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. 2020 Jan 21;25(2):287–301. doi: 10.1007/s12192-020-01073-5

Cell-free synthesis of functionally active HSPB5

Ryoji Kojima 1,✉, Keiichi Uchiya 2, Hiroyuki Manshio 1, Kastuyoshi Masuda 3
PMCID: PMC7058722  PMID: 31960264

Abstract

Human αB-crystallin (HSPB5) is frequently modified post-translationally by UV radiation, oxidation, and age-associated processes, which complicates functional analyses of the protein using natural sources. Thus, determining the biological function of HSPB5 at the molecular structure level requires unmodified protein. Here, we employed an Escherichia coli cell-free protein synthesis system to prepare unmodified, functionally active human HSPB5. An S30 extract prepared from E. coli strain BL21 (DE3) was used for HSPB5 synthesis. The efficacy of protein synthesis was assessed by monitoring influencing factors, such as the concentrations of Mg2+ and other reaction mixture constituents, and by evaluating batch and/or dialysis synthesis systems. Chaperone-like activity of synthesized HSPB5 was assayed using alcohol dehydrogenase (ADH) under thermal stress. The amount of HSPB5 synthesized using the cell-free system depended significantly on the concentration of Mg2+ in the reaction mixture. Use of condensed S30 extract and increased levels of amino acids promoted HSPB5 production. Compared with the batch system, HSPB5 synthesis was markedly increased using the dialysis system. The construction vector played a critical role in regulating the efficacy of protein synthesis. HSPB5 synthesized using the cell-free system had a native molecular mass, as determined by mass spectrometry analysis. The co-presence of synthesized HSPB5 suppressed heat-associated denaturation of ADH. Human HSPB5 synthesized using the cell-free system thus retains functional activity as a molecular chaperone.

Keywords: Human αB-crystallin (HSPB5), Cell-free protein synthesis, Chaperoning activity, E. coli S30 extract

Introduction

Human αB-crystallin (HSPB5) is a major structural protein of the vertebrate eye lens (Horwitz 2003). The structure, stability, and short-range order of crystallin are thought to contribute to the transparency of the vertebrate ocular lens (Bhat 2004). In addition to its significance in vision, human HSPB5 also reportedly plays roles in several neurologic diseases. For example, abnormal accumulation of HSPB5 has been reported in Alexander disease (Iwaki et al. 1989; Head et al. 1993), Creutzfeldt-Jakob disease (Kato et al. 1992), multiple sclerosis (van Noort et al. 1995; Stoevring et al. 2005), and Lewy body disease (Jackson et al. 1995). Thus, detailed analyses of the function of human HSPB5 are needed to more fully elucidate its pathophysiologic significance as well as its normal physiological and biological roles.

Several studies have identified HSPB5 as a small heat shock protein (sHsp). sHsps, including HSPB5, comprise a specific class of highly conserved protein families known as molecular chaperones, which transiently interact with unfolded or partially folded proteins to mediate their proper folding or contribute to the refolding or degradation of misfolded proteins (Mogk and Bukau 2017). Although the refolding and degradation of misfolded proteins by molecular chaperones such as Hsp70 occur in an ATP-dependent manner, sHsps reportedly exhibit ATP-independent activity (Haslbeck et al. 2005), indicating a unique property of sHsps. Fully elucidating the mechanism of HSPB5 chaperone activity will require identifying the conformational changes that occur during substrate recognition and binding. Previous molecular function studies have used HSPB5 isolated from natural sources such as mammalian lens (Srivastava et al. 1994), muscle (Atomi et al. 1991), and heart (Longoni et al. 1990). However, isolating sufficient quantities of unmodified HSPB5 from human lenses is challenging because the protein is often post-translationally modified by UV radiation, oxidation, and normal aging processes (Thornell and Aquilina 2015). These naturally occurring modifications make functional analyses of HSPB5 difficult. However, recently developed in vivo recombinant techniques using prokaryotes such as Escherichia coli enable the large-scale synthesis of unmodified proteins. Indeed, several studies using recombinant mutants of HSPB5 have demonstrated that the N-terminal domain (Numoto et al. 2012; Jovcevski et al. 2018), α-crystallin domain (Clark et al. 2011), and C-terminal extension (Treweek et al. 2007) play important functional roles in the chaperone and oligomerization activity.

Cell-free protein synthesis systems reportedly provide simple, rapid, robust, and convenient tools for producing proteins and that they are also suitable for synthesizing proteins prone to aggregation or those exhibiting toxic properties (Spirin 2004). In cell-free protein synthesis, an efficacy of protein synthesis is dependent on experimental conditions of regulatory factors such as Mg2+ in a reaction mixture. In fact, concentration of Mg2+ in the reaction mixture is a critical factor to allow the protein synthesis reaction to initiate and maintain (Pratt 1984; Kigawa et al. 1999). Protein extracts prepared from prokaryotic and eukaryotic cells, including ribosome proteins and tRNAs necessary for the protein synthesis are also essential components to define a protein yield (Kigawa et al. 1999; Spirin 2004). Moreover, inorganic phosphate produced during the protein synthesis reaction in the reaction mixture is an adverse factor to inhibit the efficacy of protein synthesis (Kim and Swartz 2000). A technical development in cell-free protein synthesis systems, which reduces the adverse byproduct and increases the protein yield, involved a dialysis system (Spirin et al. 1988; Spirin 2004). The dialysis system can remove inorganic phosphate from the reaction mixture and intake amino acids into the reaction mixture through a dialysis membrane (Spirin et al. 1988; Davis et al. 1996; Kigawa et al. 1999).

Recent improvements in cell-free protein synthesis systems make them especially useful for uniformly and selectively labeling proteins with stable-isotope amino acids (Kigawa et al. 1999; Terada and Yokoyama 2015) for functional studies. These improvements prevent isotopic dilution and metabolic conversion of stable isotope–labeled amino acids that can occur in cell-based protein expression systems such as those using E. coli as a host (Arata et al. 1994). The use of cell-free protein synthesis systems coupled with stable isotope labeling techniques and NMR spectroscopy (Arata et al. 1994; Kigawa et al. 1999; Terada and Yokoyama 2015) and X-ray crystallography (Kigawa et al. 2002) analysis enables the determination of molecular structures and functions of proteins at the amino acid level.

In this study, we employed a cell-free protein synthesis system using an S30 extract to produce human HSPB5 for subsequent analysis of its molecular mass and chaperone activity. We report, for the first time, the preparation of human HSPB5 exhibiting native molecular mass and chaperone activity using a cell-free protein synthesis system. The ability to synthesize HSPB5 using the cell-free protein synthesis system described here thus facilitates further atomic-level analyses of the molecular mechanism of HSPB5 chaperone activity as well as its structures of functional domains.

Materials and methods

Cloning of HSPB5 cDNA

HepG2 human hepatocyte cells were cultured to confluence in plastic dishes (10-cm diameter) in Dulbecco’s Modified Eagle’s Medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, GE Healthcare Lifescience, Logan, UT) and 2% penicillin/streptomycin (Life Technologies). After the cells reached confluence, total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. For synthesis of HSPB5 cDNA, 5 μg of total RNA was reverse transcribed in a 20-μl reaction using oligo (dT) primer and SuperScript II RNase H−Reverse Transcriptase (Life Technologies) in accordance with the manufacturers’ instructions. Polymerase chain reaction (PCR) was then carried out in a 50-μl reaction containing 2 μl of the cDNA reaction mixture, 1× high-fidelity PCR buffer, 2 mM MgSO4, 200 μM dNTP, 0.2 μM each primer, and 1 U of Platinum Taq high-fidelity polymerase (Life Technologies). The thermocycle protocol was as follows: 94 °C for 2 min, 25 cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 1 min, followed by one cycle of 68 °C for 10 min. The following primers were used for PCR: sense, 5’-GACCCCTCACACTCACCTAG-3’; antisense, 5’-CAGCACTAGTCACAAGACTT-3’. The PCR product was subjected to TA cloning (Life Technologies), and human HSPB5 cDNA was identified by sequencing the PCR product using an ABI 3700 automated DNA sequencer.

Construction of HSPB5 expression vector for cell-free protein synthesis

PCR was performed using the isolated HSPB5 cDNA as a template and the following primers with NdeI restriction endonuclease sites (sense, 5’-CATATGGACATCGCCATCCACCA-3’; antisense, 5’-CATATGGTGGGGAAACTTTCTTGTT-3’). The PCR product was subjected to TA cloning (Life Technologies) and NdeI digestion, and then the NdeI-digested PCR fragment was ligated into the NdeI-digested pIVEX2.4b Nde vector (Roche Molecular Biochemicals, Mannheim, Germany), which encodes αB-crystallin as a histidine-tagged protein (pIVEX2.4bNde-HSPB5). The pET14b vector (Merck Biosciences, Darmstadt, Germany) was also used to prepare template DNA for cell-free protein synthesis, and the PCR fragment was also inserted downstream of the histidine tag in the pET14b vector at the NdeI site (pET14b-HSPB5). In this study, among pET-based vectors, pET14b vector was chosen for cell-free protein synthesis using S30 extract derived from E. coli B strain BL21 (DE3) expressing lac repressor because of a lack of lac operator sequence in pET14b vector. Template plasmid DNAs were purified using a Qiagen Midi plasmid kit (Qiagen, Hilden, Germany) as directed by the manufacturer.

Preparation of E. coli S30 extract

An S30 extract was prepared from E. coli B strain BL21 (DE3) (F−ompT hsdSB [rB−mB−] gal dcm [DE3]) (Invitrogen, Carlsbad, CA, USA) according to the method described by Pratt (Pratt 1984). In brief, cells harvested from an overnight culture in 500 ml of LB medium were washed three times with S30 buffer (Table 1) containing 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium acetate, 1 mM dithiothreitol (DTT), and 0.5 μl/ml 2-mercaptoethanol. After final washing and centrifugation, the cells were resuspended in 12 ml of S30 buffer without 2-mercaptoethanol and disrupted using a French press at 8400 psi. After addition of 0.01 volumes of 0.1 M DTT, the resulting cell extract was centrifuged twice at 30,000×g for 30 min at 4 °C. A pre-incubation mixture (0.3 volumes) (Table 1) composed of 0.3 M Tris-acetate (pH 8.2), 9.2 mM magnesium acetate, 13.2 mM ATP (pH 7.0), 4.4 μM DTT, 63 mg/ml creatine phosphate, 775 μg/ml creatine kinase (Roche Molecular Biochemicals), and 0.4 mM amino acid mixture was added to the final supernatant, which was then incubated at 37 °C for 80 min. After incubation, the treated extract was dialyzed (SnakeSkin Pleated Dialysis Tubing, 10,000 MWCO, Pierce, Rockford, IL, USA) against 50 volumes of S30 buffer at 4 °C for 45 min, and the dialysis process was repeated twice. The resulting extract was centrifuged at 4000×g for 10 min at 4 °C, and the supernatant as the final S30 cell extract was stored at − 80 °C until use.

Table 1.

Preparation of S30 buffer and pre-incubation mixture

Stock solution Final concentration
S30 buffer:
  1.0 M Tris/acetate (pH 8.2) 10 mM
  1.4 M Magnesium acetate 14 mM
  6.0 M Potassium acetate 60 mM
  0.1 M dithiothreitol (DTT) 1 mM
  2-mercaptoethanol 0.5 μl/ml
Pre-incubation mixture:
  2.2 M Tris-acetate (pH 8.2) 0.3 M
  3.0 M Magnesium acetate 9.2 mM
  38 mM ATP (pH 7.0) 13.2 mM
  0.55 M DTT 4.4 mM
  Creatine phosphate (powder) 63 mg/ml
  12.5 μg/μ1 Creatine kinase 775 μg/mI
  50 mM Amino acid mixture 0.4 mM
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All reagents were prepared with DEPC-treated H20

Tyrosine (50 mM) was separately added to the reaction mixture

Condensed S30 extract was prepared by dialyzing the S30 extract according to the method reported by Kigawa et al. (1999). Briefly, S30 extract prepared as described above was dialyzed against 10 volumes of an equal-weight mixture of polyethylene glycol 8000 (PEG 8000; Sigma, St. Louis, MO, USA) and S30 dialysis buffer (S30 buffer without 2-mercaptoethanol) at 4 °C on a rotator, followed by further dialysis against 100 volumes of S30 dialysis buffer for 15 min at 4 °C. Aliquots (15 μl) of the condensed S30 cell extract were stored at − 80 °C. Protein content was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard.

Transcription/translation reactions in the batch and dialysis protein synthesis systems

Batch synthesis of HSPB5 was carried out according to the method reported by Kigawa et al. (1999), with slight modifications. Table 2 summarizes the reagents used for batch protein synthesis. Although Kigawa et al. (1999) used 15 μl of reaction mixture containing [14C] L-leucine in their batch system to detect the synthesized protein, we performed experiments in 50 μl of reaction mixture to evaluate the experimental conditions by Western blotting to avoid the use of a radioactive amino acid. Briefly, the reaction mixture contained 55 mM HEPES/KOH (pH 7.5), 1.7 mM DTT, 1.2 mM ATP, 0.8 mM each of CTP, GTP, and UTP, 80 mM creatine phosphate, 250 μg/ml of creatine kinase (Roche Molecular Biochemicals), 4% PEG 8000, 0.64 mM 3’,5’-cyclic AMP, 68 μM folinic acid, 210 mM potassium glutamate, 27.5 mM ammonium acetate, 4.2–16.2 mM magnesium acetate, 8.75 μg E. coli total tRNA (Sigma), 40 U RNase inhibitor (Promega, Madison, WI, USA), 15 U T7 RNA polymerase (Promega), 1 mM each of the 20 amino acids, and 2 μg pIVEX2.4bNde-HSPB5 and/or pET14b-HSPB5. A total of 15 μl of S30 extract (60 μg/μl) and/or condensed S30 extract (180 μg/μl) was used in the synthesis reaction based on the report of Kigawa et al. (1999). The reaction mixture was incubated at 37 °C for 1.5 h. In a separate experiment, the dependence on the dose of condensed S30 extract in the synthesis reaction was evaluated using a lower volume (approximately one-third) of the condensed S30 extract.

Table 2.

Preparation of reagents used for the batch system

Stock solution Final concentration in reaction mixture (50 μl)
1 M HEPES/KOH (pH 7.5) 55.0 mM *
0.1 M DTT 1.7 mM *
0.1 M ATP (lithium salt) 1.2 mM *
0.1 M CTP (lithium salt) 0.8 mM *
0.1 M GTP (lithium salt) 0.8 mM *
0.1 M UTP (lithium salt) 0.8 mM *
Creatine phosphate 80.0 mM *
12.5 μg/μl Creatine kinase 250.0 μg/ml *
40% PEG8000 4.0% *
10 mM 3’, 5’-cyclic AMP 0.64 mM *
25 mM Folinic acid 68.0 μM *
Potassium glutamate 210.0 mM *
7.5 M Ammonium acetate 27.5 mM *
0.24 M Magnesium acetate 4.2–16.2 mM **
10 μg/μ1 E.coll total tRNA 8.75 μg
40 WpIRNase inhibitor 40.0 U
15 U/μl T7 RNA polymerase 15.0 U
50 mM Amino acid mixture 1.0 mM
pIVEX2 4bNde-HAB or pET14b-HAB 2.0 μg
S30 extract or condensed S30 extract 15.0 μl
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*For convenience, these reagents were prepared in 1 ml as a “2.5 x Premix”, and then aliquots were stored at −80 °C until use

**Magnesium acetate (0.24 M) at different doses (1 μl = + 4.8 mM) was added to the reaction mixture to adjust an appropriate concentration of Mg2+. Tyrosine (50 mM) was separately added to the reaction mixture

In the dialysis protein synthesis system, a simple unit composed of a disposable dialyzer and a conical tube was constructed to allow for continuous supply of substrate and removal of byproducts from reaction mixture, as described by Spirin et al. (1988), Davis et al. (1996), and Kigawa et al. (1999). In brief, a total of 300 μl of reaction mixture (referred to as internal solution) containing the same components as used in the batch system (except for the additional amount of template DNA and condensed S30 extract) was placed in a DispoDialyzer CE tube (1 ml, 10,000 MWCO; Spectra/Por CE, Rancho Dominguez, CA, USA). The dialysis tube was inserted in a 15-ml Falcon conical centrifuge tube (Corning, Corning, NY, USA) containing a total of 3 ml of substrate mixture (referred to as external solution) consisting of the components of the internal solution without template DNA, creatine kinase, T7 RNA polymerase, RNase inhibitor, and condensed S30 extract. The external solution contained extra magnesium acetate (+ 4.2 mM) carried over from the condensed S30 extract. The reaction was carried out at 37 °C for 24 h with gentle agitation.

SDS-PAGE and Western blotting analyses of HSPB5 synthesized using the batch system

In the present experiments, we used Western blotting to evaluate the efficacy of protein synthesis because of its greater convenience and safety as compared with the use of conventional radioactive amino acids. Briefly, an aliquot (0.1 volume) of the transcription/translation reaction mixture was removed, mixed with 10 volumes of acetone, and placed on ice for 20 min. The sample was then centrifuged at 14,000×g for 10 min, and the pellet was air dried for 20 min, followed by suspension with 1× SDS sample buffer (10 volumes). An aliquot of the sample (0.1 volume) was subjected to SDS-PAGE and electroblotted onto a polyvinylidene difluoride nylon membrane (Millipore, Bedford, MA, USA). The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution. The membrane was then washed three times with TBS-0.1% Tween 20 (TBST), incubated for 1 h with horseradish peroxidase–conjugated donkey anti-rabbit IgG (GE Healthcare Lifescience), and washed with TBST. Signal was detected using enhanced chemiluminescence reagents (GE Healthcare Lifescience). Duration of exposure of the nylon membrane to X-ray film varied depending on signal intensity. QuantiScan software (Biosoft, Cambridge, UK) was used for semi-quantification of signal intensity.

Effects of different reaction mixture concentrations of RNA polymerase, phosphocreatine, amino acids, and PEG on HSPB5 synthesis

To evaluate the effect on protein synthesis of varying concentrations of reaction mixture components such as amino acids, HSPB5 was purified under denaturing conditions. Briefly, internal solution was diluted (1:10) with 10 mM Tris-HCl (pH 8.0), 100 mM NaH2PO4, and 8 M urea and then subjected to chromatography on a Ni+-NTA agarose beads (Qiagen) column (Econo-Column 0.7 × 5 cm, Bio-Rad). After sequential washing with pH 6.3 and pH 5.9 buffer containing 10 mM Tris-HCl, 100 mM NaH2PO4, and 8 M urea, denatured HSPB5 was eluted with buffer consisting of 10 mM Tris-HCl (pH 4.5), 100 mM NaH2PO4, and 8 M urea. An aliquot (100 μl) of the resulting eluate (2.5 ml) was subjected to acetone precipitation (10 volumes) and dried. An aliquot (5 μl) of the sample treated with 1× SDS sample buffer and subjected to SDS-PAGE and Western blotting, followed by densitometric analysis of signal intensity. An aliquot of the reaction mixture was also taken prior to Ni+-NTA agarose beads chromatography and analyzed as described above.

Purification, SDS-PAGE, Western blotting, and mass spectrometry (MS) analyses of HSPB5 synthesized using the dialysis system

To purify HSPB5 under native conditions, internal solution was diluted (1:10) with 20 mM Tris-HCl buffer (pH 7.5) containing 200 mM NaCl and then subjected to Ni+-NTA agarose beads chromatography. After extensive washing (1 ml × 10) with 20 mM Tris-HCl buffer (pH 7.5) containing 50 mM imidazole, HSPB5 was eluted from the column using elution buffer, which consisted of 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 200 mM imidazole. The resulting eluate was then dialyzed using a Slide-A-Lyzer Dialysis Cassette (10,000 MWCO, Thermo Fisher Scientific, Waltham, MA, USA) and lyophilized. The quality of the final product was assessed by SDS-PAGE using Coomassie blue staining, followed by Western blotting and MS analysis.

Synthesized human HSPB5B was analyzed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS (MALDI-TOF-MS) using an Autoflex Speed MALDI-TOF-MS mass spectrometer (Bruker Daltonics, Billerica, MA, USA). A non-acidic matrix, sinapinic acid (Sigma), was used for MALDI-TOF-MS analysis. In brief, a mixture (1–2 μl) consisting of equal volumes of protein and matrix solution was applied to the MALDI target plate and allowed to air dry. MALDI-TOF-MS mass spectra were then acquired in the linear mode over the m/z range 12,000 to 35,000.

Analysis of the chaperone activity of HSPB5 synthesized using the dialysis system

Inhibition of protein aggregation by HSPB5 was assayed according to the method reported by Horwitz (1992). Alcohol dehydrogenase (ADH, Sigma or ORIENTAL YEAST, Tokyo, Japan) was used as the target protein, and aggregation was monitored by measuring the apparent absorption at 360 nm caused by light scattering, using an Ultrospec 3100 pro UV/visible spectrophotometer (GE Healthcare Lifescience) equipped with a temperature-controlled cuvette holder. Assays of the chaperone activity of HSPB5 prepared under native conditions were carried out at 37 °C in a total reaction volume of 500 μl containing 2 μM ADH, 1 or 2 μM purified HSPB5 protein, 25 mM HEPES/KOH (pH 7.5), 5 mM magnesium acetate, 50 mM KCl, and 5 mM β-mercaptoethanol. The optical density in each cell was recorded every 3 min for 45 min. In addition, to compare the chaperone activity of HSPB5 prepared by the batch (HSPB5-B) and dialysis (HSPB5-D) systems, citrate synthase (CS; 0.5 μM, Sigma) (Sreelakshmi and Sharma 2006) was also used and its heat aggregation was monitored as described above, except for 39 °C as a denaturing temperature. For a preparation of HSPB5 by the batch system, HSPB5 protein from each individual 16 reaction mixture was collected and purified according to the method used for the purification of HSPB5 synthesized by the dialysis system.

Results

Effect of Mg2+ concentration on HSPB5 synthesis using the batch system

Previous studies indicated that the efficiency of cell-free protein systems depends on the concentrations of Mg2+ and PEG (Kim et al. 1996; Kigawa et al. 1999; Kim and Swartz 1999). We therefore initially optimized the Mg2+ concentration in the reaction mixture. As shown in Fig. 1a, as the Mg2+ concentration in the reaction mixture was increased (4.2, 9.0, 12.0, and 13.8 mM), the amount of HSPB5 synthesized also increased. We then compared the synthesis of HSPB5 between the S30 (untreated) and condensed S30 extracts. The amount of HSPB5 synthesized was markedly higher in the condensed S30 cell extract, as compared with the untreated S30 extract (Fig. 1b). In addition, adjustment of the reaction mixture Mg2+ concentration was also required with the condensed S30 extract (Fig. 1b).

Fig. 1.

Fig. 1

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Effect of magnesium ion concentration (a) and condensed S30 extract (b) on HSPB5 synthesis using the batch system. a HSPB5 was synthesized in a final volume of 50 μl using the batch system. Magnesium acetate (0.24 M) at different doses was added to each reaction mixture to achieve the indicated final concentration of Mg2+ (4.2 mM, 9.0 mM, 11.4 mM, or 13.8 mM). The Mg2+ concentration (4.2 mM) is carried over from 15 μl of the S30 extract. Aliquots from each reaction mixture were subjected to SDS-PAGE and Western blot analysis, as described in the “Materials and Methods.” For Western blot analysis, the X-ray film was exposed for 1 h for detection of signals. Right panel is the respective quantification graph of HSPB5’s intensity from the adjacent X-ray film. HSPB5 synthesis was dependent on the Mg2+ concentration. b Level of HSPB5 production compared between the S30 extract and condensed S30 extract. The Mg2+ concentration was adjusted as in Fig. 1a. The dose of S30 extract and condensed S30 extract was 15 μl. Upper blot: The X-ray film was exposed for 1 min rather than 1 h for detection of signals to avoid unmeasurable signal intensity derived from the condensed S30 extract due to overexposure. Lower blot: The X-ray film was exposed for longer time to detect signals from the S30 extract. Results of semi-quantification of HSPB5 synthesis by image analysis are illustrated to the right. Protein synthesis with condensed S30 extract resulted in higher HSPB5 production, as compared with the S30 extract, and the Mg2+ concentration was a crucial factor in protein synthesis for both systems

Effect of condensed S30 extract volume (protein content) on HSPB5 synthesis using the batch system

Previous reports indicated that concentrated S30 extracts provide increased protein production relative to unconcentrated extracts (Kim et al. 1996; Kigawa et al. 1999). We therefore examined the effect of using condensed S30 extract in the batch system on production of HSPB5. Figure 2 illustrates the effect of condensed S30 extract volume on HSPB5 synthesis. In an experiment examining the addition of different volumes of condensed S30 cell extract to the reaction mixture, no obvious HSPB5 production was observed at 2.5 μl of extract/50 μl of reaction mixture. However, HSPB5 production was evident with 3.5 μl of extract/50 μl of reaction mixture and plateaued at 4.5 μl of extract/50 μl of reaction mixture. The amount of HSPB5 produced was thus limited, even when the protein content of the concentrated S30 extract was increased.

Fig. 2.

Fig. 2

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Effect of condensed S30 extract protein concentration on HSPB5 synthesis using the batch system. a Different doses of condensed S30 extract were added to the reaction mixture, and then HSPB5 was analyzed as described in the “Materials and Methods.” As shown in the right panel of a, with the high dose (5–15 μl) of condensed S30 extract, no obvious changes in protein production were observed; therefore, lower doses of condensed S30 extract were used to evaluate the dose dependence (see the left panel of a). The Mg2+ concentration was adjusted to 9.0 mM, as shown in Fig. 1b, because the Mg2+ concentration carried over from the condensed S30 extract varied in each reaction mixture. b Semi-quantification of HSPB5 synthesis by image analysis. DNA (−): complete reaction components except for HSPB5 cDNA. HSPB5 protein production was dependent on the dose of condensed S30 extract

Effect of the plasmid vector on cell-free protein synthesis of HSPB5

To assess the efficiency of template plasmid vectors on protein synthesis, human HSPB5 cDNA was inserted into the pET14b plasmid vector, which produces histidine-tagged HSPB5. The level of HSPB5 produced using this plasmid was then compared with that produced using the pIVEX2.4bNde vector with condensed S30 extract. As shown in Fig. 3a, markedly less HSPB5 was synthesized using the pET14b-HSPB5 plasmid vector compared with the pIVEX2.4bNde-HSPB5 vector.

Fig. 3.

Fig. 3

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Effect of different plasmid vectors on the efficiency of HSPB5 synthesis in the batch (a) and dialysis (b, c) systems. a HSPB5 cDNA was inserted into the pET14b vector and pIVEX2.4bNde vector, and protein synthesis using the batch system was carried out with condensed S30 extract (15 μl). The Mg2+ concentration was adjusted by addition of 0.24 M magnesium acetate, as indicated in Fig. 1a. Arrows show HSPB5 protein at lower and higher molecular weight derived from pET14b-HSPB5 and pIVEX2.4bNde-HSPB5 plasmid vectors, respectively, resulting from the different amino acid sequences in each vector for the His-tag region upstream of the HSPB5 cDNA. Arrowhead indicates non-specific signals on the blot membrane. X-ray film was exposed for 1 min for detection of signals. Use of the pIVEX-2.4bNde-HSPB5 plasmid vector produced a much larger amount of HSPB5 compared with use of the pET14b-HSPB5 plasmid vector. b The pIVEX2.4bNde vector was used for protein synthesis with condensed S30 extract, and the amount of HSPB5 synthesized in the batch and dialysis systems was compared. The dialysis system provides markedly higher HSPB5 synthesis. c Schematic illustration of the batch and dialysis systems

HSPB5 synthesis in the batch and the dialysis systems using condensed S30 extract

Dialysis is often employed in cell-free protein synthesis to remove byproducts such as monophosphate, which can inhibit protein synthesis during prolonged production (Davis et al. 1996; Kigawa et al. 1999; Kim et al. 1996). Dialysis thus removed harmful byproducts from the reaction mixture and provides a steady supply of energy resources. The production of HSPB5 was markedly increased using the dialysis system as compared with the batch system (Fig. 3b).

Effects of reaction mixture concentrations of RNA polymerase, phosphocreatine, amino acids, and PEG on HSPB5 synthesis

As shown in Fig. 4a, Western blot revealed a positive band corresponding to HSPB5 and two lower bands around 20 kDa in the reaction mixture before the column purification (lane 1). These two bands were detected in the eluates by pH 6.3 and 5.9 elution buffers, except for flow-through and the eluate by pH 4.5 elution buffer. This suggests that with regard to the two lower bands, they are non-specific signal bands unrelated to HSPB5 and possibly proteins derived from S30 extract used for HSPB5 synthesis. HSPB5 was recovered at high purity using pH 4.5 elution buffer under denaturing conditions. Thus, the effects on HSPB5 synthesis of various constituents of the reaction mixture were evaluated under denaturing conditions.

Fig. 4.

Fig. 4

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Effects of T7 RNA polymerase, phosphocreatine, PEG, and amino acid content in the reaction mixture on HSPB5 protein synthesis. a HSPB5 was synthesized using the dialysis system, and, after binding to a Ni+-NTA agarose beads column, HSPB5 was eluted with buffers of different pH containing 8 M urea. Aliquots were subjected to SDS-PAGE and Western blotting. Left and right panels show SDS-PAGE and Western blotting results, respectively. Arrow shows HSPB5 on Western blot. Eluates recovered using pH 4.5 buffer were stored as the HSPB5 fraction and used to evaluate the effect of different factors. b and c HSPB5 protein was synthesized under the conditions of 2-fold (X2) or 3-fold (X3) concentrations of T7 RNA polymerase (T7), phosphocreatine (PC), PEG8000 (PEG), and/or amino acids (AA), and then the amount of HSPB5 synthesized was compared with that obtained using the original components (Ori) described in the “Materials and Methods.” Aliquots of each reaction mixture before isolation with beads (c) and of each sample (b) prepared under denaturing conditions (8 M urea, pH 4.5 buffer) as shown in Fig. 4a were subjected to SDS-PAGE and Western blotting. The right panels of b and c show semi-quantification of each blot signal intensity

As shown in Fig. 4b, no significant change in the amount of HSPB5 synthesized was observed when the concentration of T7 RNA polymerase was increased 2-fold. However, 2- and 3-fold increases in the amino acid content resulted in higher production of HSPB5 as compared with the original reaction mixture. A 2-fold increase in phosphocreatine resulted in a slight decrease in HSPB5 synthesis. Increasing the amount of PEG8000 in the reaction mixture interfered with purification of HSPB5 and resulted in a slight decrease in the amount of protein synthesized, as illustrated in Fig. 4a and c. Quantification graph in Fig. 4c showed that HSPB5 level in reaction mixture before the column purification of AAx2 (AA2) and AAx3 (AA3) seemed to be similar to original (Ori), in spite of a higher yield of HSPB5 in AAx2 and AAx3 reaction mixtures. It may be possible that in the lysis buffer containing 8 M urea before purification, a hydrophobic interaction of denatured HSPB5 protein and other denatured protein, including ribosome proteins derived from S30 extract, may interfere a precise collection of HSPB5 protein as a starting material. Especially, the hydrophobic interaction between the denatured proteins may be accelerated in the reaction mixtures (AA2 and AA3) with a large amount of HSPB5 protein. The dialysis system with 3-fold increase in the amino acid resulted in production of approximately 500 μg/ml of HSPB5.

Purification and MALD-TOF MS analysis of HSPB5 synthesized using the dialysis system

When purified under native conditions, HSPB5 was eluted in the concentration range of 100 to 200 mM imidazole (Fig. 5a), accompanied with a slight signal of HSPB5 in 250 mM imidazole, indicating that the elution buffers containing imidazole at the concentration range were enough to purify most of HSPB5 protein bound to Ni +-agarose beads. Thus, for MS and chaperone activity analyses, HSPB5 was eluted from the Ni+-NTA agarose beads column using 200 mM imidazole after extensive washing with 50 mM imidazole.

Fig. 5.

Fig. 5

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Purification (a), SDS-PAGE/Western blotting (b), and MS (c) of HSPB5 synthesized using the dialysis system. a HSPB5 synthesized using the dialysis system was isolated under native conditions. Isolation buffers containing a serial concentration of imidazole were used, and buffer containing 200 mM imidazole was chosen to purify HSPB5 after extensive washing with buffer containing 50 mM imidazole. b and c Aliquots of purified HSPB5 were subjected to SDS-PAGE and Western blotting (b) and MS (c)

As shown in Fig. 5b, SDS-PAGE and Western blot analyses revealed synthetic human HSPB5 as essentially a single band. Densitometric analysis of the purified HSPB5 indicated it was at least 95% pure. The molecular mass of human HSPB5 as calculated from the reported sequence (GenBank, ACP18852.1) is 20,158.91 Da. Given that the synthetic HSPB5 contains a His-tag sequence (MSGSHHHHHHSSGIEGRGRLIKH) at the N-terminus, with a molecular mass of 2594.86 Da, the theoretical molecular mass of HSPB5 with the His-tag is 22,752.77 Da (computed pI/Mw). As illustrated in Fig. 5c, MALD-TOF MS analysis of the synthetic HSPB5 revealed a molecular mass of 22,869 Da, almost identical to the theoretical molecular mass. The slight difference between the theoretical and measured molecular mass values could be due to the limited spectral resolution of the MS instrument used in this study.

Synthetic HSPB5 inhibits thermally induced aggregation of ADH and CS

To examine the chaperone activity of HSPB5 synthesized using the dialysis system and purified under native conditions, the changes in light scattering associated with heat-induced aggregation of ADH and CS were monitored. As shown in Fig. 6a, the optical density of ADH increased as a result of heat denaturation at 37 °C. However, treatment with HSPB5 synthesized by the dialysis system significantly suppressed the increase in the optical density of ADH, by 40 and 70% at a molar ratio of 1:0.5 and 1:1 (ADH: HSPB5), respectively. This result indicates that HSPB5 synthesized using the dialysis system retains the native molecular chaperone activity. When compared the chaperone activity of HSPB5-D with that of HSPB5-B, no significant difference was observed in the aggregation experiments with ADH and CS (Fig. 6b). This indicates that each HSPB5 protein synthesized by the batch and dialysis systems is functionally equivalent in chaperone activity. However, it is obvious that the dialysis system is more efficient in protein synthesis than the bath system.

Fig. 6.

Fig. 6

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Inhibition of heat-induced aggregation of ADH and CS by HSPB5 synthesized using the batch and dialysis systems. Light scattering resulting from heat-induced aggregation of ADH and CS was assessed by measuring the increase in optical density at 360 nm, as described in the “Materials and Methods.” a HSPB5 prepared by the dialysis system exhibited dose-dependent inhibition of the increase in optical density associated with heat-induced aggregation of ADH. b HSPB5-D and HSPB5-B prepared by the dialysis and batch systems, respectively, showed similar inhibitory activity against heat–induced aggregation of ADH (left panel) and CS (right panel). In this study, heat-induced aggregation of ADH and CS was monitored at 39 °C and incubation time for ADH and CS were 30 and 45 min, respectively

Discussion

As described in the Introduction, altered expression of HSPB5 is reportedly associated with the development of several human neurologic diseases (Iwaki et al. 1989; Kato et al. 1992; Head et al. 1993; Jackson et al. 1995; van Noort et al. 1995; Stoevring et al. 2005). Recent studies have implicated HSPB5 in regulating secretion, by astrocytes (Hong et al. 2017), and retinal pigment epithelial cells (Gangalum et al. 2016), of exosomes, small membranous vesicles that contain proteins and RNAs and play an important role in cell-to-cell communication. These findings suggest that in addition to its chaperone activity, HSPB5 has other as yet unknown biological functions, thus highlighting the need for further investigations of the molecular functions of HSPB5.

In this study, we investigated the applicability of cell-free and E. coli–based protein synthesis systems for synthesizing HSPB5 that retains chaperone activity. In the cell-free protein synthesis system, E. coli strains with different genotypes are used as sources of S30 extract. In this study, we used E. coli B strain BL21 (DE3), as this strain (Krinsky et al. 2016) and a strain genetically engineered from it, BL21 Star (DE3) (Kang et al. 2005; Kwon and Jewett 2015), are reportedly reliable sources of S30 extract for cell-free protein synthesis.

In the first experiment, we found that the concentration of Mg2+ in the reaction mixture has a significant impact on HSPB5 production (Fig. 1a). Synthesis using condensed S30 extract, though also dependent on Mg2+ concentration, produced elevated amounts of HSPB5 relative to the standard S30 extract (Fig. 1b). The addition of an optimal amount of condensed S30 extract to the reaction mixture was also found to be a critical factor in determining the amount of HSPB5 synthesized (Fig. 2a), similar to the production of chloramphenicol acetyltransferase (CAT) using a cell-free protein synthesis system (Jewett et al. 1996). The limited synthesis of HSPB5 using condensed S30 extract may be partially explained by the production of interfering byproducts and the limited energy supply in the batch system.

Among plasmid vectors used for protein expression, pET-based vectors are widely used for E. coli–based synthesis of eukaryotic proteins, and they provide for efficient protein production in cell-free protein synthesis systems (Krinsky et al. 2016; Kang et al. 2005). However, the efficiency of protein synthesis in cell-free systems can vary based on the vector used, as in the case of E. coli–based protein synthesis. We therefore examined different expression vectors to determine which provided the most efficient protein synthesis in the cell-free system. As shown in Fig. 3a, use of the pIVEX2.4bNde vector provided significantly greater synthesis of HSPB5 as compared with use of the pET14b vector. This finding indicated that expression vectors commonly used for E. coli–based protein synthesis may not be optimal for use in cell-free protein synthesis systems. Use of the pIVEX2.4bNde vector enables the synthesis of much higher amounts of human HSPB5 in the cell-free synthesis system than does the use of pET14b. Although the reason for the difference in protein synthesis between the two plasmid vectors was not determined in this study, it is possible that the 5’-promoter sequence of pET14b vector forms a stem-loop structure with the early region of the coding sequence of the pET14b-HSPB5 vector, thereby inhibiting binding of T7 RNA polymerase to binding sites and blocking mRNA/protein synthesis (Rahn and Whitfield 2003).

To increase the yield of protein production of batch systems, several studies have developed and improved continuous-exchange cell-free protein synthesis systems (i.e., dialysis systems), in which a solution containing low-molecular-weight substrates, such as amino acids, is supplied to the reaction mixture through a dialysis membrane. In these systems, byproducts that could inhibit protein synthesis derived from the synthesis reaction, such as inorganic phosphate, can be efficiently removed from the reaction mixture (Kim et al. 1996; Kigawa et al. 1999; Kim and Swartz 1999). As illustrated in Fig. 4b, increased production (approx. 3- to 4-fold) of HSPB5 was observed using the dialysis system with a 2- to 3-fold increase in the amino acid content, as compared with the original reaction mixture composition. Several studies have reported that selective addition of reagents to the reaction mixture and modifying the energy regeneration system enable enhanced protein production using cell-free synthesis systems. For instance, ATP is the most essential factor for maintaining a longevity of protein synthesis reaction due to the energy it provides. Instead of employing the conventional phosphoenolpyruvate/pyruvate kinase system for supplying energy (Pratt 1984), the pyruvate/pyruvate oxidase/acetate kinase system (Kim and Swartz 1999), creatine phosphate/creatine kinase system (Kigawa et al. 1999), and PANOx system using phosphoenolpyruvate (Kim and Swartz 2001) reportedly provide greater ATP regeneration. Other factors that can affect the efficiency of protein production using cell-free systems include PEG (Kim et al. 1996; Kigawa et al. 1999), tRNA (Kanda et al. 2000), and amino acids (Kim and Swartz 1999; Kim and Swartz 2000). In this study, increasing the amino acid concentration led to markedly elevated HSPB5 synthesis (Fig. 4). PEG is thought to play a role similar to that of polyamines, such as spermine and spermidine, which stimulate formation of the peptide initiation complex (Atkins et al. 1975; Igarashi et al. 1980). However, the presence of PEG8000 at a high concentration (8%) inhibited His-tag–based purification of HSPB5 and slightly reduced the efficiency of protein synthesis (Fig. 4). The reasons for the unfavorable effect of PEG8000 are unclear, but it is possible that PEG8000 inhibits the elution of HSPB5 from the Ni+-NTA agarose beads. Regarding the slight decrease in protein synthesis at high PEG8000 concentrations, Kigawa et al. (1999)) reported decreased production of CAT in the presence of 6% PEG8000. Kim et al. (1996) demonstrated the dependence of CAT synthesis on the phosphopyruvate concentration in the reaction mixture, but increasing the phosphocreatine concentration was found to have no significant effect on HSPB5 synthesis in the present study (Fig. 4). It is likely that the increased phosphocreatine concentration is associated with an increase in inorganic phosphate, which can affect the efficiency of protein synthesis in the early stages by reducing the free Mg+ concentration in the reaction mixture, even though it increases ATP generation.

MS analysis is a reliable method for determining protein molecular weight. MS was used to determine the native molecular mass of α-crystallin isolated from human lens and identify various post-translational modifications, such as phosphorylation, deamidation, and acetylation (Miesbauer et al. 1994). Our accurate MS data regarding the molecular mass of HSPB5 demonstrate the applicability of the cell-free protein synthesis system under the present experimental conditions.

Functional analyses of proteins synthesized using cell-based protein synthesis systems are essential to confirm the biological properties of proteins. Analyses of the chaperone activity of molecular chaperones are based on the suppression of heat-induced denaturation of native proteins. The simple light scattering method remains the most useful tool available for assessing the chaperone activity of HSPB5 isolated from mammalian lens (Horwitz 1992). Here, we report for the first time that HSPB5 synthesized using a cell-free protein synthesis system prevented the heat-induced aggregation of ADH and CS (Fig. 6), indicating that human HSPB5 synthesized under the present conditions retained its chaperone activity, even though it had a His-tag sequence at the N-terminus. A recent study also reported that HSPB5 protein bearing a GST-tag at the N-terminus (which is larger in molecular size than a His-tag) retained its chaperone activity (Arbach et al. 2017). Collectively, these results suggest that additional soluble peptide sequences at the N-terminus of HSPB5 do not block its chaperone activity.

The molecular mechanism of the chaperone activity of HSPB5 remains to be fully elucidated. To address this gap in knowledge, several studies have reported the structures of the peptide domains of HSPB5 responsible for substrate binding, oligomer formation, and subunit exchange (Haslbeck et al. 2019). The α-crystallin domain, which forms β-sandwiches consisting of β-strands (Bagnéris et al. 2009), plays a crucial role in dimer formation (Clark et al. 2011) and chaperone activity by binding to amyloid-β (Hochberg et al. 2014) and α-synuclein (Cox et al. 2016). Unlike the α-crystallin domain, the C-terminal domain containing a conserved IXI motif is unstructured, contains a highly flexible C-terminal extension (Treweek et al. 2010), and mediates oligomeric assembly via intermolecular interactions with the α-crystallin domain (Pasta et al. 2004; Jehle et al. 2010). The C-terminal domain also participates in chaperone activity and inhibition of amyloid fibril formation (Treweek et al. 2007). The N-terminal domain has also been implicated in chaperone activity, oligomer assembly (Numoto et al. 2012; Jovcevski et al. 2018), and the formation of large multimers (Jehle et al. 2011). Based on the significance of the N-terminal domain’s contribution to the formation of higher-order oligomers, it may be difficult to account for the observed chaperone activity of HSPB5 with a GST- (Arbach et al. 2017) and/or His-tag (current study) at the N-terminus. Interestingly however, it has been suggested that HSPB5 multimers are dissociated from highly ordered structures under stress/disease conditions, which exposes the N-terminus of HSPB5 outside the oligomer. Dissociated HSPB5 then binds to aggregation-prone proteins such as amyloid-β, α-synuclein, and tau (Peschek et al. 2013), thus inhibiting aggregation of these neurodegeneration-related proteins. This model of the chaperone activity of HSPB5 is also supported by a study indicating that HSPB5 with a GST-tag at the N-terminus exhibits less polydispersity than native HSPB5 and displays chaperone activity comparable with native HSPB5 (Arbach et al. 2017). These studies thus suggest that smaller oligomers (such as dimers) rather than larger oligomers and polydispersed architectures are required for chaperone activity. This may explain why HSPB5 with a His-tag at the N-terminus inhibits thermal stress–induced aggregation of ADH and CS (Fig. 6), although further experiments are needed.

Some of the structural features of the functional domains of HSPB5 have been determined based on NMR studies using HSPB5 prepared from E. coli grown in medium containing [15N]-ammonium chloride (Treweek et al. 2010; Jehle et al. 2011; Hochberg et al. 2014) to label amino acid residues. Combining stable-isotope labeling with cell-free protein synthesis has facilitated the determination of structural features of various biofunctional proteins by NMR spectroscopy (Kigawa et al. 1999; Terada and Yokoyama 2015; Kigawa 2017; Hoffmann et al. 2018). Methods for site-selective stable-isotope labeling of amino acid residues have enabled the determination of dynamic structures of proteins synthesized using cell-free systems (Hohsaka et al. 1996; Yabuki et al. 1998; Mukai et al. 2015; Adachi et al. 2019). These methods have added further information to the structural information obtained for proteins synthesized using E. coli–based protein expression systems, in which amino acid residues are not site-selectively labeled. Although most previous studies using NMR spectroscopy have explored structural features of HSPB5 labeled with stable isotope and prepared from E. coli as described above, how the chaperone activity of HSBP5 functions for amyloid-β, α-synuclein, and tau at the atomic level remains unclear. Recently, however, Liu et al. used NMR spectroscopy to demonstrate how HSPB5 recognizes α-synuclein and tau using uniformly [15N]-labeled HSPB5 prepared from E. coli (Liu et al. 2018). Thus, synthesis of HSPB5 using cell-free protein synthesis in combination with uniform, amino acid– and site-selective stable-isotope labeling methods followed by NMR spectroscopy provided an additional approach for analyzing the molecular mechanism of the chaperon activity of human HSPB5 at the atomic level. Such studies could lead to the development of pharmacologic therapies to prevent or reverse neurodegeneration. Recent research revealed that an arginine residue in the N-terminal domain RLFDQxFG motif of sHsps, including HSPB5, plays a pivotal role in regulating the size of HSPB5 oligomers (Shatov et al. 2018). Thus, the cell-free protein synthesis system described here combined with selective isotope labeling could provide a tool for determining the functions of other amino acid residues in the other domains of HSPB5.

In conclusion, the results of the present study indicate that the concentration of Mg2+ in the cell-free protein synthesis system reaction mixture is an important factor that influences the efficiency of human HSPB5 synthesis. The use of condensed S30 cell extract and incorporation of a dialysis system significantly increased synthesis of HSPB5 in the present study. The pIVEX2.4b Nde plasmid vector was found to be superior for the cell-free synthesis of HSPB5. In addition, human HSPB5 synthesized using the cell-free system described in the present report was functional with regard to chaperone activity.

Acknowledgment

We thank Mr. Hidetoshi Kamijyo for technical support.

Funding information

This work was supported in part by a grant from Meijo University Research Institute (Grant-in-Aid for Specially Promoted Research).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

2/18/2020

In the original publication, the given name of the last author was incorrectly displayed as the name must read: Katsuyoshi Masuda.

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