Journal of Scientific & Industrial Research Vol 74, June 2015, pp. 348-353

Extraction of bovine serum albumin using aqueous two-phase poly (ethylene glycol) – poly (acrylic acid) system S Settu, P Velmurugan, R R Jonnalagadda* and B U Nair Chemical Laboratory, Council of Scientific and Industrial Research-Central Leather Research Institute, Adyar, Chennai - 600 00, India Received 6 August 2014; revised 2 February 2015; accepted 12 April 2015 The partitioning behavior of bovine serum albumin in a new aqueous two-phase system (ATPS) based on low cost commercial polymers like poly (ethy1ene glycol) (PEG) and poly (acrylic acid) (PAA) has been studied. In this ATPS, PAA and PEG are enriched in the bottom and upper phase, respectively. The influence of molecular weight (MW), tie line length, pH, temperatures and NaCl concentration on the partition coefficient of protein has been studied. The protein partitioning decreases on increasing MW of PEG and temperature, whereas increases with an increasing pH and NaCl concentration. This may be attributed by strong electrostatic interactions between the proteins and polymer as well as excluded volume effects. Protein partitioning is better for PEG4000 at 0oC, pH 8.0 with 1 M NaCl with a yield 88.8%. The present study indicates that PEG-PAA ATPS may be considered as an interesting alternative for protein purification from the biological suspensions. Keywords: Aqueous Two Phase; Liquid-Liquid Extraction; Protein Separation; Poly (ethylene glycol); Poly (acrylic acid); Bovine serum albumin

Introduction Aqueous two-phase systems (ATPS) are a powerful method in biochemical research for the separation and purification of macromolecules, cells, and cell fragments. Conventional methods used for protein purification are usually expensive and difficult to scale up. For this reason, a need exists for an efficient, effective and economic large-scale bio-separation technique to achieve high purity and high recovery, while maintaining the biological activity of the protein. Hence, there is an ongoing interest in biotechnology for the development of new separation and purification methods that are both economically viable and gentle enough to preserve biological activity of proteins. ATPS is one of the novel techniques to provide secured separation and purification of bio-molecules1-3. The most commonly used and investigated ATPS are composed of either PEG–inorganic salts or PEG – dextran. It also has the disadvantage of low solubility for amphiphilic proteins and high salt concentration results in waste disposal problem7. To overcome these problems, polymer-polymer systems are being used for protein extraction. High cost of dextran leads to continuous interest for developing novel polymers4-7. ___________  Author for correspondence E-mail: [email protected]

The PEG-PAA aqueous two-phase polymer-polymer system has been developed and well characterized by our group8. The research presented here will raise the potential practical application of a previously well-characterized system. An attempt has been made to study the partitioning behavior of model protein bovine serum albumin in order to evaluate the capability of the novel aqueous two-phase poly (ethylene glycol) (PEG) – poly (acrylic acid) system for protein separation. Materials and methods Materials

Polyethylene glycol with molecular weights of 4000, 6000 and 10000 (Da) was obtained from Merck-Schuchardt (Munich, Germany). Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, MO, USA). Poly (acrylic acid, sodium salt) of average molecular weight of 100 was procured from Aldrich chemicals company, USA. For the present work the polymers were used without further purification. Milli-Q water was used throughout the experiments. Circular dichroism spectrum of BSA

Dichroic spectroscopy measurements were conducted on a JASCO J-815 CD spectrophotometer (JASCO Inc.) for pure and extracted BSA. For CD

JONNALAGADDA et al.: EXTRACTION OF BOVINE SERUM ALBUMIN

measurements, sample was used in a 0.1-cm path length quartz cuvette and the slit was varied in an automatic manner from 0.2 to 0.7 nm. Repetitive scanning of 3 cycles was used. Preparation of aqueous two-phase systems

Aqueous two-phase systems were prepared from stock solutions of poly (ethylene glycol) of molecular weight 4000, 6000 and 10000 of about 40% (w/w) and poly (acrylic acid) of about 30% (w/w). A known amount of 40% (w/w) PEG (Desired Molecular Weight) solution was taken into a glass jacketed vessel of volume 50 cm3 for the experimental determination of liquid-liquid equilibrium. The glass vessel was provided with an external jacket in which water at constant temperature (Desired Temperature) was circulated using a thermostat. The temperature was controlled to within  0.05oC. The binodal curves were determined using the turbidity method3,8. The determinations of the tie lines involve preparation of the feed samples (about 20 cm3) by mixing appropriate amounts of polymers and water in the vessel. The thermostat was set at constant temperature and the sample was stirred for 1 h. Then the mixture was allowed to settle for 4 h. After separation of the two-phases, the concentration of PEG and PAA in top and bottom phases was determined using HPLC measurements9. Determination of protein partition coefficient in ATPS

Partitioning of soluble protein bovine serum albumin was carried out in PEG+PAA+Water system. All partition experiments were carried out at different temperatures (20, 30 and 40oC) and pH (6.0, 7.0 and 8.0). Phase systems were prepared in 50 mL graduated centrifuge tubes by weighing out appropriate quantities of the PEG of desired molecular weight and poly (acrylic acid) stock solutions. The pH of the system was maintained using phosphate buffer. A known concentration of 5 mg/mL of soluble protein was added to the ATPS at the desired temperature of the system, and the contents were mixed thoroughly. Complete phase separation was achieved by centrifugation at 5000 rpm for 30 min to speed up the phase separation and left for 4 h to ensure complete equilibration. After equilibration, the volumes of top and bottom phases were measured. In order to determine the concentrations of proteins in each of the coexisting phases, samples from each solution phase were aspirated using a syringe. First, without disturbing the

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fragile liquid-liquid interface between the two phases, a sample of the top PEG-rich solution phase was carefully collected. Following the collection of the top-phase sample, the remainder of the top phase was sucked from the interfacial region using a Pasteur pipette. The interfacial sample, which typically contained a mixture of the top and bottom phases, was then discarded. Triplicate runs were carried out for each partitioning experiment. Analysis of protein

Total soluble proteins present in the sample were measured using the modified Bradford dye-binding assay. Interference from phase forming components was eliminated by taking equally diluted identical phase systems without sample as blank in the spectrophotometric assay. Results and Discussion Bovine serum albumin properties

BSA has a negative charge at pH 7.1. The key values for purification processes based on ATPS— partition coefficients and protein yields—have been determined for pure BSA solution. Effect of PEG molecular weight on the protein partitioning in ATPS

The polymer molecular weight influences the partitioning of protein by changing the polymer– protein interaction. In the present work, the influence of polymer molecular weights, effect of TLL, effects of NaCl addition, pH and the variation of temperature on protein partitioning and purification have been studied. PEG concentration of 19.83-3.17% (w/w) and PAA concentration of 1.63-15.13% (w/w) were used for the partitioning of BSA to investigate the effect of molecular mass of PEG (4000, 6000 and 10000 Da) on the partitioning at different pH (6.0, 7.0 and 8.0), temperature (0, 30 and 40oC) and NaCl concentration (0, 0.1 and 1M) (Tables 1-2). From the tables it has been observed that the partitioning of proteins in PEG + PAA + water system is dependent on the molecular weight of the PEG. The molecular weight of bovine serum albumin is 66.4 kDa. The extent of partitioning of protein is higher when the molecular mass of PEG is lower. In PEG4000+PAA+water system, the percentage yield of the extraction of bovine serum albumin at 0oC, pH 8.0 in 1M NaCl increases from 63.9 to 88.7 with an increase in TLL. On comparing the percentage extraction yields at 0oC in 1 M NaCl for different molecular weights of PEG (4000, 6000 and 10000 Da), it has been observed that the

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J SCI IND RES VOL 74 JUNE 2015 Table 1-Effect of TLL, NaCl concentration and temperatures on the yield of extracted proteins (BSA) in the PEG4000+PAA+water system (pH 8.0)

TLL (%w/w) 29.2 37.9 41.6 48.3 54.7

0.1M

%Y at 20oC

52.8±0.2 57.7±0.2 69.0±0.1 76.8±0.2 82.7±0.3

%Y at 30oC

1M

0.1M

63.9±0.1 68.7±0.1 73.2±0.2 82.9±0.2 88.7±0.1

49.1±0.1 53.9±0.2 62.4±0.2 71.4±0.1 78.4±0.2

1M

0.1M

57.2±0.1 61.5±0.1 66.8±0.2 80.5±0.2 83.8±0.1

46.3±0.2 50.1±0.2 58.9±0.1 69.4±0.1 76.3±0.1

%Y at 40oC

1M

54.4±0.2 58.7±0.1 62.7±0.1 79.0±0.2 80.5±0.1

Table 2-Effect of TLL, NaCl concentration and temperatures on the yield of extracted proteins (BSA) in the PEG10000+PAA+water system (pH 8.0) TLL (%w/w) 30.4 39.1 42.9 49.7 56.3

0.1M

%Y at 20oC

46.3±0.1 52.0±0.1 60.9±0.2 70.3±0.1 76.1±0.1

1M

0.1M

55.0±0.2 60.9±0.2 68.4±0.2 74.1±0.1 80.2±0.1

40.9±0.1 47.4±0.2 54.8±0.1 65.9±0.1 74.1±0.1

percentage yield decreases with an increase in the molecular weights of PEG. Maximum percentage yield was observed for PEG4000 + PAA + water system at 0oC, pH 8.0 in 1M NaCl (Tables for pH variation 6 and 7 for different conditions not shown). On increasing the molecular weight of PEG from 4000 to 10000, the partition coefficient of bovine serum albumin decreases from 6.4 to 3.30 at 54.7 % (w/w) TLL, at pH 8.0, 0oC with 1M NaCl concentration For a lower molecular weight PEG, the partition coefficient is high and thereby increasing the extraction efficiency of the system due to an excluded volume effect.Better partitioning of protein was achieved with lower molecular weight PEG compared to that with higher molecular weight PEG. This may be due to the fact that interfacial tension is lower when molecular weight of PEG is lower. It was reported that interfacial tension between the phases is an important determining factor that influence the partitioning behavior of particles and cells. Furthermore, K increases with an increase in TLL. As the PEG – PAA composition increases, the number of polymer units involved in the bio-molecular partitioning also increases and hence more protein molecules partition into the PEG phase due to hydrophobic interaction between the protein and PEG. Effect of NaCl on protein partitioning

%Y at 30oC

The addition of salts to these systems appears to create a Donnan-type electrical potential difference between the two coexisting phases and can therefore

1M

0.1M

51.3±0.1 55.1±0.2 62.8±0.3 69.7±0.1 77.5±0.1

34.6±0.3 40.7±0.2 48.9±0.1 57.1±0.1 71.6±0.2

%Y at 40oC

1M

45.4±0.2 49.8±0.3 56.1±0.1 65.6±0.1 70.6±0.2

influence the partitioning of the charged protein species. It appears likely that the specific interactions between the salts and proteins, in addition to the potential difference created by the salt, are responsible for the effect of different salts on protein partitioning. Also, different salts affect the water structure and hydrophobic interactions differently, and as salt concentration increases, the partition coefficient of a biomolecule with a large hydrophobic region or surface in its structure will change due to its interaction with the surrounding phases. Neutral salts are frequently used in aqueous two-phase systems to direct partitioning of target molecules between the phases. Sodium chloride of different concentrations (0-1 M) was used for the protein partitioning in PEG+PAA+Water system. The selection of NaCl is based on the reports that NaCl favors the protein transfer to the top phase (PEG rich) of the two-phase system10-11. Effect of NaCl addition on partition coefficients of BSA in PEG4000+PAA+Water systems at 0oC and pH 8.0 has been investigated. The addition of NaCl is aimed at increasing the hydrophobic resolution of the ATPS, which enhances selective recovery of BSA (a relatively hydrophobic protein) in PEG 4000. It is observed that the partition coefficient of bovine serum albumin increases from 2.75 to 6.4 by adding NaCl (0 to 1 M) to PEG-PAA aqueous two-phase system. The percentage yield of the extraction of BSA increases with increase in NaCl concentration Tables 1-2. More bovine serum albumin moves to the upper phase upon adding sodium

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chloride. Generally, different types of salts have different ions with different affinity for the two phases resulting in a driving force leading to uneven partitioning of the ions between the phases. The electrochemical driving force in partitioning has been explained by the formation of an electrostatic potential difference over the interface. This potential difference is created by the different affinities of the ions for the two phases. The electrostatic potential difference will affect the partitioning of proteins or other charged molecules present in the phase system. The non-uniform distribution of chloride ions in the two-phase system leads to a potential difference between the upper and lower phases. This causes the aqueous two-phase system to attain different charges in both the phases on increasing the sodium chloride concentration. Electrical interaction and repulsion between charged aqueous phase systems leads the movement of the model protein towards the PEG phase, resulting in the partitioning of the protein between the two-phases. Effect of temperature on protein partitioning

Temperature is an important factor in protein partitioning. The effect of temperature on protein partitioning is different for different phase systems depending on the type of polymers used. The effect of temperature on partitioning of BSA using PEG+PAA+water system has also been evaluated. The temperature is one of the important parameter to optimize the purification of the soluble protein. The effect of temperature on partition coefficient of protein in PEG4000 +PAA +water system at pH 8.0 with 1M NaCl has been investigated (Fig. 1a). It is observed that the partition coefficient decreases with an increase in temperature of the system. The partition coefficient value is higher at 0oC, pH 8.0 with 1M NaCl concentrations. The partition coefficient value for bovine serum albumin decreases from 6.4 to 3.7, with an increase in the temperature from 0 to 40oC for a PEG4000 + PAA + water system. Similar trend is observed for the PEG6000 and PEG10000 systems also (Figures not shown). The effect of partitioning of BSA at a temperature below 0oC was carried out. The observed partition coefficient value is 6.40 at 15oC, indicating no significant increase in the partition coefficient as compared to the partition coefficient value of 6.4 at 0oC. The percentage yield of the extraction Table decreases from 88.7 to 80.5 for bovine serum albumin in PEG4000 + PAA + water system on increasing the temperature from 0 to 40oC

Fig. 1- (a) Effect of temperature and (b) pH on protein partitioning in PEG4000+PAA+water system with 1 M NaCl. (c) Circular dichroism spectra of 5 µM BSA and extracted BSA from PEG phase at 30 oC in 50 mM phosphate buffer (pH 7.0).

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J SCI IND RES VOL 74 JUNE 2015

at TLL of 54.7 %, (w/w), pH of 8.0 with 1M NaCl. The yield also decreases for PEG6000 and PEG10000 on increasing the temperature of the system Tables 1-2. The partition coefficient values are found to depend on the temperature of the system. It has been reported that as the temperature increases the PEG structure becomes more extended and as a result decreases its preferential interaction with the protein thereby decreasing the partition coefficient12. In the present case, it was observed that an increase in temperature leads to a decrease in partition coefficient of the protein. Hence, the yield of extracted protein too decreases with increased temperature. Effect of pH on protein partitioning

The effect of pH on partitioning and purification of model protein (BSA) using PEG+PAA+water system has also been evaluated. The pH is also one of the important parameter to optimize the purification of the soluble protein using ATPS. The effect of pH on partitioning of protein in PEG4000+PAA+water system at 0oC with 1M NaCl has been investigated (Fig.1b). It is observed that the partition coefficient increases with increase in pH of the system from 6 to 8. The pH could affect the partitioning, either by changing the charge of the solute or by altering the ratio of the charged species present. At low pHs, the proteins have a net positive charge because the amine gains an extra proton and at high pHs, they have a net negative charge because the carboxyl loses its proton. The intermediate pH at which protein has a net charge of zero is called the isoelectric point. At higher pH, the protein is more negatively charged than at low pH, and therefore, the partition coefficient of the protein increases with increasing the pH, which may be caused by the electrostatic interactions between the protein and PEG units. Negatively charged proteins partition to the top phase (PEG rich) and positively charged protein to the bottom phase in the ATPS9. The thermal stability of globular proteins is known to increase at lower pH, but the rate of protein aggregation increases as the pH nears the isoelectric point. The globular protein aggregates and attains negative charge on increasing the pH of the system. This provides better partitioning of the proteins in the top phase (PEG rich) on increasing the pH from 6 to 8. The increase in partition coefficient value could be due to hydrophobic interaction and net charge effect, which is a function of the polymer concentration and solution pH3.

Effect of TLL on protein partitioning

The effect of TLL on the partitioning of BSA has been studied (Figs. 1a-b). In this study it has been observed that for increasing values of TLL, the partition coefficient increases. On increasing TLL, the free volume of the bottom phase decreases and promotes the partition of protein from the bottom phase to the top phase or to the interface. The effect of TLL on percentage yield for five selected ATPS illustrates an increase in the extraction of BSA Tables 2 at different molecular weight of PEG (4000, 6000 and 10000, temperature (0, 30 and 40oC) and NaCl concentration (0, 0.1 and 1 M). The results show that increasing TLL caused both the partition coefficient of protein and the estimated protein yield from the top phase to increase. CD spectral studies of BSA for stability analysis

The secondary structure of pure and extracted BSA in PEG phase has been studied using circular dichroism (Fig. 1c). Secondary structural analysis has been carried out in order to ascertain the conformational stability of BSA after the extraction in PEG-PAA system. The BSA has been separated using ultra filtration method from the PEG top phase and CD spectrum has been recorded. From the figure it has been observed that, present separation method didn’t induce significant conformational changes in the secondary structure of BSA. Secondary structural analysis results are in good agreement with pure BSA in terms of alpha helical content (pure BSA, 48.3 %, purified BSA 45.7 %). Specific binding of ligands to BSA may induce slight perturbation in the CD spectrum of BSA in the far UV region (00–50 nm) due to conformational changes around the tryptophan residues. No significant change was observed in the near UV region of the CD spectrum of extracted protein (70–300 nm) suggesting that PEG does not induce micro arrangements at the BSA tryptophan residues accessible to solvent.The advantages of this novel PEG-PAA system are due its low cost of phasing forming components as well as short time durations for equilibrations as compared to other commonly used polymer-polymer systems (PEG-Dextran and PEG-PVA). More over the extraction efficiency (% Yield) is more for PEG-PAA system (88.8%) as compared to other polymerpolymer system13-14. It is one of the most effective methods for protein purification. The major benefit beyond pure partitioning is the phase forming components could be recycled.

JONNALAGADDA et al.: EXTRACTION OF BOVINE SERUM ALBUMIN

Conclusion PEG-PAA aqueous two-phase system has been used for the partitioning of bovine serum albumin. Partitioning of the protein has been influenced by molecular weight of PEG, concentration of NaCl, temperature and pH. The study shows better partitioning of the protein for PEG4000, 1M NaCl, 0°C, and pH 8.0 with the maximum extraction yield of 88.70%. Hence, PEG-PAA aqueous two-phase system forms a suitable practical application for the extraction of globular proteins from the biological suspensions. Acknowledgement One of the authors (SS) acknowledges Council of Scientific and Industrial Research, New Delhi for Research Associateship.

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