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Designing an Antibody-based Chaperoning System through Programming the Binding and Release of Folding Intermediate Tingting Liu, Caixian Sun, Cong Li, Jinhyuk Lee, Yong-Doo Park, Yixin Zhang, and Sen Li ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00191 • Publication Date (Web): 09 Mar 2016 Downloaded from http://pubs.acs.org on March 13, 2016

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ACS Chemical Biology

Designing an Antibody-based Chaperoning System through Programming the Binding and Release of Folding Intermediate

Tingting Liu,† Caixian Sun,† Cong Li,† Jinhyuk Lee,‡,# Yong-Doo Park,ơ Yixin Zhang,§,* and Sen Li†,*

†

Department of Biochemistry and Molecular Biology, College of Life Sciences, Beijing

Normal University, Gene engineering and Biotechnology Beijing Key Laboratory, The Key Laboratory of Cell Proliferation and Regulation Biology Ministry of Education, Beijing 100875, P. R. China ‡

Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and

Biotechnology, Daejeon 305-806, Korea #

Department of Nanobiotechnology and Bioinformatics, University of Sciences and

Technology, Daejeon 305-350, Korea ơZhejiang

Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute

of Tsinghua University, Jiaxing 314006, P. R. China §

B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Dresden

01307, Germany

ACS Paragon Plus Environment


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ABSTRACT: Protein folding pathway consists of sequential intra-molecular interactions, while chaperones exert their functions either by stabilizing folding intermediates or preventing non-specific intermolecular interactions, which are often associated with aggregation involving exposed hydrophobic residues in folding intermediates. As chaperones do not possess specificity to individual client proteins, we designed an antibody-based chaperoning system to mimic the sequential binding and release of client proteins undergoing folding. Single-chain variable fragment of antibody (scFv) A4 binds to human muscle creatine kinase (HCK) and prevents it from aggregation. The slow dissociation of HCK from A4 resulted in delayed but eventually high quality refolding, as reflected by the higher recovery of enzymatic activity as well as abolished aggregation. Peptide P6, a sequence in HCK involved in A4 binding, competes with HCK, promotes its dissociation from A4 and accelerates the rate of high quality refolding. The sequential addition of A4 and P6 is essential for the chaperoning effect. The programmed binding/release method can also be applied to refold HCK from inclusion bodies. Because the association/dissociation of folding intermediate with antibody is highly specific, the method can be used to design tailored refolding systems and to investigate chaperoning effects on protein folding/aggregation in a sequence specific manner.

INTRODUCTION Protein folding is a central problem in biological chemistry at both fundamental and practical levels. During the folding process, proteins often expose hydrophobic regions, while protein misfolding and aggregation occur when these regions interact and form kinetically stable species.1,2 Proteins misfolding and aggregation have been found to be in close relation to a series of neurodegenerative diseases, such as Alzheimer's disease, Parkinson’s disease


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and prion disease.3,4 Insights into the mechanisms of protein misfolding and aggregation as well as development of new techniques of favoring productive protein folding will have many applications ranging from protein engineering to biomedical research of neurodegenerative diseases. In the past, molecular chaperones have been found to assist in the de novo folding and assembly of client proteins. One of the main roles of molecular chaperones is to prevent protein misfolding and aggregation, especially under conditions of cellular stress.5,6 Cellular chaperones could play a critical role in reducing the threat of neurodegenerative diseases by preventing misfolding and aggregation of related proteins. They might represent novel therapeutic strategies and open a new door in clinical research into the neurodegenerative diseases.7-9 Many types of chaperones have been discovered, ranging from small molecules called chemical chaperones to macromolecules such as proteins and RNA.10-14 Some antibodies have been reported to exhibit chaperone-like activity, facilitating folding and preventing the aggregation of protein antigens.15-22 Notably, the influence of antibodies on folding is strictly antigen-specific. To design an efficient antibody-based chaperoning system that can assist in the folding of a particular protein could shed new light on the study of that protein’s folding pathway and also represent an interesting approach to understanding diseases associated with protein-misfolding. In a previous study, we discovered one scFv (single-chain variable fragment) antibody from screening a high-capacity phage antibody library using human muscle creatine kinase (HCK, ATP: creatine N-phosphotransferase, EC 2.7.3.2) as the target ligand.23 scFv A4 was found to show chaperone-like activity: it can inhibit aggregation and favor the recovery of the native conformation of HCK during its refolding. However, this antibody forms a stable complex with HCK and leads to an extremely slow recovery of enzymatic activity (4 days).



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Some intracellular chaperone systems have been found to contain at least two partners, one responsible for binding with protein substrates and the other responsible for the release of folded proteins or folding intermediates. For example, in the GroEL/ES chaperone system, the chaperone GroEL binds protein substrates and assists in the folding, while GroES helps substrate release from GroEL in a state that is more committed to folding.24,25 The other chaperones, such as DsbC and some peptidyl prolyl cis/trans isomerases (PPIases) with chaperone activity, also possess the dual ability to bind and release their target proteins by weakly associating with the exposed hydrophobic region.26-28 We postulate that to design an efficient antibody-based protein-specific chaperoning system needs to include not only specific binding but also effective release mechanisms. In this work, epitopes of HCK recognized by A4 were discovered using a peptide array experiment and further analyzed using biochemical and molecular simulation methods. The peptide P6 was found to show a strong ability to compete with HCK for binding to scFv A4, promoting the release of HCK from the A4-HCK complex. Based on the specific binding of HCK to A4 and the accelerated dissociation mediated by the competing peptide P6, we were able to design an efficient chaperoning system through programming the binding/release events.

RESULTS AND DISCUSSION Epitope mapping of scFv A4. To understand the unique chaperone property of scFv A4, we identified the portion of HCK bound by scFv A4 (usually called epitopes) using a novel method. This method, called the peptide array technique, was developed to screen potential linear epitopes on proteins.29 Peptide fragments covering the whole sequence of a protein antigen were synthesized and spotted on the cellulose membrane, and then the membrane was treated with specific antibodies. After washing away the unbound antibodies and labeling the bound antibodies with HRP-conjugated secondary antibodies, the peptide segments of


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potential epitopes could be identified. For these linear peptide epitopes, antibody binding depends solely on the amino acid sequence. If an antibody can bind a linear peptide epitope, it will bind the peptide segment regardless of whether the peptide is on the surface of the protein, on the cellulose membrane, or free in solution. The result of peptide array experiment is shown in Figure 1A. The set of peptides spotted from A1 to M6 sampled the entire HCK sequence, with each spot containing a 16-mer peptide starting from the HCK N-terminus, and each subsequent peptide generated by shifting one position to the C-terminus. After antibodies and chromogenic substrates treatments, six sets of continuous spots were identified. Each set of the continuous spots corresponds to a peptide segment of HCK. Spots E4–E11 correspond to Ser128-Pro143 of HCK. Spots E20–E23 correspond to Leu142-Leu157 of HCK. Spots F19-F22 correspond to Phe169-Gln184 of HCK. Spots I22–I25 correspond to Phe264-Tyr279 of HCK. Spots I26– I29 correspond to Gly268-Cys283 of HCK. Spots K7–K14 correspond to Glu310-Val325 of HCK. These six peptide segments represent potential scFv-A4 binding sites (epitopes) on HCK. We synthesized 7 peptides: P1-P7 (Table 1). Peptides P1-P6 contain the sequences corresponding to the possible HCK epitopes. Peptide P7 was synthesized and used as a control peptide that does not interact with scFv A4. Figure 1 Table 1 Study of the interaction of peptides with scFv A4. The binding kinetics of peptides P1-P7 to scFv A4 were determined by SPR (Surface Plasmon Resonance) analysis. The association and dissociation rate constants (kon and koff, respectively) were measured and the dissociation equilibrium constant (Kd) was calculated as koff/kon (Table 2). The results show that peptides P1-P6 have relatively strong binding affinity with scFv A4 when compared to the control peptide P7. Among the six peptides, peptide P6 has the strongest binding affinity



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with scFv A4. We further tested the competition of these peptides with HCK to bind to scFv A4 using a competitive ELISA. The results (Figure 2) showed that peptides P1-P6 could compete with HCK for binding to scFv A4. As expected, among these six peptides, peptide P6 exhibited the strongest ability to decrease the binding capability of scFv A4 to HCK, in good agreement with the SPR measurements. Table 2 Figure 2 Computer Simulation. The binding of scFv A4 to HCK was further analyzed using molecular simulation method. The structure of scFv A4 was modeled using the homology modeling method. The best template structure of the scFv A4 antibody was 1MOE originating from musmusculus, with a sequence identity of 0.496 (where 1.0 indicates the same sequence). We used the structure of HCK modeled from PDB file 1QH4. The HEX program found the five lowest energy complexes: (HEX energy order 1: -681.95, 2: -681.93, 3: -679.00, 4: -676.87, and 5: -675.75 kcal/mol). In all five complexes shown in Figure 1B, the P6 segment (residue numbers from 310 to 325) was found to be buried at the interface between HCK and scFv A4. For further analysis, the first lowest energy complex was chosen for MD (molecular dynamics) simulations. The final complex generated by MD simulation is shown in Figure 1C. It can be seen that the antibody forms a tight complex with HCK as in a lock-and-key model and the P6 segment region is located in the inner pocket. Building a specific and efficient chaperoning system. Although A4 abolishes the HCK aggregation, it remains a very inefficient chaperoning system because the high affinity between A4 and HCK prevents the release of HCK from complex to refold and thus retards the reactivation of this enzyme (Figure 3A, inset). We speculated that promoting the dissociation of HCK from the complex with A4 by adding a competitor could lead to a more efficient chaperoning system. Because peptides P1-P6 have shown binding affinity with scFv


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A4, they might interact with the scFv-HCK complex and promote release of HCK from the antibody as competitors. The released HCK molecules do not aggregate but form active proteins, leading to the increase of the reactivation yield. We added each peptide (P1-P7) to the scFv-HCK complex solution at a molecular ratio of 10:1 to determine whether the peptide could compete with HCK for binding to scFv A4 and accelerate the release and eventual refolding and reactivation of HCK. The reactivation curves of HCK after peptide addition are measured and shown in Figure 3A. Peptide P6 exhibited the strongest ability to release HCK from the HCK-A4 complex and to promote HCK reactivation. The refolding of HCK alone caused high level of aggregation and reached peak activity (60%) in 1.5 hours. In contract, the reactivation of HCK in the presence of A4 abolished the aggregation and reached 60% activity after 60 hours, followed by a further increase to 70 % at 96 hours (Figure 3A, inset). Adding P6 to the HCK-A4 mixture accelerated the reactivation by a factor of more than 10, shortening the time of 50% reactivation from 40 hours to 3.2 hours. As a negative control, adding P6 alone to the assay exhibited no effect on HCK refolding (Figure S1). Peptide P3 exhibited a similar effect. It is important to note that refolding under the programmed binding/release condition did not cause protein aggregation. The other peptides (P1, P4, P5 and P2) exhibited weaker effects on the reactivation of the scFv-HCK complex than peptides P6 and P3. The control sample peptide P7 showed nearly no effect. Figure 3 The aggregation of HCK is associated with high concentration of folding intermediates. Whereas refolding of diluted sample resulted in relatively high yield, high HCK concentration led to low recovery of enzymatic activity.30 When the concentration of HCK was increased to 5.6 µM, a remarkable decrease of refolding yield has been observed (Figure 3B). When HCK was premixed with A4 followed by adding competing peptide P6, the



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refolding yield after 16 hours was increased from 27% to 58%. Moreover, the aggregation of HCK was abolished by using the A4/P6 chaperoning system (figure 3C). In the previous study, we found scFv A4 could inhibit aggregation of HCK by forming stable complex with it. HCK has been found to be a homodimer consisting of two identical subunits, with each subunit containing two domains, the N-terminal domain and the Cterminal domain connected by a long linker.31, 32 It has been suggested that the C-terminal domain of CK folds independently, prior to the folding of N-terminal domain.33,34 It was also shown that the C-terminal domain of CK was more likely to be the adhesion site during the refolding process, which causes the aggregation.35 The exposed hydrophobic side chains of Phe250, Val255 and Val347 in the C-terminal domain of the refolding intermediate were found to contribute to the aggregation of CK.36 The results in our present study show that the P6 segment region (Glu310-Val325) might be the possible epitope bound by scFv A4. Because this Glu310-Val325 epitope is in the C-terminal domain of HCK, we speculated that A4 could protect CK against aggregation by providing steric hindrance to the nonspecific intermolecular interactions between the C-terminal domains of CK, especially the hydrophobic interactions between the hydrophobic side chains after binding to this domain. The intactA4/P6 chaperoning system aid refolding and reactivation of HCK by the two-step binding-release mechanism. In the binding step shown in Figure 4, the antibody scFv A4 captures the monomeric folding intermediate of HCK and prevent aggregation between them. In the release step, the addition of peptide P6 promotes the release and final reactivation of the substrate protein. The kon and koff values of A4 to HCK are 4.36×105 M-1s-1 and 1.85×10-2 s-1 respectively, and the Kd is 42.4 nM.23 The Kd between A4 and P6 is 79.5 nM. Therefore, excess amount of P6 was required to compete with HCK for binding to A4. While the halflife of HCK-A4 complex is 37.5 s (t1/2 = ln2/koff), the value is close to the time of forming 50% of protein aggregation (about 60 s for 4 µM HCK), but much shorter than the half time


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to reach 50% of reactivation (>30 min). HCK aggregation is associated with early folding intermediates, which can be suppressed upon binding to A4. Therefore, we speculate that A4HCK half-life would be the key variable to tune for developing such chaperoning systems, especially on the aspect of suppressing protein aggregation and releasing folding intermediates. Figure 4 If the chaperoning effect of A4/P6 system can be attributed to the programmed binding/dissociation of HCK folding intermediate with antibody to mimic the native chaperone systems, the sequential addition of A4 and P6 should be essential for the observed enhancement of folding quality. The effects of preforming the A4/peptide complex on HCK folding were studied. HCK was refolded in the presence of scFv A4, which was preincubated with peptides P1-P7 at the molar ratio of 1:10. The reactivation and aggregation were measured and shown in Figure 5A and 5B. Contrary to the programmed refolding experiments (Figure 3), the peptides inhibited the effect of scFv on HCK folding, as shown by the increase in the initial reactivation yield and the aggregation level of HCK during the refolding process. These results illustrate that the presence of peptides P1-P6 could interfere with the binding of scFv A4 with the HCK refolding intermediate (illustrated in Figure 4). Premixing P6 with (and inhibiting) A4, HCK exhibited fast refolding similar to the A4-free experiment (Figure 5A), though the fast refolding is also associated with aggregation (Figure 5B). It is important to note that both the loss of chaperone-like function of A4 (Figure 5) in the non-programmed process and the gain of chaperoning effect (Figure 3) in the programmed procedure can be correlated well with the affinity and competition measurements (Table 2 and Figure 2). Figure 5



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In the past, the effects of some naturally occurring chaperones on folding of CK have been investigated. Ou et al. studied the chaperone-like effect of porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (Cyp18) on folding of rabbit muscle CK and found Cyp18 could inhibit protein aggregation. They also found Cyp18 of low concentrations were able to improve the refolding yields of CK (from 70% to 88%), while high concentration of Cyp18 caused decrease of the refolding yields.37 Zhao et al. studied the effect of protein disulfide isomerase (PDI) on folding of rabbit muscle CK. At concentrations above 1 µM, PDI acted as a protector against aggregation but an inhibitor of reactivation by forming PDI-CK complexes through hydrophobic interaction and intermolecular disulfide bonds. When DTT was added to the PDI-CK complexes, CK was released and the activity increased to nearly or slightly higher than that of the self-refolding CK.38 We think the effect and mechanism of scFv A4 on folding of HCK in our present study is similar to those of PDI and high concentration of Cyp18. They could prevent aggregation by forming complex with HCK. However, the complex is very stable, thus CK could not be released and gain their native activity. The scFv A4-P6 peptide system resembles the PDI-DTT system which consists of two components. The first component (scFv A4 or PDI) was used to inhibit aggregation, the second component (peptide P6 or DTT) was used to release CK and promote recovery of enzymatic activity. The advantage of our scFv-peptide system compared with other chaperones or chaperone systems is the high substrate specificity. It provides a novel method to develop target protein-specific chaperones. Refolding of HCK inclusion bodies. In previous research, we found that the expression of recombinant HCK in E. Coli yielded many inclusion bodies when the environmental temperature is higher than 18oC.23 In order to demonstrate that the programmed folding procedure using antibody/competitor system can be applied to protein production directly, we studied the effect of the scFv A4-P6 peptide system on the refolding of HCK inclusion bodies.


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The urea-dissolved HCK inclusion bodies were diluted in refolding buffer containing scFv A4 antibody. After 1.5 hours incubation, peptide P6 was added at a molecular ratio of 10:1 and the enzymatic activity of HCK was measured over time. The reactivation yield of HCK inclusion bodies increased with use of the scFv A4-P6 peptide system (Figure 6), demonstrating that programmed binding/release conditions could aid in the refolding and reactivation of HCK inclusion bodies. It can be seen that the reactivation yield of HCK inclusion bodies is lower than that of natively purified HCK at the same concentration (Figure 3A). The result of SDS-PAGE analysis (Figure S2) shows that the impurity of HCK inclusion bodies might cause the lower reactivation yield. On the other hand, the effect of A4-P6 chaperoning system on reactivation of HCK inclusion bodies is more remarkable compared with natively purified HCK at the same concentration. With the help of this chaperoning system, the reactivation yield of HCK inclusion bodies could increase from 48% to 67% in 18 hours. Figure 6 Developments in genetic engineering have enabled eukaryotic protein production in bacteria. However, the expression of high levels of recombinant proteins in bacteria often results in the formation of insoluble inclusion bodies.39 Refolding of solubilized inclusion body proteins into bioactive forms is usually cumbersome, requires many operational steps and typically leads to very low recovery of refolded protein.40 Therefore, there is increasing interest in the development of refolding methods and systems to favor folding relative to aggregation.41,42 Our experiment provides a novel method to develop protein-specific strategies to assist in their refolding from inclusion bodies. The first step in this strategy is to screen antibodies that can bind refolding proteins and prevent aggregation. The second step is to establish a chaperone-like system based on this antibody such as the scFv A4-P6 peptide system to assist in the final refolding and reactivation of the inclusion bodies.



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Besides the applications to assist protein folding in solution, regulating enzymes and chaperones to help protein folding in cells can lead to novel strategies to facilitate protein expression in bacteria as well as new insights into diseases related to protein misfolding.43 For example, disulfide isomerase has been designed to enhance multi-disulfide substrate protein expression in E. Coli.44 Chaperone inhibitors have been intensively studied to understand their effect on the trafficking of cystic fibrosis transmembrane receptor (CFTR) and their association with the pathology of cystic fibrosis.45 Developing efficient antibodybased substrate-specific chaperoning system could provide novel molecular tools to the broad field of protein folding. In summary, we have designed an antibody-based chaperoning system to assist the folding of HCK. By orchestrating the binding of the folding intermediate to antibody and the following dissociation via a competing peptide, the programmed chaperoning effect led to a high recovery of enzymatic activity and abolished protein aggregation. This chaperoning system can be used to promote the reactivation of denaturant-resolved inclusion bodies. This study also provides new molecular details regarding how antibodies with chaperoning effects interact with their client proteins and help these proteins to fold. In the future, tailored systems for both protein engineering and folding studies can be developed by tuning the recognition sequence (epitope) and binding affinity of antibody to client protein as well as designing more potent competitors.

METHODS Materials. The gene encoding HCK was cloned into the pET21b expression vector, expressed in E. coli BL21(DE3) and purified as previously described.46 The scFv antibody A4 specific for HCK was screened from a human source phage display library and purified using affinity chromatography as previously described.23 The purified HCK and scFv A4 were


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observed to be homogenous by SDS polyacrylamide gel electrophoresis. HCK and scFv A4 concentrations were determined by the Bradford assay using BSA as a protein standard. Guanidine chloride (GdmCl, ultra-pure grade) and BSA were obtained from Sigma (St. Louis, MO). Creatine and ATP were purchased from Fluka (Milwaukee, WI) and DTT was obtained from Promega (Madison, WI). All other chemicals were local products of analytical grade. Unfolding and refolding of HCK. HCK was denatured at 25°C for 1 hour in 3 M guanidine chloride dissolved in buffer containing 10 mM Tris-HCl and 2 mM DTT (pH 8.0). In refolding studies, the denatured HCK described above was diluted 30-fold into refolding buffer (10 mMTris-HCl, 2 mM DTT, pH 8.0) containing scFv A4 and incubated at 25oC for 1.5 hours. Peptides were then added into the scFv-HCK solution and incubated at 25oC for 12-16 hours. Measurements of enzyme activity. Refolding samples of denatured HCK were assayed for enzyme activity at different time intervals after the refolding process began. Enzyme activity was determined as described by Yao et al..47 All enzyme activities were normalized against and expressed as a percentage of a 4 µM solution of native HCK in refolding buffer. Control experiments were performed to ensure that the scFv A4 and peptides used had no influence on the assay. Enzyme activity was measured at 25°C using a GBC U/V spectrophotometer. Screening of epitopes by the peptide array experiment. 16-mer peptides generated from the HCK sequence were synthesized on a cellulose membrane as individual spots. Peptide sequences started from the HCK N-terminus and each subsequent peptide shifted one position to the right. This set of peptides completely sampled the whole HCK sequence and formed an array on the membrane. The peptide array membrane was incubated with purified scFv-A4 antibodies in MP buffer (30 mM Tris-HCl, pH 7.6, 170 mM NaCl, 6.4 mM KCl,



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0.05% Tween 20, 5% sucrose) overnight at 4oC with gentle shaking. Unbound antibodies were removed with TBS, and bound antibodies were electro-transferred onto a PVDF membrane using a semi-dry blotter. The membrane was blocked with 5% non-fat dry milk for 2 h and incubated with the primary antibody (anti-V5 antibody, Invitrogen, Grand Island, NY) overnight at 4oC. After three washes with TBS buffer, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies for 1 hour and developed using the enhanced chemiluminescence method. Surface plasmon resonance (SPR) analysis. The binding kinetics of peptides to scFv A4 were measured by SPR analysis with BIAcore 3000 (Biacore, Uppsala, Sweden) according to the manufacturer's instructions. ScFv A4 was immobilized on a CM5 sensor chip using an amine coupling kit with a contact time of 15 min at a flow rate of 5 µl/min. The rate constants of association (kon) and dissociation (koff) were obtained at five different peptide concentrations ranging from 120 to 2400 nM. Measurements were carried out at a flow rate of 40 µl/min. The dissociation constant (Kd) was calculated from the ratio of the rate constants (koff/kon). Sensorgrams were analyzed with the BIAevaluation 2.1 program. Competitive ELISA. ELISA plates (NUNC, Denmark) were coated overnight at 4 °C with 2 µM of HCK in 0.1 M carbonate buffer (pH 9.6) and blocked for 2 hours at room temperature with blocking buffer (0.1 M phosphate buffer, 0.05 % Tween 20, 150 mM NaCl, 3% BSA, pH 7.2). ScFv A4 antibodies were diluted at the desired concentration in blocking buffer and incubated with 10-fold excess synthetic peptides for one hour at room temperature with gentle rocking. Next, 100 µl of the pre-incubated antibody-peptide solutions were added to the wells and incubated at room temperature for 3 hours. The wells were washed 4 times with 1×PBS/0.05% Tween 20, and 100 µl anti-V5 antibody were added to each well. After incubation for one hour at room temperature and washing as above, 100 µl horseradish peroxidase-conjugated secondary antibodies were added. The bound HRP conjugate was


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detected by adding 100 µl of HRP substrate (tetramethylbenzidine and hydrogen peroxide). The peroxidase reaction was stopped after 5 minutes by the addition of 50 µl 0.5 M H2SO4. Optical densities at 450 nm were measured using an ELISA reader. Solubilization and refolding of HCK inclusion bodies. HCK was expressed in the insoluble form in inclusion bodies by recombinant E. coli BL21(DE3) strains grown at 37oC. The cell pellets were collected by centrifugation, resuspended in 50 ml of 10 mM Tris-HCl buffer (2 mM DTT, pH 8.0) and lysed by ultrasonication in an ice bath. Cellular lysates were centrifuged at 12,000 rpm for 15 min. The pelleted inclusion bodies were washed twice with 15 ml of washing buffer (10 mM Tris-HCl, 2 mM DTT, 0.5% Triton X-100, pH 8.0). Then, the pellets were solubilized in 10 ml denaturation buffer (10 mM Tris-HCl, 3 M guanidine chloride, 2 mM DTT, pH 8.0) at 37oC for 2 h. The solubilized HCK inclusion bodies were diluted using renaturation buffer (10 mM Tris-HCl, 2 mM DTT, pH 8.0) containing scFv A4 and incubated at 25oC. After 1.5 hours incubation, peptides were added and the enzymatic activity of HCK was measured over time. Computer Simulation. As the structure of the scFv A4 protein has not been experimentally determined, the homology modeling method PQR-SA (pseudo quadratic restraints with simulated annealing) was used to generate a structure.48 The structure of HCK was modeled from PDB file 1QH4. The structures of scFv and HCK were used to perform scFv-HCK docking using the HEX program.49 With the first lowest energy complex found by the HEX program, a 10 nanosecond (ns) molecular dynamics (MD) simulation was perform to relax the complex. The scFv-HCK interaction model was constructed in the final MD structure. MD simulation and structural property analysis were run using CHARMM (chemistry at Harvard macromolecular mechanics).50 ASSOCIATED CONTENT



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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Effect of peptide P6 on refolding of HCK; SDS-PAGE analysis result of HCK inclusion bodies (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS This work was supported by the grants of National Natural Science Foundation of China (No. 31570756) and the Special Funds for Co-construction Project of Beijing Municipal Commission of Education. Y. X. Zhang is supported by the German Bundesministerium für Bildung und Forschung (BMBF) Grant No. 03Z2E512. We thank M. Thompson for critical reading of the manuscript. REFERENCES (1) Dobson, C. M. (2003) Protein folding and misfolding. Nature 426, 884–890. (2) Wittung-Stafshede, P. (2011) Protein folding inside the cell. Biophys. J. 101, 265–266. (3) Bolshette, N. B., Thakur, K. K., Bidkar, A. P., Trandafir, C., Kumar, P., and Gogoi, R. (2014) Protein folding and misfolding in the neurodegenerative disorders: a review. Rev. Neurol. 170,151–61. (4) Chiti, F., and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366.


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FIGURE LEGENDS



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Figure 1. Study of binding of scFv A4 to HCK. (A) Screening of HCK binding epitopes to scFv A4 with peptide array. The 16-mer HCK peptides were synthesized on a cellulose membrane as individual spots. The membrane was incubated with purified scFv A4 overnight. After washing, the peptide-bound antibodies were electro-transferred onto a PVDF membrane and detected by western blotting. (B) Protein-protein interaction docking model for the scFv A4-HCK complex constructed using the HEX program. The five lowest energy complexes models are shown (Grey: HCK; Green: scFv; red: P6 peptide). (C) HCK model (left) and scFv-HCK complex model (right) after a 10 ns molecular dynamics simulation (Red: HCK; Blue: scFv; Green: P6 peptide).

Figure 2. Results of competitive ELISA. ELISA plates were coated with HCK. ScFv A4 antibodies were pre-incubated with peptides P1-P7 for one hour and added to the plates. After wash, the bound scFv antibodies were detected with the anti-V5 antibody and HRPconjugated secondary antibody. Signals were developed with the TMB substrate, followed by measurement of the absorbance values at 450 nm. Samples were tested in triplicates and mean values ± SD are shown in the graph.

Figure 3. Effect of programmed antibody-based chaperoning system on refolding of HCK. (A) Effect of scFv-peptide system on reactivation of HCK. Guanidine-denatured HCK was refolded in the presence of scFv A4 for 1.5 hours. Peptides P1-P7 were then added into the scFv-HCK solution and the enzymatic activity was measured. The inset shows the reactivation curve of HCK in Tris-HCl buffer in the absence and presence of scFv A4. The concentrations of HCK, scFv A4, and peptides were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. (B) Effect of scFv A4-P6 peptide system on reactivation of HCK of higher concentration. The inset shows the reactivation curve of HCK in the absence of scFv A4. The concentrations of HCK, scFv


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A4, and peptides were 5.6 µM, 11.2 µM and 112 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. (C) Effect of scFv A4-P6 peptide system on aggregation of HCK during folding. Guanidine-denatured HCK refolded in the absence and presence of scFv A4 and absorbance at 450 nm was measured for 15 min. Peptides P6 was added 1.5 hours later and the inset show the measured absorbance at 450 nm. The concentrations of HCK, scFv A4, and peptides were 5.6 µM, 11.2 µM and 112 µM, respectively. All experiments were performed in triplicate and the representative curves were shown.

Figure 4. Design of an antibody-based chaperoning system on folding of HCK. U: unfolded HCK subunit; I: monomeric folding intermediate of HCK; E: fully active enzyme.

Figure 5. Effects of scFv-peptide complexes on refolding of HCK. (A) Effects of scFvpeptide complexes on reactivation of HCK. ScFv A4 was pre-incubated with peptides P1-P7 for one hour. Guanidine-denatured HCK was refolded in the presence of scFv-peptide complexes and enzymatic activity was measured at different time intervals. The concentrations of HCK, scFv A4, and peptides were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. (B) Effects of scFv-peptide complexes on HCK aggregation examined by turbidity measurement. Guanidine-denatured HCK was refolded in the presence of pre-incubated seven scFv-peptide complexes, and the absorbance at 450 nm was measured for 15 minutes. The concentrations of HCK, scFv A4, and peptides were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and the representative curves were shown.

Figure 6. Effect of the antibody-based chaperoning system on reactivation of HCK inclusion bodies. Guanidine-denatured HCK inclusion bodies were refolded in the presence of scFv A4



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for 1.5 hours. Peptide P6 was then added and enzymatic activity was measured at different time intervals (curve 3). Curve 1 represents HCK inclusion bodies refolded in the absence of the scFv-peptide system. Curve 2 represents HCK inclusion bodies refolded in the presence of scFv A4 without peptide. The concentrations of the HCK inclusion bodies, scFv A4, and peptide P6 were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown.


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TABLES

Table 1. The amino acid sequence of P1-P7

Peptide

The amino acid sequence

Segment of HCK

P1

SSRVRTGRSIKGYTLP

Ser128-Pro143

P2

LPPHCSRGERRAVEKL

Leu142-Leu157

P3

FKGKYYPLKSMTEKEQ

Phe169-Gln184

P4

FKKAGHPFMWNQHLGY

Phe264-Tyr279

P5

GHPFMWNQHLGYVLTC

Gly268-Cys283

P6

EILTRLRLQKRGTGGV

Glu310-Val325

P7

ETPSGFTVDDVIQTGV

Glu46-Val61



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Table 2. Association and dissociation rate constants and affinities of seven peptides with scFv A4 measured by SPR Peptide

kon (104 M−1 s−1) koff (10−3 s−1)

Kd (nM)

P1

5.92

7.24

122.3

P2

3.54

27.7

782.5

P3

5.21

11.5

182.3

P4

4.75

19.3

406.3

P5

5.43

25.3

465.9

P6

6.24

4.96

79.5

P7

Not detected

Not detected

-


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Figure 1. Study of binding of scFv A4 to HCK. (A) Screening of HCK binding epitopes to scFv A4 with peptide array. The 16-mer HCK peptides were synthesized on a cellulose membrane as individual spots. The membrane was incubated with purified scFv A4 overnight. After washing, the peptide-bound antibodies were electro-transferred onto a PVDF membrane and detected by western blotting. (B) Protein-protein interaction docking model for the scFv A4-HCK complex constructed using the HEX program. The five lowest energy complexes models are shown (Grey: HCK; Green: scFv; red: P6 peptide). (C) HCK model (left) and scFvHCK complex model (right) after a 10 ns molecular dynamics simulation (Red: HCK; Blue: scFv; Green: P6 peptide). 75x73mm (300 x 300 DPI)



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Figure 2. Results of competitive ELISA. ELISA plates were coated with HCK. ScFv A4 antibodies were preincubated with peptides P1-P7 for one hour and added to the plates. After wash, the bound scFv antibodies were detected with the anti-V5 antibody and HRP-conjugated secondary antibody. Signals were developed with the TMB substrate, followed by measurement of the absorbance values at 450 nm. Samples were tested in triplicates and mean values ± SD are shown in the graph. 59x46mm (300 x 300 DPI)


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Figure 3. Effect of programmed antibody-based chaperoning system on refolding of HCK. (A) Effect of scFvpeptide system on reactivation of HCK. Guanidine-denatured HCK was refolded in the presence of scFv A4 for 1.5 hours. Peptides P1-P7 were then added into the scFv-HCK solution and the enzymatic activity was measured. The inset shows the reactivation curve of HCK in Tris-HCl buffer in the absence and presence of scFv A4. The concentrations of HCK, scFv A4, and peptides were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. 68x60mm (300 x 300 DPI)



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Figure 3 (B) Effect of scFv A4-P6 peptide system on reactivation of HCK of higher concentration. The inset shows the reactivation curve of HCK in the absence of scFv A4. The concentrations of HCK, scFv A4, and peptides were 5.6 µM, 11.2 µM and 112 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. 67x60mm (300 x 300 DPI)


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Figure 3 (C) Effect of scFv A4-P6 peptide system on aggregation of HCK during folding. Guanidine-denatured HCK refolded in the absence and presence of scFv A4 and absorbance at 450 nm was measured for 15 min. Peptides P6 was added 1.5 hours later and the inset show the measured absorbance at 450 nm. The concentrations of HCK, scFv A4, and peptides were 5.6 µM, 11.2 µM and 112 µM, respectively. All experiments were performed in triplicate and the representative curves were shown. 75x74mm (300 x 300 DPI)



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Figure 4. Design of an antibody-based chaperoning system on folding of HCK. U: unfolded HCK subunit; I: monomeric folding intermediate of HCK; E: fully active enzyme. 79x49mm (300 x 300 DPI)


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Figure 5. Effects of scFv-peptide complexes on refolding of HCK. (A) Effects of scFv-peptide complexes on reactivation of HCK. ScFv A4 was pre-incubated with peptides P1-P7 for one hour. Guanidine-denatured HCK was refolded in the presence of scFv-peptide complexes and enzymatic activity was measured at different time intervals. The concentrations of HCK, scFv A4, and peptides were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. (B) Effects of scFv-peptide complexes on HCK aggregation examined by turbidity measurement. Guanidine-denatured HCK was refolded in the presence of pre-incubated seven scFv-peptide complexes, and the absorbance at 450 nm was measured for 15 minutes. The concentrations of HCK, scFv A4, and peptides were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and the representative curves were shown. 72x37mm (300 x 300 DPI)



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Figure 6. Effect of the antibody-based chaperoning system on reactivation of HCK inclusion bodies. Guanidine-denatured HCK inclusion bodies were refolded in the presence of scFv A4 for 1.5 hours. Peptide P6 was then added and enzymatic activity was measured at different time intervals (curve 3). Curve 1 represents HCK inclusion bodies refolded in the absence of the scFv-peptide system. Curve 2 represents HCK inclusion bodies refolded in the presence of scFv A4 without peptide. The concentrations of the HCK inclusion bodies, scFv A4, and peptide P6 were 4 µM, 8 µM and 80 µM, respectively. All experiments were performed in triplicate and mean values ± SD are shown. 117x84mm (300 x 300 DPI)


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ToC graphic 79x49mm (300 x 300 DPI)


Designing an Antibody-Based Chaperoning ... - ACS Publications

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