Extraction of Bovine Serum Albumin Using Aqueous Two-Phase Poly (Ethylene Glycol) – Poly (Acrylic Acid) System

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 The PEG-PAA aqueous two-phase polymer-polymer Aqueous two-phase systems (ATPS) are a powerful system has been developed and well characterized by method in biochemical research for the separation and our group8. The research presented here will raise the purification of macromolecules, cells, and cell potential practical application of a previously fragments. Conventional methods used for protein well-characterized system. An attempt has been made purification are usually expensive and difficult to to study the partitioning behavior of model protein scale up. For this reason, a need exists for an efficient, bovine serum albumin in order to evaluate the effective and economic large-scale bio-separation capability of the novel aqueous two-phase poly technique to achieve high purity and high recovery, (ethylene glycol) (PEG) – poly (acrylic acid) system while maintaining the biological activity of the for protein separation. protein. Hence, there is an ongoing interest in biotechnology for the development of new separation Materials and methods and purification methods that are both economically Materials viable and gentle enough to preserve biological Polyethylene glycol with molecular weights of activity of proteins. ATPS is one of the novel 4000, 6000 and 10000 (Da) was obtained from techniques to provide secured separation and Merck-Schuchardt (Munich, Germany). Bovine serum purification of bio-molecules1-3. The most commonly albumin (BSA) was purchased from Sigma (St. Louis, used and investigated ATPS are composed of either MO, USA). Poly (acrylic acid, sodium salt) of PEG–inorganic salts or PEG – dextran. It also has the average molecular weight of 100 was procured from disadvantage of low solubility for amphiphilic Aldrich chemicals company, USA. For the present proteins and high salt concentration results in waste work the polymers were used without further disposal problem7. To overcome these problems, purification. Milli-Q water was used throughout the polymer-polymer systems are being used for protein experiments. extraction. High cost of dextran leads to continuous interest for developing novel polymers4-7. Circular dichroism spectrum of BSA ______Dichroic spectroscopy measurements were Author for correspondence conducted on a JASCO J-815 CD spectrophotometer E-mail: [email protected] (JASCO Inc.) for pure and extracted BSA. For CD JONNALAGADDA et al.: EXTRACTION OF BOVINE SERUM ALBUMIN 349

measurements, sample was used in a 0.1-cm path fragile liquid-liquid interface between the two phases, length quartz cuvette and the slit was varied in an a sample of the top PEG-rich solution phase was automatic manner from 0.2 to 0.7 nm. Repetitive carefully collected. Following the collection of the scanning of 3 cycles was used. top-phase sample, the remainder of the top phase was sucked from the interfacial region using a Pasteur Preparation of aqueous two-phase systems pipette. The interfacial sample, which typically Aqueous two-phase systems were prepared from contained a mixture of the top and bottom phases, was stock solutions of poly (ethylene glycol) of molecular then discarded. Triplicate runs were carried out for weight 4000, 6000 and 10000 of about 40% (w/w) each partitioning experiment. and poly (acrylic acid) of about 30% (w/w). A known amount of 40% (w/w) PEG (Desired Molecular Analysis of protein Weight) solution was taken into a glass jacketed Total soluble proteins present in the sample were vessel of volume 50 cm3 for the experimental measured using the modified Bradford dye-binding determination of liquid-liquid equilibrium. assay. Interference from phase forming components The glass vessel was provided with an external was eliminated by taking equally diluted identical jacket in which water at constant temperature phase systems without sample as blank in the (Desired Temperature) was circulated using a spectrophotometric assay. thermostat. The temperature was controlled to within o Results and Discussion  0.05 C. The binodal curves were determined using the turbidity method3,8. The determinations of the tie Bovine serum albumin properties lines involve preparation of the feed samples BSA has a negative charge at pH 7.1. The key (about 20 cm3) by mixing appropriate amounts of values for purification processes based on ATPS— polymers and water in the vessel. The thermostat was partition coefficients and protein yields—have been determined for pure BSA solution. set at constant temperature and the sample was stirred for 1 h. Then the mixture was allowed to settle for Effect of PEG molecular weight on the protein partitioning in 4 h. After separation of the two-phases, the ATPS concentration of PEG and PAA in top and bottom The polymer molecular weight influences the phases was determined using HPLC measurements9. partitioning of protein by changing the polymer– protein interaction. In the present work, the influence Determination of protein partition coefficient in ATPS of polymer molecular weights, effect of TLL, effects Partitioning of soluble protein bovine serum of NaCl addition, pH and the variation of temperature albumin was carried out in PEG+PAA+Water system. on protein partitioning and purification have been All partition experiments were carried out at different studied. PEG concentration of 19.83-3.17% (w/w) and temperatures (20, 30 and 40oC) and pH (6.0, 7.0 and PAA concentration of 1.63-15.13% (w/w) were used 8.0). Phase systems were prepared in 50 mL for the partitioning of BSA to investigate the effect of graduated centrifuge tubes by weighing out molecular mass of PEG (4000, 6000 and 10000 Da) appropriate quantities of the PEG of desired on the partitioning at different pH (6.0, 7.0 and 8.0), molecular weight and poly (acrylic acid) stock temperature (0, 30 and 40oC) and NaCl concentration solutions. The pH of the system was maintained using (0, 0.1 and 1M) (Tables 1-2). From the tables it has phosphate buffer. A known concentration of 5 mg/mL been observed that the partitioning of proteins in PEG of soluble protein was added to the ATPS at the + PAA + water system is dependent on the molecular desired temperature of the system, and the contents weight of the PEG. The molecular weight of bovine were mixed thoroughly. Complete phase separation serum albumin is 66.4 kDa. The extent of partitioning was achieved by centrifugation at 5000 rpm for of protein is higher when the molecular mass of PEG 30 min to speed up the phase separation and left for is lower. In PEG4000+PAA+water system, the 4 h to ensure complete equilibration. After percentage yield of the extraction of bovine serum equilibration, the volumes of top and bottom phases albumin at 0oC, pH 8.0 in 1M NaCl increases from were measured. In order to determine the 63.9 to 88.7 with an increase in TLL. On comparing concentrations of proteins in each of the coexisting the percentage extraction yields at 0oC in 1 M NaCl phases, samples from each solution phase were for different molecular weights of PEG (4000, 6000 aspirated using a syringe. First, without disturbing the and 10000 Da), it has been observed that the 350 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)

%Y at 20oC %Y at 30oC %Y at 40oC TLL (%w/w) 0.1M 1M 0.1M 1M 0.1M 1M

29.2 52.8±0.2 63.9±0.1 49.1±0.1 57.2±0.1 46.3±0.2 54.4±0.2 37.9 57.7±0.2 68.7±0.1 53.9±0.2 61.5±0.1 50.1±0.2 58.7±0.1 41.6 69.0±0.1 73.2±0.2 62.4±0.2 66.8±0.2 58.9±0.1 62.7±0.1 48.3 76.8±0.2 82.9±0.2 71.4±0.1 80.5±0.2 69.4±0.1 79.0±0.2 54.7 82.7±0.3 88.7±0.1 78.4±0.2 83.8±0.1 76.3±0.1 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)

%Y at 20oC %Y at 30oC %Y at 40oC TLL (%w/w) 0.1M 1M 0.1M 1M 0.1M 1M

30.4 46.3±0.1 55.0±0.2 40.9±0.1 51.3±0.1 34.6±0.3 45.4±0.2 39.1 52.0±0.1 60.9±0.2 47.4±0.2 55.1±0.2 40.7±0.2 49.8±0.3 42.9 60.9±0.2 68.4±0.2 54.8±0.1 62.8±0.3 48.9±0.1 56.1±0.1 49.7 70.3±0.1 74.1±0.1 65.9±0.1 69.7±0.1 57.1±0.1 65.6±0.1 56.3 76.1±0.1 80.2±0.1 74.1±0.1 77.5±0.1 71.6±0.2 70.6±0.2 percentage yield decreases with an increase in the influence the partitioning of the charged protein molecular weights of PEG. Maximum percentage species. It appears likely that the specific interactions yield was observed for PEG4000 + PAA + water between the salts and proteins, in addition to the system at 0oC, pH 8.0 in 1M NaCl (Tables for pH potential difference created by the salt, are variation 6 and 7 for different conditions not shown). responsible for the effect of different salts on protein On increasing the molecular weight of PEG from partitioning. Also, different salts affect the water 4000 to 10000, the partition coefficient of bovine structure and hydrophobic interactions differently, serum albumin decreases from 6.4 to 3.30 at 54.7 % and as salt concentration increases, the partition (w/w) TLL, at pH 8.0, 0oC with 1M NaCl coefficient of a biomolecule with a large hydrophobic concentration For a lower molecular weight PEG, the region or surface in its structure will change due to its partition coefficient is high and thereby increasing the interaction with the surrounding phases. Neutral salts extraction efficiency of the system due to an excluded are frequently used in aqueous two-phase systems to volume effect.Better partitioning of protein was direct partitioning of target molecules between the achieved with lower molecular weight PEG compared phases. Sodium chloride of different concentrations to that with higher molecular weight PEG. This may (0-1 M) was used for the protein partitioning in be due to the fact that interfacial tension is lower PEG+PAA+Water system. The selection of NaCl is when molecular weight of PEG is lower. It was based on the reports that NaCl favors the protein reported that interfacial tension between the phases is transfer to the top phase (PEG rich) of the two-phase an important determining factor that influence the system10-11. Effect of NaCl addition on partition partitioning behavior of particles and cells. coefficients of BSA in PEG4000+PAA+Water Furthermore, K increases with an increase in TLL. As systems at 0oC and pH 8.0 has been investigated. The the PEG – PAA composition increases, the number of addition of NaCl is aimed at increasing the polymer units involved in the bio-molecular hydrophobic resolution of the ATPS, which enhances partitioning also increases and hence more protein selective recovery of BSA (a relatively hydrophobic molecules partition into the PEG phase due to protein) in PEG 4000. It is observed that the partition hydrophobic interaction between the protein and PEG. coefficient of bovine serum albumin increases from 2.75 to 6.4 by adding NaCl (0 to 1 M) to PEG-PAA Effect of NaCl on protein partitioning aqueous two-phase system. The percentage yield of The addition of salts to these systems appears to the extraction of BSA increases with increase in NaCl create a Donnan-type electrical potential difference concentration Tables 1-2. More bovine serum albumin between the two coexisting phases and can therefore moves to the upper phase upon adding sodium JONNALAGADDA et al.: EXTRACTION OF BOVINE SERUM ALBUMIN 351

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 Fig. 1- (a) Effect of temperature and (b) pH on protein extraction Table decreases from 88.7 to 80.5 for partitioning in PEG4000+PAA+water system with 1 M NaCl. (c) Circular dichroism spectra of 5 µM BSA and extracted bovine serum albumin in PEG4000 + PAA + water BSA from PEG phase at 30oC in 50 mM phosphate buffer o system on increasing the temperature from 0 to 40 C (pH 7.0). 352 J SCI IND RES VOL 74 JUNE 2015

at TLL of 54.7 %, (w/w), pH of 8.0 with 1M NaCl. Effect of TLL on protein partitioning The yield also decreases for PEG6000 and The effect of TLL on the partitioning of BSA has PEG10000 on increasing the temperature of the been studied (Figs. 1a-b). In this study it has been system Tables 1-2. The partition coefficient values observed that for increasing values of TLL, the are found to depend on the temperature of the partition coefficient increases. On increasing TLL, the system. It has been reported that as the temperature free volume of the bottom phase decreases and increases the PEG structure becomes more extended promotes the partition of protein from the bottom and as a result decreases its preferential interaction phase to the top phase or to the interface. The effect of with the protein thereby decreasing the partition TLL on percentage yield for five selected ATPS coefficient12. In the present case, it was observed that illustrates an increase in the extraction of BSA Tables 2 an increase in temperature leads to a decrease in at different molecular weight of PEG (4000, 6000 and o partition coefficient of the protein. Hence, the yield 10000, temperature (0, 30 and 40 C) and NaCl of extracted protein too decreases with increased concentration (0, 0.1 and 1 M). The results show that temperature. increasing TLL caused both the partition coefficient of protein and the estimated protein yield from the top Effect of pH on protein partitioning phase to increase. The effect of pH on partitioning and purification of model protein (BSA) using PEG+PAA+water system CD spectral studies of BSA for stability analysis has also been evaluated. The pH is also one of the The secondary structure of pure and extracted BSA important parameter to optimize the purification of in PEG phase has been studied using circular the soluble protein using ATPS. The effect of pH on dichroism (Fig. 1c). Secondary structural analysis has partitioning of protein in PEG4000+PAA+water been carried out in order to ascertain the system at 0oC with 1M NaCl has been investigated conformational stability of BSA after the extraction in (Fig.1b). It is observed that the partition coefficient PEG-PAA system. The BSA has been separated using increases with increase in pH of the system from 6 to 8. ultra filtration method from the PEG top phase and The pH could affect the partitioning, either by CD spectrum has been recorded. From the figure it changing the charge of the solute or by altering the has been observed that, present separation method ratio of the charged species present. At low pHs, the didn’t induce significant conformational changes in proteins have a net positive charge because the amine the secondary structure of BSA. Secondary structural gains an extra proton and at high pHs, they have a net analysis results are in good agreement with pure BSA negative charge because the carboxyl loses its proton. in terms of alpha helical content (pure BSA, 48.3 %, The intermediate pH at which protein has a net charge purified BSA 45.7 %). Specific binding of ligands to of zero is called the isoelectric point. At higher pH, BSA may induce slight perturbation in the CD the protein is more negatively charged than at low pH, spectrum of BSA in the far UV region (00–50 nm) and therefore, the partition coefficient of the protein due to conformational changes around the tryptophan increases with increasing the pH, which may be residues. No significant change was observed in the caused by the electrostatic interactions between the near UV region of the CD spectrum of extracted protein and PEG units. Negatively charged proteins protein (70–300 nm) suggesting that PEG does not partition to the top phase (PEG rich) and positively induce micro arrangements at the BSA tryptophan charged protein to the bottom phase in the ATPS9. residues accessible to solvent.The advantages of this The thermal stability of globular proteins is known to novel PEG-PAA system are due its low cost of increase at lower pH, but the rate of protein phasing forming components as well as short time aggregation increases as the pH nears the isoelectric durations for equilibrations as compared to other point. The globular protein aggregates and attains commonly used polymer-polymer systems negative charge on increasing the pH of the system. (PEG-Dextran and PEG-PVA). More over the This provides better partitioning of the proteins in the extraction efficiency (% Yield) is more for PEG-PAA top phase (PEG rich) on increasing the pH from 6 to 8. system (88.8%) as compared to other polymer- The increase in partition coefficient value could be polymer system13-14. It is one of the most effective due to hydrophobic interaction and net charge effect, methods for protein purification. The major benefit which is a function of the polymer concentration and beyond pure partitioning is the phase forming solution pH3. components could be recycled. JONNALAGADDA et al.: EXTRACTION OF BOVINE SERUM ALBUMIN 353

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