Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems

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Abstract

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50–55°C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60°C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45°C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.

Introduction

Study of the protein folding problem is as old as Anfinsen’s experiments [1]. Extensive studies on the intermediate state have been carried out in recent years because of its intriguing features and practical importance in the refolding process. The protein in this state is thought to have a secondary structure like the native state, but not the close packed tertiary structure [2], [3]. Recently, a systematic approach to the protein folding process was reported in a series of works related to the evaluated values for proteins in various conformations [4], [5], [6]. The variation of the surface properties of proteins (e.g., surface net and local hydrophobicity) during the denaturation and refolding processes can be evaluated quantitatively using the aqueous two-phase partitioning method [4], [5], [6]. The local hydrophobicity of a protein was found to play an important role not only in the protein denaturation or aggregate-formation processes [4], [5], [6], but also in the interaction with liposomes [7], [8], [9], heat shock proteins (GroEL and GroES) [10], and hydrophobic ligands [11]. Based on these evaluated properties, the efficiency of the protein refolding process can be improved.

It is also possible to use molecular chaperones, which are defined as a family of unrelated cellular proteins that mediate the correct assembly of other polypeptides [12], in order to improve the efficiency of the protein refolding process. Some heat shock proteins (HSPs), which are produced by cells under various stresses, are classified in terms of molecular chaperones. DnaK, DnaJ, GroEL and GroES are typical representatives of HSPs, produced in E. coli cells under heat stress conditions. HSPs are known to play important roles in protein folding in vivo and in vitro. Recently, more attention has been paid to the use of various synthetic polymers in vitro [13], [14], which is one of the most useful approaches for the modification and stabilization of the protein structure and for enhancement of the refolding of the unfolded protein. In a previous study, the separation process was successfully achieved as a novel separation process for reactivated proteins by combining the functions of HSPs with aqueous two-phase partitioning systems (Fig. 1a) [15]. The efficiency of protein recovery in the active state can be improved by using the functions of such a natural chaperone machinery.

It has been reported that many other ligands, such as amphiphilic polymers and polyols, could also have such chaperone-like functions [15], [16], [17], [18], [19], [20]. The stimuli-responsive polymer has similar functions to those of the natural chaperone machinery, which can react and adapt itself to environmental stimuli. Recently, most research attention has been focused on the polymers that can spontaneously and reversibly change their structure and properties in response to external chemical and/or physical stimuli such as pH and temperature. These polymers, called smart polymers [21] or stimuli-responsive polymers, sense a stimulus as a signal, judge the magnitude of this signal and then alter their function in response to the corresponding stimuli. It is thought that stimuli-responsive ligands which assist and enhance protein refolding can be designed and used as an artificial chaperone instead of natural chaperones.

It is also interesting and reasonable to use a stimuli-responsive polymer (Fig. 1b) both as an artificial chaperone and as well as a modifier of the top phase of an aqueous two-phase system in order to improve the previous separation process using both GroEL functions and aqueous two-phase systems [15]. The final purpose of this study was to develop a protein refolding process using aqueous two-phase systems modified with a stimuli-responsive polymer, which has a chaperone-like function. The polymer, which has a PEG chain and a stimuli-responsive hydrophobic head (poly(propylene oxide)-phenyl group), was first synthesized for the design of an artificial chaperone. A carbonic anhydrase from bovine (CAB) was selected as a model protein. The effect of the addition of the polymer on CAB refolding was investigated at various temperatures. A possible mechanism for protein folding assisted by a stimuli-responsive polymer is elaborated. Based on these results, the possibility of developing a protein refolding process using aqueous two-phase systems modified with a stimuli-responsive polymer was investigated.

Section snippets

Materials

Carbonic anhydrase from bovine (CAB, EC 4.2.1.1, 28,800 molecular mass) was purchased from Sigma (St. Louis, MO, USA). Guanidine hydrochloride (GuHCl) used as a denaturant of CAB was purchased from Wako Pure Chemical Industries (Osaka, Japan). The phase-forming polymers in aqueous two-phase systems such as dextran 100–200k (100,000–200,000 molecular mass) and poly(ethylene glycol) (PEG) 1540, 4k, 8k (1500, 3000, 8000 molecular mass) were purchased from Wako. Triton X-405 was purchased from

Effect of the addition of functional ligands on CAB refolding

The effect of the addition of stimuli-responsive ligands (PPO-Ph-PEG) on the refolding of CAB denatured with GuHCl was investigated in order to check the possibility of the application of the unfolding, refolding, and separation processes in aqueous two-phase systems.

Conclusion

The possibility of the stress-mediated refolding of CAB was investigated using a stimuli-responsive polymer (PPO-Ph-PEG) in order to achieve protein unfolding and refolding in protein separation using aqueous two-phase systems (Fig. 1). In the presence of PPO-Ph-PEG, the refolding yield of CAB increased 1.7 times and aggregate formation was suppressed when suitable heating to increase the local hydrophobicity of both PPO-Ph-PEG and CAB was selected. Based on the model for CAB refolding, CAB

Nomenclature

    LH

    Local hydrophobicity (=Δ ln Kpr) determined from the partitioning behavior of proteins in aqueous two-phase systems [25]

    OD340

    Optical density of the solution at 340 nm as a measure of the aggregate formed in solution

    Ry

    CAB refolding yield

List of abbreviations

    ANS

    8-Anilino-1-naphthalene-sulfonate

    ATPS

    Aqueous two-phase systems

    CAB

    Carbonic anhydrase from bovine

    GroELp

    GroEL purified using a previous method [15]

    GuHCl

    Guanidine hydrochloride

    p-NPA

    p-Nitrophenylacetate

    PEG

    Poly(ethylene glycol)

    Ph-PEG

    Phenyl-poly(ethylene glycol)

    PO-Ph-PEG

    (Propylene oxide)-phenyl-poly(ethylene glycol)

    PPO

    Poly(propylene oxide)

    PPO-Ph-PEG

    Poly(propylene oxide)-phenyl-poly(ethylene glycol)

Acknowledgements

This work was partly supported by a Grant-in-Aid for Scientific Research (No. 11750693) from the Ministry of Education, Science, Sports and Culture of Japan. We acknowledge the experimental contributions of Mr. Takuya Hashimoto.

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