Bacterial Overexpression and Denaturing Purification of VPS34-Binding Domain of Beclin 1

Abstract

As a scaffolding subunit of the PIK3C3/VPS34 complex, Beclin 1 recruits a variety of proteins to class III phosphatidylinositol-3-kinase (VPS34), resulting in the formation of a distinct PIK3C3/VPS34 complex with a specific function. Therefore, the investigation of a number of Beclin 1 domains required for the protein-protein interactions will provide important clues to understand the PIK3C3/VPS34 complex, of which Beclin1-VPS34 interaction is the core unit. In the present study, we have designed a bacterial overexpression system for the Beclin 1 domain corresponding to VPS34 binding (Vps34-BD) and set up the denaturing purification protocol due to the massive aggregation of Vps34-BD in Escherichia coli. The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.

Introduction

Macroautophagy (hereafter referred to as autophagy) is a lysosome-dependent catabolic pathway to degrade cellular contents in response to a variety of cellular stresses, such as nutrient deprivation, protein aggregates, dysfunctional subcellular organelles, and infected pathogens [5,11,15]. Typically, autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which sequester the cytoplasmic contents to move into the lysosome for degradation [6]. Extensive biochemical and genetic studies in yeast and Drosophila have demonstrated a set of genes (ATG, AuTophagy-related Gene) required for autophagy, of which the PIK3C3/VPS34 complex plays important roles for autophagy initiation [12,21,22]. In response to autophagy-inducing conditions, such as nutrient starvation, the PIK3C3/VPS34 lipid kinase complex marks the initial autophagosomal membrane with phosphatidylinositol-3-phosphate (PI3P) to recruit ATG5-ATG12 conjugates and LC3-II for autophagosome biogenesis. Notably, the PIK3C3/VPS34 complex is known to exist in many different forms to contribute a variety of cellular functions, such as multivesicular body pathway, retrograde trafficking from endosomes to Golgi, phagosome maturation, as well as autophagy [1,3]. All PIK3C3/VPS34 complexes include VPS34 (a catalytic subunit)-Beclin 1 interaction, in which Beclin 1 functions as a scaffolding subunit recruiting various proteins, including Bcl-2 [16], ATG14L [19], UVRAG [10], Ambra-1 [2], Bif-1 [20], and Rubicon [24], to determine the function of the resulting PIK3C3/VPS34 complex. Beclin 1 contains an N-terminal Bcl-2-homology 3 (BH3) domain, a central coiled-coil domain (CCD), and an evolutionarily conserved domain (ECD) [18]. The N-terminal BH3 domain is involved in the interaction with Bcl-2 family proteins to inhibit autophagy. The ECD plays an important role in association with the endoplasmic reticulum and mitochondrial membranes and is essential for interaction with VPS34. The CCD is a protein-binding platform for Atg14L, UVRAG, Bif-1, and Rubicon to form the distinct PIK3/VPS34 complex. However, the molecular basis of such interactions is largely unknown. Therefore, an understanding of the biochemical features of binding domains on Beclin 1 would be helpful to get insight of the molecular mechanisms underlying PIK3C3/VPS34 complex formation and the regulation of the complex activity.

In this study, we have prepared the bacterial overexpression system to obtain the VPS34-binding domain of Beclin 1 (hereafter referred to as Vps34-BD) because VPS34-Beclin 1 interaction is a core unit of the PIK3C3/VPS34 complex. However, the bacterial Vps34-BD was found to be unstable and present as an inclusion body. We have tested denaturing purifications as well as renaturation methods to obtain the Vps34-BD from the insoluble aggregates, and finally set up the optimized purification protocol for soluble and functional Vps34-BD from E. coli.

 

Materials and Methods

Materials

The reagents for bacteria culture, protein purification, and antibodies used in this study were obtained as follows: LB agar high salt (Duchefa, Netherlands), LB broth high salt (Duchefa, Netherlands), kanamycin monosulfate (Duchefa, Netherlands), isopropyl-b-D-thiogalactoside (Fisher Scientific, USA), Ni-NTA Superflow Agarose (ThermoFisher Scientific, USA), gluthathione (GSH)-Sepharose (Elpis Biotech, Republic of Korea), Flag-M2 bead (Sigma-Aldrich, USA), imidazole (Sigma-Aldrich, USA), His-probe (Santa Cruz, USA), anti-GST antibody (Santa Cruz, USA), and HRP-conjugated anti-rabbit IgG antibody (Bethyl, USA). PCR reagents, such as thermostable DNA polymerase Pfu-Turbo, dNTP mixture, and DMSO, were purchased from Agilent Technologies (USA), Takara (Japan), and Duchefa (Netherlands), respectively. All gene cloning reagents, such as restriction enzymes (EcoRI and XhoI) and T4 DNA l igase, were obtained from NEB Biolabs. All other reagents were obtained from Sigma unless described otherwise.

Preparation of the Bacterial Vps34-BD Expression Construct

The gene corresponding to Vps34-BD on Beclin 1 (amino acid 266-337) was obtained by polymerase chain reaction (PCR) by a specific primer set (forward: 5’-GAATTCGGCGGCGGTAATGTCTTCAATGCCACC-3’; reverse: 5’-CGTGGTGGTGCTCGAGTCATTACAGATAGGAATGATTTCC-3’) containing 5’-EcoRI and 3’-XhoI cloning sites, respectively, using mouse Beclin 1 full-length cDNA as a template. The PCR product and the modified pET28 bacterial expression vector (pET28-6His/SBP), in which the genes encoding streptavidin-binding peptide (SBP) and three glycine residues (3Gly) were inserted at the 3’-region of T7-tag on the original pET28a vector (Merk Biosciences, Germany), were double-digested by EcoRI and XhoI. The digested DNAs were purified by gel extraction after resolving on 1% agarose gel, and then they were ligated at 16℃ overnight with a molar ratio of 1:5 (vector:insert). The ligated product was transformed into E. coli competent cells (DH5α) and the transformants were selected on LB agar plates containing 50 μg/ml of kanamycin. The colonies were selected and screened to isolate the desired Vps34-BD expression construct (pET28-6His/SBP-Vps34-BD) by restriction enzyme mapping and by DNA sequencing.

Expression and Purification of Recombinant Vps34-BD (6His/SBP-Vps34-BD)

The bacterial Vps34-BD expression construct, pET28-6His/SBP-Vps34-BD, was transformed into an expression host, BL21(DE3), which has relatively low protease activity, and a single colony of these cells w as g rown i n LB m edium c ontaining 50 μg/ml kanamycin (LB-Kan) at 37℃ overnight. Then, 1 ml of the overnight culture (1/200 volume of large culture) was transferred to grow in 200 ml of LB-Kan broth until the cell density reached to OD600 between 0.5 and 1.0. Thereafter, 0.5 mM of IPTG and additional 50 μg/ml of kanamycin were added to induce the protein expression at either 18℃ overnight or 37℃ for an hour as indicated. The resulting cells were harvested by centrifugation (1,500 ×g, 10 min, and 4℃) and the cell pellets were stored at -80℃ until use. Bacterial 6His/SBP-Vps34-BD protein was purified under the native condition as follows. Frozen cell pellets were thawed on ice and resuspended in 10 ml of PBST-Ni buffer (PBS containing 0.5% Triton X-100, 2 mM EDTA, 0.5 M NaCl, 2 mM β-mercaptoethanol, 20 mM imidazole, and protease inhibitor cocktail (Roche, Switzerland)) on ice for 5 min and then the cell lysate was ultrasonicated (20k Hz pulse for 5 min). The cell lysates were centrifuged for 10 min at 18,000 ×g and the supernatant was directly loaded onto the Ni-NTA agarose bead (200 μl, 50% slurry), which was pre-equilibrated with PBST-Ni, and incubated at 4℃ for 2 h on a rotating incubator. The Ni-NTA beads were harvested by centrifugation (100 ×g for 2 min) and extensively washed with PBST-Ni containing 50 mM imidazole. The beads were transferred into the column (Bio-Rad Laboratories, USA) and then protein elution was carried out in PBS buffer containing 0.1% Triton X-100 and 250 mM imidazole. Finally, it was dialyzed overnight against buffer containing 20 mM Tris, pH 8.0, and 10% glycerol at 4℃ overnight. The 6His/SBP-Vps34-BD protein was also purified under denaturing conditions. The purification process was essentially identical as described above except as follows. First, instead of supernatant, the pellets after ultrasonication in the native purification were harvested to dissolve in 5 ml of the denaturing buffer (PBS containing 8 M urea, 0.1% Triton X-100, and 0.5 M NaCl) at room temperature for 10 min on a rocker, and followed by a brief ultrasonication at 20k Hz pulse for 1 min. The following processes were performed at room temperature to prevent the precipitation of high concentration of urea and the denatured proteins, unless otherwise described. After centrifugation at 18,000 ×g for 10 min, the supernatant was directly loaded onto the Ni-NTA column, which was pre-equilibrated with the same denaturing buffer, and the column was washed with a series of denaturing-washing buffers (the denaturing buffers containing 50 mM imidazole and the decreasing concentrations of urea, 8 M, 6 M, 4 M, 2 M, and finally no urea) for on-column renaturation. The 6His/SBP-Vps34-BD protein was eluted by the PBS buffer containing 0.1% Triton X-100 and 500 mM imidazole, and followed by dialysis against buffer containing 50 mM Tris, pH 8.0, and 10% glycerol. In parallel, the 6His/SBP-Vps34-BD protein-bound beads were extensively washed with the denaturing-washing buffer containing 4 M urea, and then the proteins were eluted by the denaturing-buffer containing 3 M urea, 0.05% Triton X-100, and 500 mM imidazole. The eluate was subjected to a series of dialyses against a buffer containing 50 mM Tris, pH 8.0, 10% glycerol, and the decreasing concentrations of urea (1.5 M, 1.0 M, 0.5 M, 0.25 M, 0.125 M, and finally no urea) for renaturation at room temperature (in case of urea-containing buffer, 2 h) or 4℃ (in case of the buffer without urea, overnight). After the final dialysis, the supernatant was harvested by a short centrifugation at 18,000 ×g to remove any insoluble precipitate and the clear supernatant was collected. The protein concentration was determined by the Lowry method using bovine serum albumin (BSA) as a standard protein.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis

The samples were mixed with SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 1% β-mercaptoethanol, 2% SDS, 0.02% bromophenol blue, and 10% glycerol), and then boiled at 100℃ for 5 min. The samples were resolved on 15% SDS-PAGE, and then the separated proteins were electrophoretically transferred to a PVDF membrane by wet-transfer units (Bio-Rad). The membranes were incubated with TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20) containing 5% skim milk at room temperature for 1 h. The membrane was reacted with polyclonal rabbit anti-His probe (1:500 dilution in TBST) at 4℃ overnight, and then further incubated with HRP-conjugated secondary antibody (1:10,000 dilution in TBST containing 5% skim milk) at room temperature for an additional 1 h. Antibody detection was carried out using an ECL detection kit (ThermoFisher Scientific, USA) and visualized by exposure to film in cassette with developer.

Assay of the Bacterial 6His/SBP-Vps34-BD Binding to VPS34 Protein

To test the functionality of the 6His/SBP-Vps34-BD protein obtained from the denaturing conditions, VPS34 protein binding was examined by GST pull-down assay using GST-VPS34, which was prepared by the transfected HEK293 cells as previously described [7]. As indicated, mammalian GST-VPS34 protein and bacterially produced 6His/SBP-Vps34-BD were mixed in the final volume of 150 μl with MLB buffer containing 10 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 0.1% NP-40, 50 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail, in which GSH-Sepharose beads were added. The binding reaction was carried out at 4℃ overnight and the beads were extensively washed with MLB. In parallel, the full-length Flag-Beclin 1 protein (Flag-Beclin 1), which includes Vps34-BD between amino acids 266-337, were similarly prepared from the transfected HEK293 cells [7] and used for GST-VPS34 pull-down assay as a control. The proteins on the GSH-beads were eluted by adding SDS-PAGE sample buffer and they were resolved by 15% SDS-PAGE. The pull-down proteins were analyzed by western blots using anti-GST, anti-His, and anti-Flag antibodies for GST-VPS34, 6His/SBP-Vps34-BD, and full-length Flag-Beclin 1 detection, respectively.

 

Results

Construction of Bacterial Vps34-BD Expression System

To obtain Vps34-BD of Beclin1 in a large quantity from E. coli, we have selected the pET28 bacterial expression vector, which has a strong bacteriophage T7 promoter under the control of IPTG. Additionally, this vector carries the genes encoding six histidines (6His) and T7-tag at the 5’-region of the target gene location. Therefore, the target gene is produced as an N-terminal 6His-tagged fusion protein, which is easily purified by a single-step affinity purification using Ni-NTA beads. Additionally, we have modified this original vector to add the genes encoding SBP and three glycines between 6His and the target gene (pET28-6His/SBP vector, Fig. 1A). SBP was added to increase the molecular weight of the target gene product (Vps34-BD) because the molecular weight of Vps34-BD (71 amino acids) on Beclin 1 is too small to handle on SDS-PAGE analysis. Additionally, three glycines (3Gly) were added to provide a flexible arm, which was expected to facilitate independent folding/function of N-terminal tags (6His-, T7-, and SBP-tags) and the target protein (Vps34-BD). The gene (213 bp) encoding the domain required for VPS34 binding (Beclin 1 266-337) [18] was obtained by PCR using a full-length mouse Beclin 1 cDNA as a template and subcloned into the modified pET28-6His/SBP bacterial expression vector (Fig. 1B).

Fig. 1.Construction of the bacterial Vps34-BD overexpression system. (A) A schematic diagram of the bacterial Vps34-BD overexpression construct, pET28-6His/SBP-Vps34-BD. The gene encoding VPS34-binding domain on Beclin 1 is inserted into the modified pET28-6His/SBP vector at 5’-EcoRI and 3’-XhoI sites. Vps34-BD is expressed as an N-terminal fusion protein containing six histidines (6His), T7, and SBP (streptavidin-binding peptide). Three glycines (3xGly) are aimed to add between these tags and Vps34-BD to increase flexibility of the fusion protein. (B) Analysis of the Vps34-BD expression construct by restriction enzyme digestions. The constructed expression vectors were isolated and double-digested by the restriction enzymes used in the subcloning reaction, 5’-EcoRI and 3-XhoI. The digested products were resolved by 1% agarose gel electrophoresis and visualized on an UV transilluminator by ethidium bromide (EtBr) staining. The 1 kb and 100 bp DNA ladders were loaded to estimate the gene size.

Expression of Vps34-BD in E. coli and Purification under the Native Condition

The pET28a-6His/SBP-Vps34-BD expression construct was transformed into the E. coli expression host, BL21(DE3). Since protein induction at a low temperature is sometimes helpful to enhance the solubility of the overexpressed protein in E. coli by controlling the folding rate [8], the protein expression was induced at 18℃ overnight in the presence of 0.5 mM IPTG. The cells were harvested by centrifugation and 6His/SBP-Vps34-BD protein was purified under the native condition as described in Materials and Methods. The purified protein was resolved on 15% SDS-PAGE and visualized by Coomassie staining (Fig. 2A).

Fig. 2.Expression of the recombinant Vps34-BD (6His/SBP-Vps34-BD) in E. coli. (A) Purification of 6His/SBP-Vps34-BD under the native condition. BL21(DE3) harboring the Vps34-BD expression construct was grown and the protein expression was induced by adding 0.5 mM IPTG at 18℃ overnight. 6His/SBP-Vps34-BD was purified under the native condition as described in Materials and Methods. After elution (E), the remaining bead-bound fraction (B) was also tested to know the elution efficiency. (B) Monitoring of 6His/SBP-Vps34-BD expression in a time-dependent manner. In contrast to Fig. 2A, 6His/SBP-Vps34-BD expression was induced by adding 0.5 mM IPTG at 37℃. The aliquots of the cells were taken every hour as indicated and then dissolved in SDS-PAGE sample buffer to obtain the total cell lysates. (C) Solubility of bacterially expressed 6His/SBP-Vps34-BD. The cell pellets after 0.5 mM IPTG induction at 37℃ for an hour were resuspended in PBST-Ni buffer. The aliquot of the resuspension was mixed with the same volume of 2× SDS-PAGE buffer (designated as T). The remaining resuspension was ultrasonicated and then centrifugated at 18,000 ×g for 10 min to separate the soluble (S) and insoluble (P) fractions. All samples were resolved by 15% SDS-PAGE and the proteins were visualized by Coomassie blue staining. The total cell lysates before (B.I.) and after (A.I.) IPTG induction were prepared and dissolved in SDS-PAGE sample buffer to determine the induction efficiency of 6His/SBP-Vps34-BD in the experimental condition.

Additionally, the total cell lysates, which were prepared by dissolving the aliquots of bacterial cells before and after IPTG induction with SDS-PAGE sample buffer, were analyzed to determine the expression level of the target protein under our induction condition. Unfortunately, 6His/SBP-Vps34-BD protein was not successfully overexpressed in this experimental setting. As shown in Fig. 2A, there was no significant change between the total cell lysates obtained before and after IPTG induction. Unexpectedly, there were many nonspecific proteins on the eluate and even on the beads after elution, but there was no 6His/SBP-Vps34-BD protein as evidenced by western blotting using anti-His antibody (data not shown). It is not likely due to the nature of N-terminal tags on the 6His/SBPVps34-BD construct because the same bacterial expression construct containing Beclin 1 CCD (pET28-6His/SBP-CCD) was successfully obtained under the same experimental conditions (data not shown), suggesting that Vps34-BD is unstable or forms an inclusion body. To solve the problem, we examined the Vps34-BD expression condition. To this end, the induction temperature was shifted to 37℃ and the aliquots of the bacterial cells after induction were taken every hour. As shown in Fig. 2B, the expression of 6His/SBP-Vps34-BD already reached to the maximum at 1 h in the presence of 0.5 mM IPTG, and it was rapidly decreased to almost undetectable level at 5 h after induction. These data suggest that 6His/SBP-Vps34-BD protein is quite unstable and is quickly degraded in E. coli. Therefore, 6His/SBP-Vps34 expression should be tightly controlled for the short time period (less than an hour). Next, we tested the solubility of 6His/SBP-Vps34-BD protein because the unstable protein in E. coli tends to be aggregated to form inclusion bodies [8]. The cell pellets after 1 h of IPTG induction were resuspended in the lysis buffer containing non-denaturing detergent (0.5% Triton X-100), followed by ultrasonication to get a soluble fraction. Additionally, the resulting pellets were dissolved in SDS-PAGE sample buffer to prepare the insoluble fraction. As shown in Fig. 2C, a majority of 6His/SBP-Vps34-BD protein was observed in the pellet fraction, indicating that the target protein expressed in E. coli was present as an inclusion body. Therefore, we need to set up the purification strategy under the denaturing conditions to obtain bacterially produced 6His/SBP-Vps34-BD.

Purification of 6His/SBP-Vps34-BD under the Denaturing Condition

It is important to renature the protein obtained by the denaturing purification because the protein is only functional when in its native structure. Therefore, the renaturation method for the denatured proteins is an important issue to be considered in the denaturing purification process [8]. Several approaches have been reported to refold the denatured protein into an active protein with the native structure, such as size-exclusion chromatography [9], reversed micelle systems [17], zeolite absorbing systems [14], and the natural GroEL-GroES chaperone system [23]. However, these methods are time-consuming and require many additional steps/materials. To set up a simple renaturation protocol, we first tested an on-column renaturation method to obtain the functional 6His/SBP-Vps34-BD. The cells after IPTG induction were dissolved in the denaturing lysis buffer containing 8 M urea, followed by ultrasonication to break down the macromolecules, such as chromosomal DNA. After centrifugation, the supernatant was directly loaded on Ni-NTA beads and then the bound proteins were renatured by washing the beads with a series of denaturing-washing buffers containing decreasing concentrations of urea (stepwise decreases from 8 M urea to no urea by 2 M steps). The proteins were finally eluted by the PBS buffer containing 0.1% Triton X-100 and 500 mM imidazole. However, as shown in Fig. 3A, 6His/SBP-Vps34-BD was not observed in the eluate, and a majority of the protein still remained on the Ni-NTA beads. It was not due to the elution condition because 6His/SBP-Vps34-BD was not eluted even in the presence of 1 M imidazole (data not shown). These data indicate that the on-column renaturation method was not successful to renature the protein. Next, we tried to elute the target protein under the denaturing conditions, and then renature the protein by a series of dialyses. To find the optimum condition, we examined elution efficiency under the denaturing conditions (Fig. 3B). Although several nonspecific proteins were co-eluted with 6His/SBP-Vps34-BD, elution under the denaturing conditions successfully recovered the target protein from the beads. The protein band corresponding to the molecular weight of 6His/SBP-Vps34-BD on SDS-PAGE was confirmed by western blotting using anti-His antibody (Fig. 3B bottom panel). To eliminate nonspecific proteins in the eluate, the beads were washed in the more stringent conditions with increasing concentration of imidazole. As shown in Fig. 3C, we found that 50 mM imidazole was the minimum concentration to eliminate nonspecific proteins from the beads in the denaturing purification conditions. Finally, the eluted 6His/SBP-Vps34-BD was slowly dialyzed against a series of buffers containing decreasing concentrations of urea as described in Materials and Methods (Fig. 3D). Although some 6His/SBP-Vps34-BD was precipitated during the stepwise dialyses for renaturation, a significant amount of the protein was observed in the soluble fraction in this experimental setting. Using this denaturation/renaturation purification protocol, we could obtain 0.12 mg of 6His/SBP-Vps34-BD from 200 ml of bacteria cultures. The purification protocol, including the reagents, is summarized in Table 1 and the purification table for the yield is shown in Table 2.

Fig. 3.Purification of 6His/SBP-Vps34-BD under the denaturing conditions. Because bacterially expressed 6His/SBP-Vps34-BD protein was present in an inclusion body, the cells after IPTG induction were lysed with the denaturing buffer containing 8 M urea and the supernatant was directly loaded onto Ni-NTA beads. (A) On-column renaturation of denatured 6His/SBP-Vps34-BD. The denatured proteins bound on the beads were on-column renatured by washing the beads with a series of the denaturing buffers containing the stepwise decreasing concentration of urea. Elution (E) was carried out with the PBS buffer containing 0.1% Triton X-100 and 500 mM imidazole. After elution, the bead-bound fraction (B) was also prepared to determine the elution efficiency. (B) Elution efficiency of on-column renatured 6His/SBP-Vps34-BD. The denatured 6His/SBP-Vps34-BD was on-column renatured and eluted (designated as Rn-E) as described in (A). In parallel, the denatured proteins on the beads were washed with the denaturing-washing buffer containing 25 mM imidazole and directly eluted by the denaturing-elution buffer containing 3 M urea and 500 mM imidazole (Dn-E). The samples were resolved by 15% SDS-PAGE and the proteins were visualized by Coomassie blue staining (Top panel). Western blot analysis on the same gel running was also performed using anti-6His antibody (Bottom panel). (C) Optimization of washing condition. To eliminate the nonspecific proteins co-eluted with the denatured 6His/SBP-Vps34-BD from Ni-NTA beads, the beads were extensively washed with the different denaturing-washing buffers containing the increasing concentration of imidazole as indicated. The bound proteins were eluted by the denaturing-elution buffer (E) and the remaining bead-bound fractions (B) were prepared. (D) Renaturation of the denatured 6His/SBP-Vps34-BD by a series of dialyses. The denatured 6His/SBP-Vps34-BD was eluted by the denaturing-elution buffer (Dn-E), and then subjected to renaturation by a series of dialyses against the buffers containing 50 mM Tris, pH 8.0, 10% glycerol, and stepwise decreasing concentrations of urea as described in Table 1. After step dialyses, the eluate was centrifugated to separate the soluble supernatant (S) and the potential protein aggregates (P) during the renaturation process. B.I., the total cell lysates before IPTG induction; A.I., the total cell lysates after IPTG induction.

Table 1.The denaturing purification protocol of 6His/SBP-Vps34-BD from E. coli.

Table 2.aProtein concentrations were determined by the Lowry method using BSA as a standard protein b6His/SBP-Vps34-BD purification was performed in 200 ml LB culture. Protein expression was induced at 37℃ for an hour, and then the cell pellets were harvested by centrifugation. cCell pellets were lysed in the native conditions with buffer containing a non-denaturing detergent, Triton X-100, and soluble cell lysates were harvested by centrifugation. dThe remaining pellets after obtaining the soluble cell lysates were dissolved in the denaturing buffer containing 8 M urea and then ultrasonicated to remove the macromolecules, such as chromosomal DNA. Inclusion bodies were obtained by centrifugation. ePurity is approximately 90% as evidence by the ratio of 6His/SBP-Vps34-BD to the total proteins shown in the Coomassie-stained gel results.

Functional Assay of the Purified 6His/SBP-Vps34-BD

The successful refolding process allows the denatured protein to function. In this aspect, we have examined the functionality of the purified 6His/SBP-Vps34-BD by testing VPS34 protein binding. The bacterially produced 6His/SBP-Vps34-BD, which was obtained by the denaturing purification protocol shown in Table 1, was incubated with GST-VPS34 protein coupled with GSH-Sepharose beads. In parallel, the full-length Beclin 1 protein (Flag-Beclin1), which includes Vps34-BD between amino acids 266-337, was prepared from the transfected HEK293 cells, and then it was used for GST-Vps34 pull-down assay as a control. As shown in Fig. 4, 6His/SBP-Vps34-BD was shown to interact with GST-VPS34, comparable to full-length Flag-Beclin 1. This result indicates that 6His/SBP-Vps34-BD purified from the denaturing conditions was successfully renatured to be functional.

Fig. 4.Binding assay of bacterially produced 6His/SBP-Vps34-BD onto VPS34 protein. The renatured 6His/SBP-Vps34-BD was incubated with or without GST-VPS34, in which GSH-Sepharose beads were added. The binding reaction was carried out in the presence of 0.1% NP-40 at 4℃ overnight. In parallel, the same experiment was carried out using a full-length Flag-Beclin 1 protein (Flag-Beclin 1, which was obtained from the transfected HEK293 cells) for a positive control. The pull-down proteins by GST-VPS34-coupled GSH-beads were eluted with SDS-PAGE sample buffer and resolved by 15% SDS-PAGE. The pull-down proteins were analyzed by western blotting using anti-GST, anti-His, and anti-Flag antibodies for GST-VPS34, 6His/SBP-Vps34-BD, and full-length Flag-Beclin 1 detection, respectively.

 

Discussion

There are some efforts to obtain the full-length Beclin 1 or the fragments of Beclin 1 from E. coli [4,13]. Notably, a recent study [4] has shown the three-dimensional structure of C-terminus of Beclin 1 (248-450) that includes ECD (244-337), a target region of Beclin 1 in this study. The authors have purified the C-terminal half of Beclin 1 (248-450) from E. coli as a soluble protein in a large quantity and obtained the protein crystal satisfactory to X-crystallography at 1.6 Å resolution. Among the C-terminal half of Beclin 1, ECD (244-337) is required for VPS34 binding, in which the domain (266-337) is a minimal region indispensable for VPS34 binding. In the present study, we have tried to obtain Beclin 1 (266-337) as an essential domain for VPS34 binding, and shown the bacterial overexpression/purification method. Interestingly, in contrast to the C-terminal half of Beclin 1 (248-450), which was reported to be successfully expressed in E. coli, Beclin 1 ECD (266-337) is quite unstable and tends to form an inclusion body in E. coli. We have established the optimized expression condition and set up the denaturing purification protocol for soluble/functional protein preparation. It is not due to the expression system used for Beclin 1 ECD because expression of the Beclin 1 CCD, which contains ATG14L (or UVRAG) binding regions, was successful in E. coli under exactly the same experimental conditions used in this study (data not shown). It seems the unique nature of the Beclin 1 ECD is that it is highly hydrophobic. In line with this notion, we have observed that the Beclin-1-binding domain on VPS34 protein is also quite hydrophobic and mostly exists as an inclusion body in bacterial expression condition, suggesting the hydrophobic interactions between Beclin 1 and VPS34 (unpublished data). Therefore, it is worth establishing the expression and purification method to obtain the Beclin ECD. We expect the bacterially purified VPS34-binding domain on Beclin 1 to provide a useful tool to investigate the molecular basis of Beclin 1-VPS34 interaction.