Synthetic Nanochaperones Facilitate Refolding of Denatured Proteins

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Synthetic Nano-Chaperones Facilitate Refolding of Denatured Proteins Fei-He Ma, Yingli An, Jianzu Wang, Yiqing Song, Yang Liu, and Linqi Shi ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b05947 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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Synthetic Nano-Chaperones Refolding of Denatured Proteins

Facilitate

Fei-He Ma, † Yingli An, † Jianzu Wang, † Yiqing Song, † Yang Liu, *,† Linqi Shi *,†

†Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology and Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, China

Address correspondence to Linqi Shi ([email protected]) and Yang Liu ([email protected]). ABSTRACT The folding process of a protein is inherently error-prone, owing to the large number of possible conformations that a protein chain can adopt. Partially folded or misfolded proteins typically expose hydrophobic surfaces and tend to form dysfunctional protein aggregates. Therefore, materials that can stabilize unfolded proteins and then efficiently assist them refolding to its bioactive form are of significant interest. Inspired by natural chaperonins, we have synthesized a series of polymeric nano-chaperones that can facilitate the refolding of denatured proteins with a high recovery efficiency (up to 97%). Such nano-chaperones possess phase-separated structure with hydrophobic micro-domains on the surface. This structure allows nano-chaperones stabilizing denatured proteins by binding them to the hydrophobic micro-domains. We have also investigated on the mechanism of the

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nano-chaperones in assistant of the protein refolding, and established the design principles of nano-chaperones in order to achieve effective recovery of a certain protein from their denatured forms. With a carefully designed composition of the micro-domains according to the surface properties of the client proteins, the binding affinity between the hydrophobic micro-domain and the denatured protein molecules can be tuned precisely, which enables the self-sorting of the polypeptides and the refolding of the proteins into their bioactive states. This work provides a feasible and effective strategy to recover inclusion bodies to their bioactive forms, which is potential to reduce the cost of the manufacture of recombinant proteins significantly.

KEYWORDS:

self-assembly,

nano-chaperone,

protein

refolding,

molecular

chaperone, inclusion body

In the past three decades since the first recombinant therapeutic protein of human insulin in 1982 for the treatment of diabetes,1 biopharmaceutical drugs ― including peptides, recombinant therapeutic proteins, enzymes, monoclonal antibodies and antibody-drug conjugates ― have transformed the pharmaceutical industry. To date, there are over 400 marketed recombinant products (peptides and proteins) and other 1300 are undergoing clinical trials.2 The global market for biopharmaceuticals is estimated to be worth approximately €50-60 billion3 with a growing rate of approximately 9% annually.4 As such, recombinant protein production is crucial for both the development of therapeutic proteins and the structural determination of drug targets.

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The enteric bacterium E. coli is one of the most extensively used expression system for the production of recombinant proteins that do not require post-translational modification.5-7 However, high-level expression of recombinant proteins in E. coli often results in the intracellular accumulation of partially folded proteins and subsequent formation of inclusion bodies, which are insoluble and devoid of biological activities.8,9 Although protein expression in the form of inclusion bodies is often considered undesirable, their formation can be advantageous. The major advantages associated with the formation of inclusion bodies are (i) expression of a very high level of target protein, (ii) easy isolation of the inclusion bodies from cells, (iii) lower degradation of the expressed protein, (iv) resistance to proteolytic attack by cellular proteases, and (v) homogeneity of the protein of interest in inclusion bodies (lesser contaminants) which helps in reducing the number of purification steps to recover pure protein.10 Since the purification is very costly and can account up to 80% of the total production cost in current manufacture of recombinant proteins,11 expressed as inclusion bodies is potentially a cost-effective method due to the straight forward purification of the protein of interest from the cell, as long as an effective method to recover bioactive proteins from inclusion bodies could be developed. Traditionally, it is achieved by solubilizing inclusion bodies using a high concentration of denaturants and then removing the denaturants slowly to allow the refolding of the proteins. To date, protein refolding can be achieved in many methods, from simple dilution of solubilized protein solutions to some complex strategies including matrix-assisted methods.12-15 However, most of the methods are 3


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cumbersome and offer low refolding yields at around 15-25% of the total protein, which accounts for the major cost in the production of recombinant proteins.16 Thus, more efficient methods are still demanded for the conversion of inclusion bodies into correctly folded products.

In traditional refolding methods, solubilization of inclusion body results in the loss of secondary structure of the protein, leading to the exposure of the hydrophobic surfaces.17 Driven by the hydrophobic interactions, these denatured proteins often form aggregations as the removal of the denaturants, which is considered to be the main reason for the low efficiency in the recovery of biofunctional proteins from inclusion body. In living systems, such issue in folding of nascent proteins is overcome by employing molecular chaperones,18,19 which assist protein folding primarily by preventing aggregation via recognizing specific residues exposed by non-native proteins.20,21 Furthermore, some chaperonins provide a physical environment to promote the folding of a single protein molecule.22,23 Inspired by natural chaperonins, researchers have constructed varied systems to mimic their functions. Such approaches involve a capturer and a stripper to realize the bind with the unfolded protein intermediates and the release of the refolded proteins.24-28 Although these approaches may achieve high refolding yields, they are usually complicated and only suitable for certain proteins.

Inspired by the functionality of natural chaperonins, we herein demonstrated a rational design of nano-chaperones that can assist the refolding of bioactive proteins

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from their denatured forms with high recovery efficiency. The nano-chaperones were prepared by fabricating polymeric micelles with charged and hydrophobic micro-domains on their surface (Figure 1A), allowing the capture of solubilized proteins and the subsequent refolding to their native states. Exemplified with the refolding of a positively charged protein, aggregated proteins were first solubilized using high concentration of denaturants (6 M guanidine hydrochloride (GdnCl) and dithiothreitol (DTT) in 1xphosphate buffered solution (PBS)). Nano-chaperones were then added into the solution to capture the denatured proteins through hydrophobic interactions and allowed them refolding to their native forms (Figure 1B). To achieve an efficient refolding, it is important for the surface charge of the nano-chaperones to match with that of the recovered proteins, where positively charged nano-chaperones can only promote the refolding of positively charged proteins and vice versa. This is because the electrical repulsion weakens the binding affinity between the nano-chaperone and denatured proteins, providing sufficient flexibility for the denatured proteins to refold to native state and detach from the chaperonins (Figure 1B, I). In contrast, mismatch of the surface charge further enhances the binding affinity, which is too strong for the denatured proteins to refold and detach from the nano-chaperone (Figure 1B, II).

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Figure 1. Protein refolding assisted by nano-chaperones. (A) Schematic representation of the fabrication of pos-nChap-1 by self-assembling two types of amphiphilic di-block copolymers to form micelles with poly(ε-caprolactone) (PCL)as the core and poly(ethylene glycol) (PEG) and poly(ε-caprolactone)-block-poly(N-isopropylacrylamide-co-n-tert-butylacrylamide-co-N-(3-methacrylamidopropyl) guanidinium chloride) (P(NI-co-Ntb-co-GUA)) chains as the shell, following by increasing the temperature above lower critical solution temperature (LCST) to achieve the surface structure with PEG for stabilizing nano-chaperones and hydrophobic micro-domains for binding

denatured

proteins.

(B)

Schematic

illustration

of

the

mechanism

of

nano-chaperones-assisted protein refolding. Using positively charged proteins as model, the protein aggregates was solubilized using high concentration of denaturants (6 M GdnCl and DTT in 1xPBS). Nano-chaperones were then added into the solution to capture the denatured proteins through hydrophobic interactions (I & II) and prevent them from aggregation during the dilution of the denaturants. A highly efficient refolding can be only achieved when the surface charge of the nano-chaperones matches with that of the recovered proteins, where positively charged chaperonins can only promote the refolding of positively charged proteins (I) and vice versa. In 6


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contrast, mismatch of the surface charge results in the enhancement of the binding affinity, which is too strong for the denatured proteins to refold and detach from the nano-chaperone (II).

RESULTS Synthesis and characterization of nano-chaperones. Nano-chaperones were synthesized by self-assembling two types of amphiphilic di-block copolymers to form micelles with PCL as the core and two types of polymer chains as the shell, following by increasing the temperature above LCST to form a surface structure with micro-phase separation (Figure 1A). The surface structure composes hydrophobic micro-domains for binding client proteins, as well as hydrophilic PEG chains that stabilize the nano-chaperone in aqueous media. More importantly, such PEG chains effectively prevent the adsorbed proteins from interacting with each other, providing a micro-environment for client proteins to achieve refolding. By tuning the type and ratio of the monomers during polymerization, the compositions of the block copolymers can be easily controlled, allowing the fabrication of nano-chaperones with different surface charge and hydrophobicity. To investigate the relationships between surface properties and refolding capability, four types of nano-chaperones were synthesized from co-assembly of PCL-b-PEG with four types of block copolymers (Figure S1-S7, Table S1) respectively, resulting in two positively charged nano-chaperones with different hydrophobicity (denoted as pos-nChap-1 and more hydrophilic pos-nChap-2) and two negatively charged ones (neg-nChap-1 and more hydrophilic neg-nChap-2) (Table S2).

The successful synthesis of the nano-chaperones was firstly confirmed using 7


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dynamic light scattering (DLS) and transmission electron micrograph (TEM). For instance, DLS measurements (Figure 2A) showed that pos-nChap-1 had a narrow size distribution with the hydrodynamic diameter of 82±1.1 nm at 15 oC, and decreased to 67±1.7

nm

when

heating

to

o

40

C

due

to

the

collapse

of

the

PCL-b-P(NI-co-Ntb-co-GUA) chains on the surface. The particle size of pos-nChap-1 returned to 85±1.5 nm when decreasing the temperature to 15 oC, indicating a fully reversible thermo-responsiveness. Further temperature-dependent transmittance measurements showed that the LCST of PCL-b-P(NI-co-Ntb-co-GUA) was approximate 31

o

C (Figure 2B). Moreover, TEM observation (Figure 2C) of

pos-nChap-1 showed a spherical structure with microphase separation on the surface, which confirmed the formation of hydrophobic domains on the surface of the nano-chaperones.29 Similar results were also achieved from pos-nChap-2, neg-nChap-1 and neg-nChap-2 (Figure S8). To further confirm the surface structure, static light scattering (SLS) measurements was employed to assess the mean square radius of gyration (Rg) of the nano-chaperones, which were summarized in Table S3 along with their hydrodynamic radius (Rh). Obviously, increasing the temperature above LCST leads to the decrease of Rh and increase of Rg for all nano-chaperones, indicating the collapse of the polymer chains on the surface and the formation of hydrophobic structures around the core of the nano-chaperones, which is consistent with the TEM observation.

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Figure 2. Characterization of nano-chaperones. (A) Hydrodynamic size distributions of

pos-nChap-1 measured by dynamic light scattering (DLS) at 15 oC (82±1.1 nm), 40 oC (67±1.7 nm) and cooling to 15 oC (85±1.5 nm), showing that the pos-nChap-1 has a narrow size distribution and a fully reversible thermo-responsiveness. The scattering angle was 90o. (B) Temperature-dependent

transmittance

measurements

showed

that

the

LCST

of

PCL-b-P(NI-co-Ntb-co-GUA) was approximate 31 oC. (C) TEM images of pos-nChap-1 showing uniform spheres with diameters of 65 nm. The high-resolution image (the right side of C) revealing the microphase separation structure on the surface. All the TEM samples were prepared at 40 oC.

Nano-chaperones assisted refolding of denatured lysozyme. To investigate the capability of nano-chaperones in refolding proteins, denatured lysozyme (isoelectric point (pI) = 9.32 in the native form) was employed as a model for positively-charged proteins. The refolding capability of the positively charged nano-chaperone, pos-nChap-1, was firstly studied. This is achieved by monitoring the recovered enzyme activity (refolding yield) as a function of time at 40 oC in the present of 9


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nano-chaperones. The refolding capacity of pos-nChap-1 was also investigated at the same time by fixing the concentration of pos-nChap-1 at 0.5 mg ml-1 and varying the concentration of denatured lysozyme from 0.05 mg ml-1 to 2.5 mg ml-1 (weight ratio from 10:1 to 1:5, w/w). Figure 3A summarizes the refolding yields after 4h incubation with different ratios of the nano-chaperone (green bars) in comparison with the refolding yields of denatured lysozyme without the nano-chaperone (blue bars). Clearly, refolding yields of lysozyme with pos-nChap-1 are significantly higher than that without the nano-chaperones, indicating the effective assistant of the nano-chaperone in protein refolding. Particularly for the ratio of 10:1, the refolding yield reached to 97% of denatured lysozyme. As increasing the concentration of denatured protein, the refolding yield decreased and eventually became the same as the refolding yield without the nano-chaperone, indicating their limit capacity. Since the nano-chaperone has limited surface area, this result suggests that the surface binding is essential for the nano-chaperone to assist the refolding of denatured proteins.

To investigate the surface effects on the protein refolding, four types of nano-chaperones with different surfaces were studied respectively, and the refolding profiles of lysozyme under different conditions were summarized in Figure 3B. Without any assistant, the refolding of lysozyme achieved equilibration after 2h with a low refolding yield of 34%. However, introduction of nano-chaperones altered the refolding kinetics significantly. For the positively charged ones (pos-nChap-1 and pos-nChap-2), the refolding process equilibrated at 4h and the refolding yields 10


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reached up to 97% for pos-nChap-1 and 86% for pos-nChap-2, suggesting a significant enhancement in refolding efficiency. Further characterization of the recovered lysozymes with circular dichroism spectroscopy (CD) resulted nearly the same spectra as that of native lysozyme, confirming the effective refolding (Figure 3C). Considering the surface structure of the nano-chaperones, we believe that the hydrophobic surface domains are essential for the nano-chaperones to achieve an effective protein refolding. To clarify the role of the hydrophobic domains of nano-chaperones, we examined the refolding kinetics of lysozyme assisted by nano-chaperones at a temperature (15 oC) lower than the LCST to eliminate the influences of the hydrophobic domains. As shown in Figure 3D, no significant recovery (< 5%) of active lysozyme could be observed from all the groups with nano-chaperones. Compared to the refolding yield of lysozyme without any assistants (45%), all the nano-chaperones without the hydrophobic surface domains showed strong inhibition against the protein refolding. This could be attributed to the ineffectiveness in capturing the denatured proteins, as well as the excluded volume effects that constraint on protein refolding.30 Considering the differences in the structures and the refolding yields at different temperatures (40 oC and 15 oC), one can safely conclude that the hydrophobic domains are essential for the nano-chaperones to achieve an effective refolding of denatured proteins.

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Figure 3. Nano-chaperone-assisted refolding of denatured lysozyme. (A) Histogram

summarizing the refolding yields of lysozyme after 4h incubation with (green bars) and without (blue bars) pos-nChap-1. The refolding capacity of pos-nChap-1 was investigated by fixing the concentration of pos-nChap-1 at 0.5 mg ml-1 and varying the concentration of denatured lysozyme from 0.05 mg ml-1 to 2.5 mg ml-1 (weight ratio from 10:1 to 1:5, w/w). (B) The refolding kinetics of denatured lysozyme (0.05 mg ml-1) in the presence or absence (Control) of nano-chaperones (0.5 mg ml-1) as a function of time at 40 oC. (C) Circular dichroism spectroscopy (CD) and SDS-PAGE analysis (insert) of the recovered lysozymes in the presence (insert: 1, pos-nChap-2; 2, pos-nChap-1; 3, neg-nChap-2; 4, neg-nChap-1) or absence (Control) of nano-chaperones. Insert: M, protein marker; C, control; N, native lysozyme. (D) The refolding kinetics of denatured lysozyme (0.05 mg ml-1) in the presence or absence (Control) of nano-chaperones (0.5 mg ml-1) as a function of time at 15 oC. Data represent mean ± standard error of the mean (s.e.m.) from three independent experiments.

Studies on the adsorption and desorption of the client proteins with nano-chaperones. Despite the effective recovery of denatured lysozyme achieved by 12


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positively

charged

nano-chaperones,

introduction

of

negatively

charged

nano-chaperones (neg-nChap-1 and neg-nChap-2) inhibited lysozyme refolding, resulting in no active lysozyme recovered from the denatured proteins. This result indicates that the surface charge of nano-chaperones is also crucial for the efficient refolding of the client proteins. Considering that the surface charge affects the binding affinity between nano-chaperone and client proteins significantly, further studies on the adsorption and desorption of the client proteins with nano-chaperones were achieved by monitoring their binding affinities as a function of time using a quartz crystal microbalance (QCM). As shown in Figure 4A, the resonant frequency (F) of pos-nChap-1 decreased significantly (∆F = -12 Hz) as the injection of denatured lysozyme, indicating the adsorption of denatured lysozyme by the pos-nChap-1. Interestingly, the resonant frequency was then increased gradually and saturated at about ∆F = -7 Hz, suggesting the desorption of the protein from the nano-chaperone.31,32 Such two-step change in ∆F depicts mechanism of the protein refolding mediated by the nano-chaperone. As the introduction of denatured lysozymes, pos-nChap-1 quickly captures the protein and bind them on the surface. This step effectively prevents the denatured lysozymes from aggregation, and also provide an appropriate micro-environment for the polypeptide chain to self-sort and refold into its native state. Since native lysozyme is a positively charged protein, the surface of the protein become positive and hydrophilic as its refolding, which effectively decrease its binding affinity to pos-nChap-1 due to the electrostatic repulsion. As a result, the refolded protein desorbs from the nano-chaperone and 13


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restore its original biofunctions. Similar 2-step process was also observed from pos-nChap-2 in the refolding of denatured lysozyme. In contrast, introducing denatured lysozyme to neg-nChap-1 and neg-nChap-2 respectively resulted in continuous decrease of the resonant frequency with ∆F = -94 Hz for neg-nChap-1 and

∆F = -44 Hz for neg-nChap-2 at 30-min post injection. The much lower ∆F indicates a significantly stronger binding between the negatively charged nano-chaperones and the denatured lysozymes. Moreover, no increase in ∆F was observed during the whole process, suggesting no protein desorption. The stronger adsorption between lysozyme and the negatively charged nano-chaperones is attributed to the hydrophobic interactions combined with the electrostatic attraction, which enhances the binding affinity. However, such strong binding may interfere the refolding of the polypeptide and the subsequent release from the nano-chaperones, resulting in inhibition of the lysozyme refolding (Figure 3B).

Figure 4. Studies on the adsorption and desorption of the client proteins with nano-chaperones. (A, B) Time courses of frequency change of QCM responding to the addition

of denatured (A) or native (B) lysozyme in aqueous solution with different nano-chaperones respectively. For the measurement, nano-chaperones were immobilized on the QCM electrode. Denatured lysozyme or native lysozyme (0.05 mg ml-1 in pH = 7.5, 1xPBS at 40 oC) was injected 14


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into the cells at 20 min and the frequency change was monitored as a function of time.

Such differences of nano-chaperones in the adsorption of lysozyme was also studied using DLS. As summarized in Table 1, negligible change in the Rh of pos-nChap-1 or pos-nChap-2 could be observed as the introduction of denatured lysozyme, indicating that the denatured lysozyme was bond to the hydrophobic micro-domains of the nano-chaperones and their aggregation was effectively inhibited (Figure S9). In contrast, significant increase in Rh were observed from neg-nChap-1 and neg-nChap-2, suggesting the adsorption of a large amount of denatured lysozyme on their surface. Considering the much longer range of the electrostatic attraction compared to that of hydrophobic interaction, the increase in Rh may be caused by the multiple-layer absorption of denatured lysozyme, which is unfavorable for protein refolding. Further investigation on the interaction of the nano-chaperones with native lysozymes (Figure 4B) showed the binding affinity between native lysozyme and the positively charged nano-chaperones is significantly lower (∆F = -1.5 Hz for pos-nChap-1 and 0 Hz for pos-nChap-2) than those of negatively-charged nano-chaperones (∆F = -6 Hz for neg-nChap-1 and -3.5 Hz for neg-nChap-2), suggesting easier release of lysozyme from the positively charged nano-chaperones after refolding to the native state. Comparing with the QCM results assessed from these nano-chaperones, it is clear that the electrostatic repulsion between the nano-chaperone and its client protein is essential for the successful refolding and the subsequent release. Based on this finding, we could foresee that negatively charged nano-chaperones would promote the refolding of negatively charged proteins to their 15


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bioactive state with a high efficiency.

Table 1 DLS[a] measurement results of nano-chaperones in the absence or presence of denatured lysozyme (D_lysozyme) at different time. pos-nChap-1

pos-nChap-2

neg-nChap-1

neg-nChap-2

Rh (nm)

Rh (nm)

Rh (nm)

Rh (nm)

No D_lysozyme

52.6±1.1

51.5±1.3

43.6±1.2

38.4±1.0

Add D_lysozyme 0h

53.5±1.5

52.5±1.6

63.9±2.3

51.8±2.1

Add D_lysozyme 1h

53.3±1.3

52.8±1.4

64.2±2.7

51.9±2.8

Add D_lysozyme 2h

53.4±1.7

52.6±2.1

64.9±3.1

52.4±2.6

[a]

All the measurements were performed at 40 oC using a laser lighting scattering spectrometer

(BI-200SM, 532 nm) equipped with a digital correlator (BI-9000AT). Data represent mean ± s.e.m. from three independent experiments.

Refolding of CAB with negatively charged nano-chaperones. To verify our hypothesis, a negatively charged enzyme, carbonic anhydrase B (CAB) (pI = 5.9 in the native form), was employed, and the protein refolding assisted by different nano-chaperones was studied in a similar method described in the methods section. As shown in Figure 5A, denatured CAB with negatively charged nano-chaperones showed much higher refolding yields (58% for denatured CAB with neg-nChap-1, and 52% for those with neg-nChap-2) than those with positively charge nano-chaperones (21% for pos-nChap-1 and 24% for pos-nChap-2). Comparing the refolding yields of lysozyme and CAB (Figure 5B), it is clear that nano-chaperones can only enhance the refolding of denature proteins when the nano-chaperone is electrostatically repulsive with the native form of its client protein, which is in 16


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agreement with our hypothesis.

Figure 5. Refolding of CAB with negatively charged nano-chaperones. (A) The refolding yields of CAB (0.05 mg ml-1) in the presence or absence (Control) of nano-chaperones (0.5 mg ml-1) as a function of time at 40 oC. Negatively charged nano-chaperones (neg-nChap-1 and neg-nChap-2) enhanced the refolding of CAB significantly. (B) Histogram comparing the refolding yields of lysozyme (blue bars) and CAB (green bars) assisted by different nano-chaperones after 4h incubation. It is clear that nano-chaperones can only assist the refolding when the nano-chaperone is electrostatically repulsive with the native form of its client protein. Data represent mean ± s.e.m. from three independent experiments.

DISCUSSION The folding process of protein is inherently error-prone, owing to the large number of possible conformations a protein chain can adopt.22,33 Therefore, materials that can efficiently assist denatured protein refolding to its bioactive form de novo are of significant interest. In living systems, molecular chaperones play a central role in the conformational quality control of the proteome by interacting with, stabilizing and remodeling a wide range of non-native proteins.18,19 However, different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins.5,22 For example, the bacterial chaperonin GroEL and its cofactor GroES 17


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constitute the paradigmatic molecular machine that can accelerate the folding of some substrate proteins.22,23 On the other hand, trigger factor and SecB chaperone exhibit anti-folding activity due to the high binding-affinity between the chaperones and their substrate proteins.34,35 Therefore, the follow design criteria must be meet for an artificial chaperone in order to promote protein refolding – (a) artificial chaperone can bind and stabilize denatured proteins and prevent their aggregation, and (b) the binding affinity is weak enough to allow the refolding and release of the client proteins.

Towards an effective assistance in refolding denatured proteins to their bioactive forms, we designed and synthesized a series of nano-chaperones via self-assembly of block copolymers (Figure 1A). Such nano-chaperones possess a phase-separated surface with many hydrophobic micro-domains surrounding by hydrophilic PEG chains (Figure 2C, Table S3). With this surface structure, nano-chaperones can provide micro-environments to denatured protein molecules, allowing them binding to the micro-domains via hydrophobic interactions and inhibiting the adsorbed proteins from interacting with each other. Since the binding affinity between the nano-chaperone and its client protein can be easily tuned by changing the composition of the block copolymer, denatured proteins can self-sort their polypeptide chains, refold to their native states, and release from the nano-chaperones. Such capability of the nano-chaperones in assisting the protein refolding was investigated by recovering bioactive proteins from denatured lysozyme and denatured CAB, respectively. As a result, nano-chaperones enhanced the recovery efficiency significantly, achieving high 18


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refolding yields up to 97% for lysozyme (Figure 3A) and 58% for CAB (Figure 5A) respectively.

More importantly, several surface properties, including hydrophobicity and surface charge, affect the recovery efficiency of nano-chaperones significantly. Exemplified with lysozyme, only positively charged nano-chaperones could recover bioactive proteins from denatured lysozyme with high refolding yield. In contrast, nano-chaperones with negatively charged surface inhibited the refolding of lysozyme, resulting an even lower refolding yield than that without nano-chaperones (Figure 3B). The underneath mechanism behind this observation was studied using QCM analysis, revealing that a successful nano-chaperone-assisted refolding is achieved by a two-step process – denatured protein molecules firstly adsorb on the surface of nano-chaperones via hydrophobic interactions, and then detach after refolding to its bioactive form (Figure 4A). More importantly, QCM analysis also indicated a strong binding between the negatively-charged nano-chaperone and its client protein, which inhibited the protein refolding due to the failure of the client protein to detach from the nano-chaperone (Figure 4A). Considering that the surface charge of native lysozyme is positive, the high binding affinity could be attributed to the electrostatic forces between lysozyme and the negatively-charged nano-chaperones. With these findings, we concluded that nano-chaperones must be electrostatically repulsive to the native form of their client proteins in order to achieve a high refolding yields. Further validation

with

a

negatively-charge

enzyme

CAB

confirmed

that

only

negatively-charged nano-chaperones could recover bioactive CAB from its denatured 19


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forms, whereas positively-charged nano-chaperones failed to achieve the refolding of CAB (Figure 5A). This finding offers an practical method to optimize the structure of nano-chaperones to achieve a high recovery efficiency in refolding a specific protein.

CONCLUSIONS In summary, we have demonstrated a rational design of nano-chaperones that can assist the refolding of aggregated protein to their bioactive form with a high recovery efficiency. Similar to natural chaperonins, nano-chaperones assist the protein refolding in a two-step mechanism by first adsorbing the client protein on the surface micro-domain, following by releasing the protein after the refolding to its bioactive form. By adjusting the surface charge and hydrophobicity of the nano-chaperone, the refolding yield of a given protein can be easily optimized (up to 97% for lysozyme). Considering that more efficient and universal methods for the manufacture of recombinant proteins are highly demanded, we foresee the nano-chaperone will provide a practical and convenient strategy for recovering bioactive proteins from protein aggregates and inclusion bodies, which may revolutionize current standard processes for the production of recombinant proteins. For future applications, we believe that the designed nano-chaperones can recognize and capture some certain misfolded protein and then efficiently refold it to bioactive forms, which may be very useful in the treatment of protein aggregation diseases.

MATERIALS AND METHODS For further details, please see supporting information.

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Refolding of denatured lysozyme and CAB. All the refolding experiments were performed by diluting denatured proteins into the refolding buffer in the presence or absence of nano-chaperones. Generally, denatured protein was first solubilized by dissolving in phosphate buffer (pH=7.5, 20 mM) in the present of 6 M GdnCl, 50 mM glutathione reduced form (GSH) and 15 mM glutathione oxidized form (GSSG) with a final lysozyme concentration of 1.67 mg mL-1. In the meanwhile, refolding solutions were achieved by dispersing nano-chaperones in phosphate-EDTA buffer (pH =7.5, 20mM phosphate, 5 mM EDTA, 0.5 mg/mL nano-chaperone). To perform the protein refolding, 60 µL of the denatured lysozyme solution was added into 1940 µL of the refolding solution and incubate at 40 oC for 4h. The refolding yields of the denatured protein were examined by measuring the enzyme activity.

Assay for the Enzyme Activity of refolded lysozyme and CAB. The activity of lysozyme was monitored at 25 oC by using micrococcus lysodeikticus as the substrate. Briefly, freeze-dried micrococcus lysodeikticus cells were resuspended at 0.3 mg mL-1 in 66 mM PBS (pH = 6.6). Lysozyme solution (50 µL, 0.05 mg mL-1) was added to micrococcus lysodeikticus cell suspension (450 µL), and cell lysis was monitored at 25 °C by measuring the decrease in apparent absorbance of the solution at 450 nm (OD450). The decrease in OD450 for the first 1 minute was used as the measure of lysozyme activity. Relative enzymatic activity was defined as slope for first 1 min of sample divided by slope for first 1 min of native lysozyme (50 µL, 0.05 mg mL-1).

The activity of CAB was monitored at 25 oC by using p-Nitrophenyl acetate (pNPA) as the substrate. Briefly, CAB solution (600 µL, 0.05 mg mL-1) was quickly mixed with a solution of pNPA in MeCN (20 µL, 80 mM) and the increase of absorbance at 400 nm was monitored as a

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function of time. The increase of absorbance at 400 nm for the first 1 minute was used as the measure of CAB activity. Relative enzymatic activity was defined as slope for first 1 min of sample divided by slope for first 1 min of native CAB (600 µL, 0.05 mg mL-1).

QCM measurements. QCM measurements were performed on a Q-Sence E4 system (Q-Sence, Sweden) according to the procedure reported previously.36-38 The four different nano-chaperones were immobilized on the QCM electrode via an amine coupling procedure. Typically, the gold sensor chips were cleaned twice with piranha solution (fresh mixture of H2O2 (aq) and H2SO4; 30% H2O2 (aq)/ H2SO4 = 1:3 (v/v)). Next, the sensor chip was immersed in a solution of 3,3'-Dithiodipropionic acid (1.0 mL, 1 mM in water) and then incubated for 60 min. The resulting chip was washed with pure water and then the carboxylic acid groups on the gold surface were activated by loading 2.0 mL solution of EDC (50 mg/mL) and NHS (50 mg/mL) to form N-hydroxysuccinimidyl esters. After rinsing the activated chip with water, the chip was immersed into a solution of nano-chaperones (2.0 mL, 0.5 mg mL-1, 10 mM pH = 7.5 PBS) for 24 h at 30 °C. Then the chip was rinsed with deionized water, dried with nitrogen gas and put into the standard flow module before measurements. The sensor chip surface was washed with PBS buffer (10mM, pH=7.5) for 1h at a flow rate of 30 µL/min and then equilibrated at 10 µL/min until the baseline was stable. Then, native or denatured lysozyme in the flow buffer (0.05 mg mL-1) were injected for 60 min at 10 µL/min followed by continuous flow of the same buffer. All the experiments was operated at 40 oC.

ASSOCIATED CONTENT Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. The detailed synthetic routes and characterization of block copolymers, Zeta potential, LCST, DLS, SLS and TEM images of the nano-chaperones with different formulations, assay for the enzyme activity of refolded proteins, Far-UV circular dichroism and SDS-PAGE analysis of refolded lysozyme, DLS study of the formation of aggregated lysozyme by the dilution method. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (L.S.). *E-mail: [email protected] (Y.L.). Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS Financial supported by National Natural Science Foundation of China (No. 51390483 & 91527306 & 51673100), Thousand Talents Program for Young Professionals, and The Fundamental Research Funds for the Central Universities is greatly acknowledged.

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38. Qu, A.; Huang, F.; Li, A.; Yang, H.; Zhou, H.; Long, J.; Shi, L. The Synergistic Effect Between KLVFF and Self-Assembly Chaperones on Both Disaggregation of Beta-Amyloid Fibrils and Reducing Consequent Toxicity. Chem. Commun. 2017, 53, 1289-1292.

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Synthetic Nanochaperones Facilitate Refolding of Denatured Proteins

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