsin is a typical enzyme to obtain antibody fragments from an immunoglobulin, but it would hydrolyze the Fc fragment into pieces [21]. Thus, papain was used to produce antibody fragments, and the optimal conditions established in our preliminary experiments were used to digest IgY (Fig. S1). IgY could be hydrolyzed almost completely (over 96%) after 5 h incubation with the activated papain at a ratio of 25:1 (protein: papain). Non-reducing SDS-PAGE analysis (Fig. 1) indicated that IgY protein was broken into four parts (lane 2). After digestion, the intact IgY band located on the top of the gel disappeared, and two visible bands appeared at around 66 kDa and 40 kDa area. Between the two bands, there was a wide band around 45 kDa, which might be caused by excessive digestion at multiple cleavage sites. Also, a light band appeared at the 33 kDa area. Suzuki and Lee [19] also observed similar phenomena. They reported that the fragments distributed around 66 kDa, 43 kDa and 33 kDa were Fc, and a wide band at around 45
kDa was Fab. Also, two undesirable bands were observed at around 20 kDa and 15 kDa areas. It is assumed that the weak bands around 20 kDa would be from the single-chain CH2 domain.
3.2 The effect of ultrafiltration and (NH4)2SO4 precipitation in removing low-molecular peptides Ultrafiltration is one of the widely applied methods in purifying proteins [26] to reduce sample volume as well as removing low-molecular-weight proteins or salts. Two membranes with a cut-off size of 50 kDa and 30 kDa were used to remove low-molecular peptide from the papain hydrolysate of IgY. Fig. 1 indicated that ultrafiltration did not affect removing the low-molecular peptides but ultrafiltration with 30 kDa membrane helped retaining more target proteins in the retentate. Salting out of proteins is another widely used method to separate proteins [31]. In this study, treating the low-molecular peptides with > 45% saturated ammonium sulfate precipitated Fc and Fab fragments and helped remove low-molecular-weight peptides (Fig. 2). The fragments with the molecular weight around 20 kDa were precipitated when the concentration of (NH4)2SO4 was increased to 50-75% saturation, indicating that higher salt concentration is required to precipitate smaller peptides than larger ones [31, 32]. So, the mixture of Fab and Fc could be collected using comparatively low salt concentrations and the small IgY fragments were successfully removed by adding 45% saturated (NH4)2SO4.
3.3 Further separation of Fab and Fc fragments using DEAE sepharose ion exchange chromatography Ion exchange chromatography (IEC) has been widely used in separating Fab from Fc fragments digested from IgG [15, 21, 24] Ion exchange chromatography separates proteins based on the differences in charge on the surface of molecules [33]. However, the co-purification of Fab and Fc fragments of IgY could be achieved only
using a subsequent affinity chromatography as reported by [19]. The different separating efficiency between IgG and IgY may be caused by the different pI and the pI of IgY is more acid than IgG [34]. However, the pI of Fab and Fc fragments is 7.0-9.5 and 5.5-6.0, respectively [23], and thus it is speculated that DEAE-sepharose ion-exchange chromatography alone can separate the Fab and Fc fragment easily. The pH and salt concentration gradient are two important factors that influence the separation efficiency of ion-exchange chromatography [35]. The chromatographs at 0.21 M, 0.4 M and 0.7 M NaCl in Fig. 3A. Two peaks around 60 mL were separated when the NaCl concentration in eluent reached 0.21 M, but the two peaks became closer as the concentration of NaCl in the eluent buffer increased. The non-reducing SDS-PAGE analysis of each peak under different conditions indicated that the sample obtained under 0.4 M and 0.7 M NaCl did not show a significant difference (Fig. 3B) and Fab and Fc fragments could not be separated further. However, when the concentration of NaCl decreased to 0.21 M, all low-molecular (around 20 kDa) fragments existed in lane 4 and 7 were washed off in advance. To elute the bound proteins on ion exchange resins, a salt gradient from 0 to 0.2-0.5 M is often used. In general, the proteins possessing different binding-ability to column ligand requires an appropriate salt concentration to wash out [35]. As a result, 0.21 M NaCl in Tris-HCl buffer was selected as the eluting solution. At a fixed concentration of NaCl in the eluting solution, the pH effect of the equilibrated solution on the purification effect was studied. Chromatographs at pH 7.6, pH 8.0 and pH 8.5 indicated that the peak around 10 ml was gradually resolved as the pH of the buffer increased (Fig. 4A). Also, the area of the other two peaks increased significantly and the peaks appeared later. As it showed in Fig. 4B, wide band around 50 kDa (Fab, in lanes 3, 6 and 9) gradually disappeared with the decreased of pH from pH 8.5 to 7.6; when the pH reached to 7.6, no apparent band was shown at 50 kDa area (Fig. 4B, lanes 8 and 9). Therefore, separation of Fab and Fc fragments could be easily accomplished by a one-step adjustment of the equilibration buffer. In this process, Fab was eluted out in the first peak, while Fc was eluted in the third peak because of the significant difference in the pI of Fab (7.0-9.5) and Fc (5.5-6.0)
fragments. When the pH of the equilibration buffer was 7.6, all Fc fragment could bind to the ligand and could be eluted out in the third peak, while Fab fragment can flow out in advance. As a result, a pH of equilibration buffer at 7.6 and a salt concentration of 0.21 M in elution buffer were chosen to conduct the column separation.
3.4 Purification of Fab and Fc fragments Fig. 5A indicated that the second peak was resolved after the hydrolysate was treated with ammonium sulfate. This phenomenon suggested that the contaminating peptides mainly existed in the second peak. The corresponding SDS-PAGE pattern of each peak (Fig. 5B) showed that the combination of salting out and IEC could successfully separate the Fab fragment from the Fc fragment. The Fab and Fc fragments were identified with a specific antibody on Western blots (Fig. 5C). Fab fragment was detected as wide band in lane 2 around 45 kDa while the bands around 66 kDa and 33 kDa were identified as Fc. However, the band around 40 kDa was uncertain and thus was subsequently analyzed using a MALDI-TOF MS. The result in Table 1 showed two major peptides with MW of 1829.9644 Da and 2634.1833 Da, which were identified as the characteristic peptides of IgY Fc fragment from the Mascot database (Chain A, Igy Fcu3-4 with Accession No of 2w59 A). The analysis showed two major amino acid sequences that are NTGPTT located near the initial part of the CH4 domain and VLPEER located near the end of the CH4 domain. This result suggested that the fragment around 40 kDa was the CH4 domain of Fc that was produced due to its alternative cleavage sites. Also, there existed one apparent band around 20 kDa, which could not be identified using anti-Fab Western Blotting nor anti-Fc Western Blotting (Fig. S2). Suzuki and Lee (2004) suggested that there was one N-glycosylation located in the CH2 domain. The presence of glycan could cause steric hindrance to inhibit the digestion in the CH2 domain of IgY. Thus, it was assumed that papain should have digested IgY into Fab, Fc, and additional CH2 fragments.
The purity of Fc and Fab fragments obtained from DEAE-Sepharose ion-exchange chromatography (Fig. 5B) indicated that Fab sample in lane 2 was mostly distributed at 50 kDa, and its purity was 88.7%, while Fc sample in lane 3 possessed 3 bands at around 66 kDa, 40 kDa and 33 kDa, and the purity was 90.1%. The use of 45% saturated (NH4)2SO4 enabled to remove low-molecular-weight fragments at around 20 kDa. The Fab and Fc fragments mixture could be effectively separated using a DEAE-Sepharose ion-exchange chromatography column. This is the first study showing that Fab and Fc fragment could be purified simultaneously using the combination of salting out and DEAE-Sepharose ion-exchange chromatography (Fig. 6). The yield of Fab and Fc fragments collected from the 100 mg IgY digest was 2.23 mg and 8.04 mg, respectively (Table 2).
4 Conclusion This study demonstrated that high purity Fab and Fc fragments from the papain digest could be separated without using affinity and size exclusion column combination. The combination of salting out and ion-exchange chromatography is an easy and economical method to separate Fab and Fc, providing technical support for further studying IgY`s active fragment. The importance of using the active fragments of IgY has been widely recognized in the pharmacokinetics research field. Thus, an easy and economical purification method can provide technical support for the research of IgY fragments. Acknowledgments
This research was supported by Excellent Youth Foundation of Hubei Province Natural Science Foundation (No. 2018CFA073) and the Fundamental Research Funds for the Central Universities (Project code No. 2662015PY080)
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Figure captions Fig 1. The effect of ultrafiltration on IgY digest. Lane 1: protein marker; lane 2: papain digest of IgY; lane 3: membrane cut-off size 50 kDa; lane 4: membrane cut-off size 30 kDa.
Fig 2. The SDS-PAGE analyses of ammonium sulfate on the IgY digested sample. Lane 1: marker; lanes 2-8: precipitant obtained from 75%, 70%, 65%, 60%, 55%, 50%, 45% saturated ammonium sulfate.
Fig 3. The effect ionic strength on the separation of Fab and Fc fragments. (A): anion-exchange chromatograms under 0.7 M, 0.4 M and 0.21 M NaCl in elution buffer; (B): SDS-PAGE pattern of peaks obtained at different ion strength of buffer. Lane 1: protein marker; lanes 2-4: fractions obtained under 0.7M NaCl; lanes 5-7: fractions obtained under 0.4 M NaCl; lanes 8-10: fractions obtained under 0.21 M NaCl.
Fig 4. The effect of buffer pH on the separation of Fab and Fc fragment. (A): anion-exchange chromatograms under pH 7.5, pH 8.0 and pH 8.5 of equilibration buffer; (B): SDS-PAGE pattern of peaks obtained at different pH of equilibration buffer. Lane 1: protein marker; lanes 2-3: fractions obtained under pH 8.5; lanes 4-6: fractions obtained under pH 8.0; lanes 7-9: fractions obtained under pH 7.6.
Fig 5.
(A) Anion-exchange chromatograms of IgY digests after salting out; (B) The
SDS-PAGE analysis of peaks obtained from anion-exchange chromatography: Lanes 1-3: lane 1, protein marker; lane 2, the first peak; lane 3, the third peak; (C) WB analysis of peaks obtained from anion-exchange chromatography: lane anti-Fab, the first peak; lane anti-Fc, the third peak.
Fig 6. The scheme of the entire purification process
Table captions Table1 Mascot search results of the unknown fragment Table2 the purity and yield of Fab and Fc fragments obtained by DEAE FF Table1 Mascot search results of the unknown fragment Reference a
C. I. %c
Accession No d
m/z
Sequence
77
100
2w59 A
1829.9644
160AVPATEFVTTAVLPEER176
51
100
2w59 A
2634.1833
112NTGPTTPPLIYPFAPHPE
Ion Score b
Chain A, Igy Fcu3-4 Chain A, Igy Fcu3-4
ELSLSR135
a Protein recommended by the Mascot database. b Protein ion score which is reliable above 40. c Protein score which is reliable above 95. d Serial number of the protein in the database. Table2 the purity and yield of Fab and Fc fragments obtained by DEAE FF Sample
Weight a (mg)
Protein content b (%)
Purity c (%)
Protein weight (mg)
IgY Fab Fc
501.5±2.12 17.7±0.57 42.9±1.55
73.98±1.87 47.59±2.91 70.04±3.39
91.08±1.32 88.7±3.32 90.1±2.97
336.9±10.93 7.5±0.78 27.1±2.53
a The weight of freeze-dried powder b Bradford kit c Non-reducing SDS-PAGE
Total yield from intact IgY (%) 2.23±0.14 8.04±0.45
CRediT authorship contribution statement Xin Zhou: Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing - original draft, Writing - review & editing. Yanru Wang: Resources, Data curation. Dong Uk Ahn: Formal analysis, Data curation, Writing Review & Editing, Supervision. Zhaoxia Cai: Resources, Methodology, Data curation, Supervision, Writing - original draft.
Conflict of interest The authors have declared no conflict of interest
Highlight:
Fab and Fc fragments can be obtained simultaneously by using the combination method of salting out and DEAE Sepharose ion exchange column.
The purity was improved by optimizing the conditions of pH and ionic strength
The high purity of Fab and Fc fragments was determined by SDS-PAGE, 88.7% and 90.1%, respectively.
25
S1570-0232(19)31654-X https://doi.org/10.1016/j.jchromb.2020.122011 CHROMB 122011
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
9 November 2019 25 January 2020 30 January 2020
Please cite this article as: X. Zhou, Y. Wang, D. Uk Ahn, Z. Cai, An Easy and Simple Separation Method for Fc and Fab Fragments from Chicken Immunoglobulin Y (IgY), Journal of Chromatography B (2020), doi: https:// doi.org/10.1016/j.jchromb.2020.122011
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© 2020 Published by Elsevier B.V.
An Easy and Simple Separation Method for Fc and Fab Fragments from Chicken Immunoglobulin Y (IgY)
Xin Zhou1, Yanru Wang1, Dong Uk Ahn2, Zhaoxia Cai1*
1. College of Food Science and Technology, Huazhong Agricultural University, National Research and Development Center for Egg Processing, Wuhan, Hubei, 430070, PR China 2. Department of Animal Science, Iowa State University, Ames, IA 50011, USA
Abstract Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.
Keywords:
IgY;
Fab
fragment;
Anion-exchange chromatography;
Fc
fragment;
(NH4)2SO4
precipitation;
1 Introduction Chicken IgY, a type of immunoglobulin mainly existing in egg yolk, is composed of two identical Fab segments and one Fc segment [1, 2]. IgY has many advantages over mammalian IgG, 1) it can be obtained easily from egg (100-150 mg IgY/each egg yolk)[3]; 2) has high avidity to mammalian antigens; (3) has less cross-reactivity with mammalian IgG; 4) does not activate or recognize the mammalian complements, and 5) does not react with rheumatoid factors (RF) [4]. Recent studies indicated that the fragments, as well as intact immunoglobulins, have significant effects on immune responses [5]. Fab fragment is composed of partial H-chain (VH+CH1) and entire L-chain (VL+CL1), and has the antigen-binding sites. Due to the cleavage of Fc fragment (antigen portion), Fab fragment has been developed to an antimicrobial agent that performs better than an intact antibody [6], and is widely used in diagnostics and biopharmaceutical research [7-9]. Besides, Fab fragment can be used as a ligand in purification study due to its affinity-binding ability [10]. Crystallizable fragment (Fc) is vital to trigger immune defense through the interactions with specific Fc receptors, which contains two or three domains of the entire heavy chain, CH2, CH3 and CH4. Nhg et al. (2018) and Smith et al. (2014) reported that the glycosylation of Fc can provide a strategy to engineer antibodies with high therapeutic efficacy and pharmacokinetics [11, 12]. Many researchers also used the Fc region to study the mechanism of IgY transport into egg yolk [13, 14]. Vermeer, Norde, & Van (2000) showed that stable Fab and Fc fragments could behave as independent subunits within an intact immunoglobulin and suggested that the functions of Fab and Fc could be studied independently using the isolated Fab and Fc fragments [15]. Thus, it is meaningful to collect Fab and Fc fragments separately. Due to the advanced recombinant DNA technologies, Fab and Fc fragments can be produced through the transient expression of appropriate constructs in mammalian cells or Escherichia coli cells [16-18]. However, it is very expensive and sometimes the
synthesized Fc could be inactive [14]. Some data shows that the Fc fragment of IgY obtained by papain digest mainly refers to Fc3-4 (residues 346-566), and its size is 53 kDa while the value of Fab is 44 kDa [19, 20]. Pepsin is typically used to produce active fragments, but it hydrolyzes Fc fragment into pieces [21]. Protein A and G have been commonly used as immunoglobulin-binding proteins (IBPS) to purify antibodies like IgG and IgA through binding to the Fc domain. However, due to the differences in amino acid sequence and conformation, IBPS has no affinity to IgY. Recently, Jiang, Diraviyam, & Zhang (2016) developed a new ligand, human mycoplasma protein (protein M), as an IBPS to purify IgY through the interactions with the Fab domain. However, the pH of the elution buffer used in the affinity purification was about 3 that reduced the activity of fragments. Also, the affinity column used was not stable for the purification and had a low capacity [22]. Due to similar molecular sizes, the Fab and Fc fractions collected from the size exclusion chromatography contained contaminants [17]. Gradiflow is a preparative instrument to purify Fab from IgY digest. However, the purified Fab was still contaminated with low-molecular-weight fragments [23]. Ion exchange chromatography (IEC) has been broadly used to purify Fab fragment from IgG [24, 25], but the purification of high-purity immunoglobulin fragments could be achieved only when a subsequent multi-column chromatography was used [19]. The objectives of this study were 1) to establish the optimal conditions for purifying Fab and Fc fragments from the papain hydrolysate, and 2) to identify separated Fab and Fc fractions using Western blot and MALDI-TOF-MS.
2 Materials and Methods 2.1 Materials Fresh eggs were purchased from Wuhan Jiufeng farm (Jiufeng Farm, Wuhan, China). Anti-chicken IgG (Fc) (rabbit) antibody and anti-chicken IgG (Fab) (rabbit) antibody were purchased from Rockland immunochemical (Gilbertsville, PA).
Chicken IgY standard was purchased from Bio Unit (Shanghai, China). Papain was purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents used in SDS-PAGE were purchased from Guge Biotechnology (Wuhan, China). The Bradford kit was purchased from Bio Time Biotechnology (Shanghai, China). The deionized distilled water was produced by a water purification system from Bio-Legend Bio-Tech Co., Ltd. (Shanghai China). Other chemical reagents were all analytical grade.
2.2 Papain digestion of IgY IgY was purified using the salting-out method of previous works [26, 27]. The purified IgY was dissolved in 0.1 M PBS (pH 7.0) at a concentration of 1.25 mg/mL and then the IgY content was determined using the Bradford method. Papain solution (0.2 mg/mL) was prepared by dissolving papain powder in 0.1 M, pH 7.0 phosphate-buffered saline (PBS) containing 20 mM ethylenediaminetetraacetic acid disodium salt (EDTA-Na) activated by adding 100 mM (final concentration) cysteine and then incubated at 37 oC for 3 h. The digestion of IgY was performed by combining 4 mL of IgY solution with 1 mL of activated papain (0.2 mg/mL) and incubating at 37 oC for 5h. The reactions were stopped by adding 5 mL of 60 mM iodoacetamide and placing it on ice for 1h. After concentrating hydrolysate using an ultrafiltration unit (50 kDa and 30 kDa cut-off sizes), 100% saturated ammonium sulfate (NH4)2SO4 was added to the final concentrations of 75%, 70%, 65%, 60%, 55%, 50% and 45% saturation to precipitate Fc and Fab fragments. The solution was centrifuged at 10,000 × g for 15 min at 4 oC, the precipitant collected, redissolved with distilled water, desalted using an ultrafiltration unit, and then freeze-dried (Alpha2-4LD, Christ, Germany).
2.3 Separation of IgY fragments using DEAE sepharose ion exchange chromatography The freeze-dried IgY digest was dissolved in 0.1M Tris-HCl (pH 8.0) at 6 mg/mL and then loaded on a DEAE-Sepharose Fast Flow (HiTrap, 5 mL) column (GE Healthcare Bio-Sciences AB, Björkgatan 30, SE75184 Uppsala, Sweden). The chromatographic elution was carried on a liquid chromatography system AKTATM pure instrument (GE Healthcare Bio-Sciences AB, Björkgatan 30, SE75184 Uppsala, Sweden) at a flow rate of 1 mg/mL, and the effluent was monitored at 280 nm. The pH and ionic strength are two important factors that influence the separation efficiency of ion-exchange chromatography [28]. Thus, the pH effect of the equilibrating solution and the ionic strength of the eluting solution on the separation IgY fragments were studied. The loaded column was washed with 10 mM Tris-HCl buffer (pH 8.5, 8.0 and 7.6) for 25 min, and then eluted with a linear gradient of 0 to 0.21 M, 0.4 M and 0.7 M NaCl in 10 mM Tris-HCl buffer, during 120 min. All eluents were collected, desalted, and then freeze-dried. All samples were then analyzed by SDS-PAGE. All experiments were conducted in triplicate. Later, the mixture of Fab and Fc obtained in step 2.2 was separated by DEAE chromatography under the optimal conditions. Each peak was collected, desalted by dialysis, and then freeze-dried.
2.5 Yield and Purity of Fab and Fc Fragments The yield of Fab and Fc fragments was calculated according to the formula given below: 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑜𝑤𝑑𝑒𝑟 × 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 × 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑝𝑢𝑟𝑖𝑡𝑦
Yield (100%)=
𝑤𝑒𝑖𝑔ht 𝑜𝑓 𝐼𝑔𝑌 × 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑜𝑓 𝐼𝑔𝑌
∗ 100%
Protein concentration was calculated by measuring absorbance at 595 nm using the Bradford kit following the manufacture`s instruction. The purity of Fab and Fc fragments was checked by non-reducing SDS-PAGE. Western blot was used to identify the target fragments. The separation process was triplicated.
2.6 SDS-PAGE and Western Blotting All samples were analyzed using 4-12% SDS-PAGE according to the previous work with some modifications [29]. The SDS-PAGEs were run both under reducing and non-reducing conditions. For the natural gel electrophoresis, a non-reducing protein loading buffer was added to the sample directly at a ratio of 3:1. The image of the stained protein was taken using the Gel DOC 2000 imaging system (Bio-Rad, Hercules. CA, USA). Western-blotting analysis of Fab and Fc fragments were conducted as described by Bozena Kubickova [30]. The proteins in the gel were transferred to polyvinylidene fluoride membranes (PVDF). The transfer was done for 20 min at 200 mA. Subsequently, the transferred membrane was blocked in 5% non-fat milk powder solution (dissolved in phosphate-buffered saline with Tween-20, PBST) for 1 h at 37 oC. The primary antibodies used were anti-chicken IgG (Fc) rabbit IgG (1:1000 diluted in blocking buffer) and anti-chicken IgG (Fab) rabbit IgG (1:1000 diluted in blocking buffer). The immune reaction was performed in an incubation solution that contained specific primary antibodies and kept shaking overnight at 4 oC. After washing 5-6 times with PBST, the membrane was reacted with the secondary rabbit anti-chicken IgY antibody conjugated to horseradish peroxidase (HRP) for 2 h at 37 oC. After the completion of the secondary antibody incubation, the membrane was washed 5-6 times with PBST to remove excessive secondary antibody and then exposed to ECL reagent for 5 min.
2.7 Mass spectrometry The target bands in the SDS-gel were cut and then reacted with trypsin for 20 h at
37
oC.
One
μL
enzymatic
hydrolysate
was
mixed
with
0.6
μL
saturatedα-Cyano-4-hydroxyzine amic acid (CHCA) matrix solution (diluted in 50% acetonitrile, ACN 0.1% trifluoroacetic acid, TFA), deposited on a sample plate, and then allowed to air-dry. The mass spectrometric analysis was performed using a 5800 MALDI-TOF/TOF MS (AB SCIEX, USA). The mass spectrometer was operated in the positive ion mode and pulsed nitrogen laser was operated at 349 nm with Nd: YAG duration pulses. The acceleration voltage was set as 2 kV. The resolution of the mass spectrometer was 100 ppm at m/z 800-4000 Da. The result was analyzed by the Mascot database (http://www.matrixscience.com).
3 Results and Discussion 3.1 Papain digest of IgY Pepsin is a typical enzyme to obtain antibody fragments from an immunoglobulin, but it would hydrolyze the Fc fragment into pieces [21]. Thus, papain was used to produce antibody fragments, and the optimal conditions established in our preliminary experiments were used to digest IgY (Fig. S1). IgY could be hydrolyzed almost completely (over 96%) after 5 h incubation with the activated papain at a ratio of 25:1 (protein: papain). Non-reducing SDS-PAGE analysis (Fig. 1) indicated that IgY protein was broken into four parts (lane 2). After digestion, the intact IgY band located on the top of the gel disappeared, and two visible bands appeared at around 66 kDa and 40 kDa area. Between the two bands, there was a wide band around 45 kDa, which might be caused by excessive digestion at multiple cleavage sites. Also, a light band appeared at the 33 kDa area. Suzuki and Lee [19] also observed similar phenomena. They reported that the fragments distributed around 66 kDa, 43 kDa and 33 kDa were Fc, and a wide band at around 45
kDa was Fab. Also, two undesirable bands were observed at around 20 kDa and 15 kDa areas. It is assumed that the weak bands around 20 kDa would be from the single-chain CH2 domain.
3.2 The effect of ultrafiltration and (NH4)2SO4 precipitation in removing low-molecular peptides Ultrafiltration is one of the widely applied methods in purifying proteins [26] to reduce sample volume as well as removing low-molecular-weight proteins or salts. Two membranes with a cut-off size of 50 kDa and 30 kDa were used to remove low-molecular peptide from the papain hydrolysate of IgY. Fig. 1 indicated that ultrafiltration did not affect removing the low-molecular peptides but ultrafiltration with 30 kDa membrane helped retaining more target proteins in the retentate. Salting out of proteins is another widely used method to separate proteins [31]. In this study, treating the low-molecular peptides with > 45% saturated ammonium sulfate precipitated Fc and Fab fragments and helped remove low-molecular-weight peptides (Fig. 2). The fragments with the molecular weight around 20 kDa were precipitated when the concentration of (NH4)2SO4 was increased to 50-75% saturation, indicating that higher salt concentration is required to precipitate smaller peptides than larger ones [31, 32]. So, the mixture of Fab and Fc could be collected using comparatively low salt concentrations and the small IgY fragments were successfully removed by adding 45% saturated (NH4)2SO4.
3.3 Further separation of Fab and Fc fragments using DEAE sepharose ion exchange chromatography Ion exchange chromatography (IEC) has been widely used in separating Fab from Fc fragments digested from IgG [15, 21, 24] Ion exchange chromatography separates proteins based on the differences in charge on the surface of molecules [33]. However, the co-purification of Fab and Fc fragments of IgY could be achieved only
using a subsequent affinity chromatography as reported by [19]. The different separating efficiency between IgG and IgY may be caused by the different pI and the pI of IgY is more acid than IgG [34]. However, the pI of Fab and Fc fragments is 7.0-9.5 and 5.5-6.0, respectively [23], and thus it is speculated that DEAE-sepharose ion-exchange chromatography alone can separate the Fab and Fc fragment easily. The pH and salt concentration gradient are two important factors that influence the separation efficiency of ion-exchange chromatography [35]. The chromatographs at 0.21 M, 0.4 M and 0.7 M NaCl in Fig. 3A. Two peaks around 60 mL were separated when the NaCl concentration in eluent reached 0.21 M, but the two peaks became closer as the concentration of NaCl in the eluent buffer increased. The non-reducing SDS-PAGE analysis of each peak under different conditions indicated that the sample obtained under 0.4 M and 0.7 M NaCl did not show a significant difference (Fig. 3B) and Fab and Fc fragments could not be separated further. However, when the concentration of NaCl decreased to 0.21 M, all low-molecular (around 20 kDa) fragments existed in lane 4 and 7 were washed off in advance. To elute the bound proteins on ion exchange resins, a salt gradient from 0 to 0.2-0.5 M is often used. In general, the proteins possessing different binding-ability to column ligand requires an appropriate salt concentration to wash out [35]. As a result, 0.21 M NaCl in Tris-HCl buffer was selected as the eluting solution. At a fixed concentration of NaCl in the eluting solution, the pH effect of the equilibrated solution on the purification effect was studied. Chromatographs at pH 7.6, pH 8.0 and pH 8.5 indicated that the peak around 10 ml was gradually resolved as the pH of the buffer increased (Fig. 4A). Also, the area of the other two peaks increased significantly and the peaks appeared later. As it showed in Fig. 4B, wide band around 50 kDa (Fab, in lanes 3, 6 and 9) gradually disappeared with the decreased of pH from pH 8.5 to 7.6; when the pH reached to 7.6, no apparent band was shown at 50 kDa area (Fig. 4B, lanes 8 and 9). Therefore, separation of Fab and Fc fragments could be easily accomplished by a one-step adjustment of the equilibration buffer. In this process, Fab was eluted out in the first peak, while Fc was eluted in the third peak because of the significant difference in the pI of Fab (7.0-9.5) and Fc (5.5-6.0)
fragments. When the pH of the equilibration buffer was 7.6, all Fc fragment could bind to the ligand and could be eluted out in the third peak, while Fab fragment can flow out in advance. As a result, a pH of equilibration buffer at 7.6 and a salt concentration of 0.21 M in elution buffer were chosen to conduct the column separation.
3.4 Purification of Fab and Fc fragments Fig. 5A indicated that the second peak was resolved after the hydrolysate was treated with ammonium sulfate. This phenomenon suggested that the contaminating peptides mainly existed in the second peak. The corresponding SDS-PAGE pattern of each peak (Fig. 5B) showed that the combination of salting out and IEC could successfully separate the Fab fragment from the Fc fragment. The Fab and Fc fragments were identified with a specific antibody on Western blots (Fig. 5C). Fab fragment was detected as wide band in lane 2 around 45 kDa while the bands around 66 kDa and 33 kDa were identified as Fc. However, the band around 40 kDa was uncertain and thus was subsequently analyzed using a MALDI-TOF MS. The result in Table 1 showed two major peptides with MW of 1829.9644 Da and 2634.1833 Da, which were identified as the characteristic peptides of IgY Fc fragment from the Mascot database (Chain A, Igy Fcu3-4 with Accession No of 2w59 A). The analysis showed two major amino acid sequences that are NTGPTT located near the initial part of the CH4 domain and VLPEER located near the end of the CH4 domain. This result suggested that the fragment around 40 kDa was the CH4 domain of Fc that was produced due to its alternative cleavage sites. Also, there existed one apparent band around 20 kDa, which could not be identified using anti-Fab Western Blotting nor anti-Fc Western Blotting (Fig. S2). Suzuki and Lee (2004) suggested that there was one N-glycosylation located in the CH2 domain. The presence of glycan could cause steric hindrance to inhibit the digestion in the CH2 domain of IgY. Thus, it was assumed that papain should have digested IgY into Fab, Fc, and additional CH2 fragments.
The purity of Fc and Fab fragments obtained from DEAE-Sepharose ion-exchange chromatography (Fig. 5B) indicated that Fab sample in lane 2 was mostly distributed at 50 kDa, and its purity was 88.7%, while Fc sample in lane 3 possessed 3 bands at around 66 kDa, 40 kDa and 33 kDa, and the purity was 90.1%. The use of 45% saturated (NH4)2SO4 enabled to remove low-molecular-weight fragments at around 20 kDa. The Fab and Fc fragments mixture could be effectively separated using a DEAE-Sepharose ion-exchange chromatography column. This is the first study showing that Fab and Fc fragment could be purified simultaneously using the combination of salting out and DEAE-Sepharose ion-exchange chromatography (Fig. 6). The yield of Fab and Fc fragments collected from the 100 mg IgY digest was 2.23 mg and 8.04 mg, respectively (Table 2).
4 Conclusion This study demonstrated that high purity Fab and Fc fragments from the papain digest could be separated without using affinity and size exclusion column combination. The combination of salting out and ion-exchange chromatography is an easy and economical method to separate Fab and Fc, providing technical support for further studying IgY`s active fragment. The importance of using the active fragments of IgY has been widely recognized in the pharmacokinetics research field. Thus, an easy and economical purification method can provide technical support for the research of IgY fragments. Acknowledgments
This research was supported by Excellent Youth Foundation of Hubei Province Natural Science Foundation (No. 2018CFA073) and the Fundamental Research Funds for the Central Universities (Project code No. 2662015PY080)
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using
a
human
mycoplasma
protein,
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Figure captions Fig 1. The effect of ultrafiltration on IgY digest. Lane 1: protein marker; lane 2: papain digest of IgY; lane 3: membrane cut-off size 50 kDa; lane 4: membrane cut-off size 30 kDa.
Fig 2. The SDS-PAGE analyses of ammonium sulfate on the IgY digested sample. Lane 1: marker; lanes 2-8: precipitant obtained from 75%, 70%, 65%, 60%, 55%, 50%, 45% saturated ammonium sulfate.
Fig 3. The effect ionic strength on the separation of Fab and Fc fragments. (A): anion-exchange chromatograms under 0.7 M, 0.4 M and 0.21 M NaCl in elution buffer; (B): SDS-PAGE pattern of peaks obtained at different ion strength of buffer. Lane 1: protein marker; lanes 2-4: fractions obtained under 0.7M NaCl; lanes 5-7: fractions obtained under 0.4 M NaCl; lanes 8-10: fractions obtained under 0.21 M NaCl.
Fig 4. The effect of buffer pH on the separation of Fab and Fc fragment. (A): anion-exchange chromatograms under pH 7.5, pH 8.0 and pH 8.5 of equilibration buffer; (B): SDS-PAGE pattern of peaks obtained at different pH of equilibration buffer. Lane 1: protein marker; lanes 2-3: fractions obtained under pH 8.5; lanes 4-6: fractions obtained under pH 8.0; lanes 7-9: fractions obtained under pH 7.6.
Fig 5.
(A) Anion-exchange chromatograms of IgY digests after salting out; (B) The
SDS-PAGE analysis of peaks obtained from anion-exchange chromatography: Lanes 1-3: lane 1, protein marker; lane 2, the first peak; lane 3, the third peak; (C) WB analysis of peaks obtained from anion-exchange chromatography: lane anti-Fab, the first peak; lane anti-Fc, the third peak.
Fig 6. The scheme of the entire purification process
Table captions Table1 Mascot search results of the unknown fragment Table2 the purity and yield of Fab and Fc fragments obtained by DEAE FF Table1 Mascot search results of the unknown fragment Reference a
C. I. %c
Accession No d
m/z
Sequence
77
100
2w59 A
1829.9644
160AVPATEFVTTAVLPEER176
51
100
2w59 A
2634.1833
112NTGPTTPPLIYPFAPHPE
Ion Score b
Chain A, Igy Fcu3-4 Chain A, Igy Fcu3-4
ELSLSR135
a Protein recommended by the Mascot database. b Protein ion score which is reliable above 40. c Protein score which is reliable above 95. d Serial number of the protein in the database. Table2 the purity and yield of Fab and Fc fragments obtained by DEAE FF Sample
Weight a (mg)
Protein content b (%)
Purity c (%)
Protein weight (mg)
IgY Fab Fc
501.5±2.12 17.7±0.57 42.9±1.55
73.98±1.87 47.59±2.91 70.04±3.39
91.08±1.32 88.7±3.32 90.1±2.97
336.9±10.93 7.5±0.78 27.1±2.53
a The weight of freeze-dried powder b Bradford kit c Non-reducing SDS-PAGE
Total yield from intact IgY (%) 2.23±0.14 8.04±0.45
CRediT authorship contribution statement Xin Zhou: Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing - original draft, Writing - review & editing. Yanru Wang: Resources, Data curation. Dong Uk Ahn: Formal analysis, Data curation, Writing Review & Editing, Supervision. Zhaoxia Cai: Resources, Methodology, Data curation, Supervision, Writing - original draft.
Conflict of interest The authors have declared no conflict of interest
Highlight:
Fab and Fc fragments can be obtained simultaneously by using the combination method of salting out and DEAE Sepharose ion exchange column.
The purity was improved by optimizing the conditions of pH and ionic strength
The high purity of Fab and Fc fragments was determined by SDS-PAGE, 88.7% and 90.1%, respectively.
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