Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand
© Ding and Cao; licensee BioMed Central Ltd. 2013
Received: 21 March 2013
Accepted: 10 December 2013
Published: 17 December 2013
Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production.
A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE.
A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.
Since the late 1960s, affinity purification methods have been developed and continuously improved. Affinity precipitation was reported as a potential technology for the purification of proteins during early stages of downstream processing . This technology could be achieved by using reversibly soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material, such as fermentation broths or plasma. The applications of the technology depend upon the design of efficient synthetic soluble-insoluble polymers, such as pH-, temperature- and light-response polymers. Zhou et al.  purified lipase using a thermo-response polymer with hydrophobic butyl groups as a ligand. Chen and Hoffman  synthesized a copolymer of N-isopropyl acrylamide and N-acryloxysuccinimide and immobilized Protein A on this copolymer, which was used to purify IgG by thermo-precipitation. Ling and Zhu  purified BSA using a thermo-sensitive copolymer consisting of N-vinyl-2-caprolactam (NVCL) and methacrylic acid, with copper as the ligand. In our group, a thermo-response polymer was synthesized using N-methylol acrylamide (N-MAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers , and the polymer was applied to purify lysozyme with an immobilized Cibacron Blue F3GA ligand.
Human Serum Albumin (HSA), the most abundant protein in human plasma, has been one of the most extensively studied proteins over several decades [6, 7]. It is synthesized in the liver and presents in the blood with a concentration around 40 mg/ml. HSA is the major transport protein in plasma, involved in the distribution and metabolism of many biologically active compounds such as fatty acids, amino acids, natural products and drugs because of the high affinity between HSA and these biological substances . HSA is therefore widely used in pharmaceuticals. Plasma fractionation , aqueous two-phase systems , affinity beads , cation exchange method  and affinity membranes , among other methods, have been used to isolate albumin from blood plasma or serum. Affinity precipitation can reduce the volume of crude material significantly and improve purification efficiency in the early stage of protein purification. Dyes [14–17] and metal ions [18–20] are generally used as the affinity ligand to purify HSA.
In this study, a thermo-response copolymer, PNBN, was synthesized using NIPA, BA and N-MAM, and then L-thyroxin was coupled to the polymer to obtain an affinity matrix, PNBN-T. The thermo-response polymer with the L-thyroxin ligand was used for HSA purification by affinity precipitation. L-thyroxin is a synthetic form of a hormone normally secreted by the follicular cells of the thyroid gland and is typically used as a drug to treat hypothyroidism, transported in human serum . Compared with dye or metal ion ligands, it is a relatively safe affinity ligand. As far as we know, the polymer safety is unlikely to be problematic, for the following reasons. First, these monomers are widely used in polymers or copolymers that have been applied for several decades so their safety has been confirmed many times. Moreover, the recovery of PNBN-T is so high that there are only trace amounts of the polymer existing in the purified materials and this could be removed in the next purification step.
According to Petitpas and Petersen , there are structural interactions between L-thyroxin (PDB ID code 1HK1) and HSA. The phenolic hydroxyl of L-thyroxin contributes specific hydrogen bond interactions with the side chains of Y150 and R257 on HSA. The outer ring of L-thyroxin allows the phenolic hydroxyl to form hydrogen bonds with the side chains of Y411 and S489 on HSA, while the inner ring forms van der Waals contact with the side chains of Q390, N391, L394, A406, and R410. The L-thyroxin carboxylate group forms a hydrogen bond to a water molecule stabilized by D301 from a symmetry-related HSA molecule. We thus speculated that L-thyroxin could be used as a novel affinity ligand for HSA purification.
In this study, L-thyroxin was immobilized as an affinity ligand on a thermo-response polymer comprising NIPA, BA and N-MAM monomers to form an affinity polymer, which was used for HSA purification by affinity precipitation. The adsorption to and elution of HSA from the affinity polymer were investigated and several interesting results were obtained. This paper is the first to report HSA purification by affinity precipitation with a thermo-response copolymer with immobilized L-thyroxin as a novel ligand. Using this system, HSA was purified to high purity in a single step.
Results and discussion
Synthesis of PNBN
A thermo-response polymer, PNBN, was randomly copolymerized using NIPA, BA and N-MAM as monomers. Among the three monomers, NIPA provides thermo-responsiveness, the hydroxyl groups on N-MAM can be used to immobilize a hydrophobic ligand, and BA is used to control hydrophobicity. Considering the recovery and thermo-response character of the polymer and biomolecular stability, NIPA (7.77 mmol/g), N-MAM (0.44 mmol/g) and BA (0.49 mmol/g) were selected as the final ratio. The molecular weight of the polymer measured by gel permeation chromatography (GPC) was 6.52 × 104 Da. The lower critical solution temperature (LCST) and recovery of corresponding polymer were 31.0°C and 99.6% of its initial amount after recycling three times.
Immobilization of L-thryoxin on PNBN
When the initial amount of ECH was varied from 1.91 to 5.10 mmol, the densities of the ligand immobilized onto the copolymer increased from 56.10 to 62.35 μmol/g. The ligand density increased by less than 10% with a 2.5 fold increase in ECH, which indicates that the amount of ECH used did not markedly affect ligand density. The reason for this may be that the polymer could only bind to a limited amount of ECH. Figure 1(b) shows that the ligand density underwent a clear increment from 39.10 to 66.30 μmol/g with an increasing initial amount of L-thyroxin. At the same time, the yield of activated L-thyroxin after conjugation to the polymer decreased from 70.18% to 50.54%. The desired ligand density could thus be controlled according to these results.
Recovery and LCST of PNBN-T
Recovery is the most important parameter during affinity precipitation by reversible soluble-insoluble polymers. Measurements of LCST and recovery of PNBN-T were performed five times. NaCl addition can increase the recovery of thermo-response copolymers  so NaCl was also added to a final concentration of 0.5 mol/L. The maximal recovery achieved was 99.6% and the LCST was 31.0°C.
Adsorption of HSA
Adsorption kinetics curve
As shown in the literature , the molecular weight of HSA is about 67,000 Da so the adsorption capacity was equivalent to 0.21 μmol/g affinity polymer when the adsorption reached equilibrium. Compared with the ligand density of 60.0 μmol/g affinity polymer, this is apparently a relatively low binding efficiency. It is probably that the affinity interaction between HSA and L-thyroxin occurs via multi-site binding because it is difficult to understand the low binding efficiency if only mono-site binding occurs. Further study into this aspect should be conducted in the future. The steric hindrance caused by the large size of HSA is also a feasible reason for the low apparent binding efficiency. In subsequent experiments, the reaction time was controlled at 120.0 min to ensure adsorption.
Effect of ligand density, pH and ionic strength
The pH of the solution has an important effect on the adsorption equilibrium of HSA. In this study, HSA adsorption capacity was slightly decreased from pH 3.0 to 7.0. With an increase in pH, the adsorption capacity decreased sharply, as shown in Figure 3(b). The reason for this decrease in capacity is that the HSA molecules become negatively charged at higher pH values (above the isoelectric point), and the ligands on the polymer are also negatively charged. Electrostatic forces dominate at low salt concentrations, explaining the adsorption curve versus pH behaviors described above.
It can be seen in Figure 3(c) that protein adsorption capacity decreased with increasing ionic strength. NaCl was used to increase the recovery of the affinity polymer at high ionic strength and under this condition, the presence of NaCl reduced the attraction between the HSA and L-thyroxin molecules on the polymer. In subsequent experiments, NaCl was not used for adsorption of HSA.
Fitting Equation (1) to the experimental data, we obtained Qm as 14.87 mg/g affinity polymer and Kd as 0.11 mg/ml. From the molecular weight of HSA, this Qm value is equivalent to 0.22 μmol/g affinity polymer and Kd is equivalent to 1.64 μmol/ml. According to Petitpas , HSA have several binding sites for the ligand L-thyroxin. As described above and shown in Figure 3(a), the HSA adsorption capacity increased proportionally with ligand densities on polymers below 60.0 μmol/g and increased gradually above that value. This phenomenon may be caused by ligands from different polymer molecules binding onto different sites of each HSA molecule. PNBN-T was synthesized for affinity precipitation, which is usually used to purify proteins from large volumes of dilute solution. Both adsorption ratio and capacity are significant factors in this process. The adsorption isotherm was therefore drawn according to these influencing factors. Adsorption follows the Langmuir isotherm and appears to reach Langmuir saturation under the conditions used. Compared with the ligand density of 60.0 μmol/g affinity polymer, adsorption showed a relatively low utilization of ligand. This behavior may be caused by the large size of HSA, resulting in steric hindrance.
Circular dichroism (CD) measurements
where Cp is the molar concentration of the protein, n is the number of amino acid residues, and l is the path length. From Equation (2), the α-helix ratio in the secondary structure of HSA was shown to decrease from 15.44% (A) to 14.26% (B), 12.44% (C) or 9.08% (D) as in Figure 5 by adding different concentrations of L-thyroxin. These results indicate that the ligand underwent interactions with HSA, and that increasing the amount of L-thyroxin added caused greater changes in the secondary structure of HSA.
ForteBio octet system assay
Desorption of HSA
Efficiency of HSA desorption
Different desorbed conditions
1.0 mol/L NaSCN
0.1 mol/L EDTA
pH7.4 Tris–HCl+0.5 mol/L Urea
pH7.4 Tris–HCl+1.0% Triton 100
pH7.4 Tris–HCl+20.0% Glycol
Affinity precipitation of HSA from human serum
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and CD measurement were used to analyze the purified HSA.
Recycle of PNBN-T
To investigate the reusability of the polymer, the adsorption-desorption cycle of HSA was repeated five times using the same polymer sample. The HSA adsorbed onto the polymer was eluted by 1.0 mol/L NaSCN. The results show that the affinity polymers could be repeatedly used for HSA adsorption without any noticeable reduction in recovery. The elution recovery reached 93.8% and the adsorption capacity of the polymer decreased by only 3.8% after five repeated adsorption–desorption cycles. The high recovery from the polymer shows great potential for its application to affinity precipitation.
A thermo-response polymer PNBN was synthesized, and then the ligand L-thyroxin was coupled to the polymer as an affinity ligand. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6%. An optimal adsorption condition could be identified with a suitable ligand density and pH range. The interactions between HSA and L-thyroxin were confirmed by circular dichroism. A ForteBio Octet system showed the experimental value of Kd was consistent with the theoretical value. The total elution recovery of HSA was 93.6% using 1.0 mol/L NaSCN. When the affinity polymer was applied to the purification of HSA from human serum, the purified HSA showed a single band of interest in SDS-PAGE. Thus, this new purification method for HSA could obtain a high purity of HSA in a single step.
Azobisisobutyronitrile (AIBN), N-isopropyl acrylamide (NIPA), butyl acrylate (BA) and N-methylol acrylamide (N-MAM) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Human serum was obtained from Shanghai Yaji Biological Technology Co., Ltd. (Shanghai, China). Pure HSA and L-thyroxin were purchased from Sigma (St. Louis, MO, USA). All other reagents were of reagent grade.
The synthesis procedure followed Shen’s method , developed by our group, with some modifications. A mixture containing specified amounts of the three monomers (NIPA, BA and N-MAM), plus AIBN as a polymerization initiator and ethanol as a solvent were transferred into a flask under a nitrogen atmosphere maintained for 10 min. The reaction was carried out for 24 h at 60°C in a constant temperature bath, and then the ethanol was removed by vacuum distillation. The residue was dissolved in acetone and precipitated by adding an excess of hexane. Finally, the precipitates were collected and dried.
Immobilization of ligand on PNBNpolymer
Affinity precipitation of HSA
Affinity precipitation of HSA from human serum
Recycle of affinity polymer
After HSA desorption experiments were completed, the affinity polymer was recovered and cleaned with 1.0 mol/L NaSCN. The cleaned polymer was reused in the next cycle of purification experiments.
Testing the LCST
Testing the recovery
Determination of the ligand density by high-performance liquid chromatography
CD spectra assay
ForteBio Octet system assay
SDS-PAGE analysis  was performed following the method of Laemmli with a 10.0% separating gel to check the purity of the enzyme sample obtained by affinity precipitation. The gel was stained with 0.25% Coomassie Brilliant Blue R-250.
This work was supported by Natural Scientific Foundation of China (No. 21376078) and the National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204). We would like to acknowledge Zihan Wei for his technical assistance in CD and Octet experiments, as well as in scientific discussions, and Dr Junfen Wan for critical review of the manuscript.
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