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Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli
Accepted Manuscript Title: Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli Authors: Wen-Chi Tseng, Tsuei-Yun Fang, Yuan-Chieh Hsieh, Chien-Yu Chen, Meng-Che Li PII: DOI: Reference:
S0168-1656(17)30155-4 http://dx.doi.org/doi:10.1016/j.jbiotec.2017.04.001 BIOTEC 7844
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
25-1-2017 22-3-2017 1-4-2017
Please cite this article as: Tseng, Wen-Chi, Fang, Tsuei-Yun, Hsieh, Yuan-Chieh, Chen, Chien-Yu, Li, Meng-Che, Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2017.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli Wen-Chi Tseng*1, Tsuei-Yun Fang**2, Yuan-Chieh Hsieh1, Chien-Yu Chen 1, Meng-Che Li 1 1
Department of Chemical Engineering, National Taiwan University of Science and
Technology, Taipei, Taiwan; 2Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan *Corresponding author: Wen-Chi Tseng Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan E-Mail: [email protected] Fax: 886-2-2462-2586 Tel: 886-2-2730-1078 ** Corresponding author: Tsuei-Yun Fang Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung 202, Taiwan E-Mail: [email protected] Fax: 886-2-2462-2192 Tel: 886-2-2462-2586 Highlights
Low temperature cultivation was found to favor the production of the soluble form of cyanophycin.
The soluble form of cyanophycin showed upper critical solution temperatures (UCST) below 5 oC in phosphate buffered saline whereas the insoluble form exhibited a UCST around 28-31 oC at pH 3.
The particle size of the insoluble form of CGP was around 300 – 500 nm as revealed by transmission electron microscopy.
Abstract
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Cyanophycin, also known as cyanophycin granule polypeptide (CGP), is a non-ribosomal polypeptide consisting of aspartic acid as a backbone with arginine and lysine as the side chains. CGP has soluble (sCGP) and insoluble (iCGP) forms based on its aqueous solubility. In order to investigate the role of lysine in its physical properties, CGP was prepared with recombinant Escherichia coli cultivated at different temperatures, and the purified sCGP and iCGP were further fractionated with different ethanol concentrations and pHs, respectively. Low temperature cultivation was found to favor the production of more sCGP. The ratio of iCGP/sCGP increased from 0.12 to 0.35 when the cultivation temperature being raised from 17 to 37 oC. After fractionation the same fraction of either sCGP or iCGP contained an approximate content of lysine, and therefore showed expectantly similar physical properties, irrespective of the cultivation temperatures. A high arginine/lysine ratio was found to result in low solubility and high molecular weight. Fractions of sCGP showed upper critical solution temperatures (UCST) below 5 oC in phosphate buffered saline whereas iCGP exhibited a UCST around 28-31 oC at pH 3. The particle size of iCGP was around 300 – 500 nm as revealed by transmission electron microscopy. The thermal responses of CGP present a potential in biomedical applications, such as drug delivery.
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Keywords: cyanophycin; upper critical solution temperature (UCST); biopolymer; polypeptide
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Introduction Cyanophycin, multi-L-arginyl-poly-L-apspartic acid, is a non-ribosomal polypeptide which was originally found as granules of inclusion body in cyanobacteria under nutrient limitations, and also named as cyanophycin granule polypeptide (CGP) (Allen et al., 1980; Mackerras et al., 1990; Stevens and Paone, 1981). Its structure contains a backbone of poly(aspartate) which β-carboxylic group is linked to the amine group of arginine also by a peptide bond (Simon et al., 1980; Simon and Weathers, 1976). Unlike most proteins, the synthesis of CGP is directed by a single enzyme, cyanophycin synthetase, not by ribosomes (Hai et al., 1999; Simon, 1976; Ziegler et al., 1998). CGP can be produced by recombinant strains when the gene coding CGP synthetase is introduced with an expression vector (Aboulmagd et al., 2000; Frey et al., 2002; Tseng et al., 2012; Ziegler et al., 2002; Ziegler et al., 1998).
Some previous studies have proposed the applications of CGP. The peptides from CGP after partial digestion can serve as nutrition supplements (Obst and Steinbüchel, 2004; Sallam et al., 2009; Solaiman et al., 2011), and complete digestion of CGP can be employed for the preparation of arginine (Shin and Lee, 2014). Recently, the film formed by crosslinked CGP has been shown to be a biocompatible material with minimal activation of macrophages, suggesting its potential use in biomedical applications (Tseng et al., 2016).
Compared with the typical CGP isolated from cyanobacteria, the recombinant CGP has a lower molecular weight with a narrower polydispersity, and sometimes possesses a low
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percentage of lysine, around 10% of the total amino acids, instead of arginine as the side chain (Steinle et al., 2008). In addition to lysine, metabolic engineering could also facilitate the incorporation of other amino acids with a structure similar to arginine, such as ornithine and citrulline, by changing the metabolisms with genetic manipulation (Steinle et al., 2008; Steinle et al., 2010). Under certain culture conditions, a high lysine content up to around 25% of total amino acids has been found without the need of metabolic engineering (Tseng et al., 2012). Original CGP with arginine as the side chain is insoluble at the physiological condition, and becomes soluble in an acidic condition of pH as low as 2 or in an alkali condition of pH above 9 (Lang et al., 1972). When the substitution of lysine occurs, the recombinant CGP becomes soluble (Wiefel and Steinbüchel, 2014). Therefore, the recombinant CGP can be classified as soluble (sCGP) and insoluble (iCGP) forms according to the solubility in aqueous solution at physiological pH. On the other hand, a previous study has shown an increase in the yield of CGP during purification by raising the purification temperature, presumably due to the enhanced solubility at a high temperature (Steinle and Steinbüchel, 2010).
The changes in solubility at different pH values and temperatures suggest that CGP might characteristically possess pH and thermal responses. Because the incorporation of lysine will lead to the changes in solubility, understanding the role of lysine can be achieved through investigating a quantitative correlation of lysine contents with both thermal and pH responses of CGP. However, unlike most synthetic polymers which defined structures can be altered by
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polymerization reaction, the lysine levels of CGP might result from random substitutions of arginine under the enzymatic reaction of CGP synthetase, and therefore lack a precise control during the bacteria cultivation. In this study, different pH values and ethanol concentrations were employed to fractionate CGP according to its solubility, and the amino acid contents of each fraction were examined as well as the property of thermal responses. First, CGP was prepared with recombinant Escherichia coli cultured at different temperatures to investigate the effect of temperature on the production ratio of iCGP to sCGP. After purification, both insoluble and soluble forms were fractionated in the presence of different pH values and ethanol concentrations, respectively. Each fraction was subjected to amino acid analysis in order to determine the amino acid compositions, and the solubility was also measured at different pH values. Thermal responses were examined by the changes in turbidity with temperature. The size of CGP agglomerate was observed under transmission electron microscopy.
Materials and Methods
Materials
Isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin, and chloramphenicol was obtained from Ameresco (Solon, OH). Bacto yeast extract and tryptone were from BectonDickinson (Franklin Lakes, NJ). Phenylisothiocyanate (PITC) and amino acid standards were from Pierce (Rockford, IL). Other chemicals were from Sigma-Aldrich (St. Louis, MO) and
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were used as received. Water was deionized by a Milli-Q water purification system (Bedford, MA).
Preparation of CGP
Recombinant CGP was prepared according to the method as previously described (Tseng et al., 2012). Briefly, Escherichia coli BL21-CodonPlus(DE3)-RIL (Agilent/Stratagene, Santa Clara, California) harboring CGP synthetase gene cphA on pET21b was grown at 17, 22, 27, 32, and 37 oC, respectively, in the terrific medium which consisted of 12 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 2.31 g/L KH2PO4 and 12.54 g/L K2HPO4 supplemented with 100 μg/mL ampicillin plus 34 μg/mL chloramphenicol. When the cell density reached 3-4 absorbance at 600 nm, IPTG was added at a final concentration of 1 mM to the culture for the induction of cphA expression. After further cultivation to the period when the cell density remained almost unchanged within 2 h, the cells were harvested, and the CGP was purified by acid hydrolysis as follows. The harvested cells were disrupted in 0.1 N HCl under constant stirring for 12 h at 40 oC followed by centrifugation at 12500 xg for 30 min to remove the cell debris. After the cell lysate was titrated to pH 7, the precipitate obtained by centrifugation is iCGP, and the supernatant was further mixed with ethanol at a final concentration of 70% (v/v) to form precipitates of sCGP. Both forms were lyophilized prior to further processing.
SDS-PAGE analysis of CGP synthetase
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A small portion of the harvested cells cultured at each different temperatures was employed to analyze the expression of CGP synthetase. The cells were disrupted by the BugBuster protein extraction reagent instead of acid hydrolysis. After centrifugation both debris pellet and supernatant lysate were analyzed on SDS-PAGE.
Fractionation of purified CGP
Because the iCGP was harvested at pH 7 and sCGP was precipitated with 70% ethanol, pH values from 2 - 7 and ethanol concentrations from 15% - 75% (v/v) were employed to subdivide the purified sCGP and iCGP mainly based on solubility, respectively. One gram of sCGP was first redissolved in deionized water at a concentration of 5 g/L. Then a calculated amount of 95% ethanol was added drop-wise to the desired final concentrations of 15%, 30%, 45%, 60%, and 75% (v/v). At the conclusion of each desired ethanol concentration, the precipitate was collected after centrifugation at 12500 xg for 30 min. Ethanol was subsequently added to the remaining supernatant till a next desired concentration was reached. Again, the precipitate was separated by centrifugation from the supernatant to which ethanol was added for further fractionation. The fractionation procedures were repeated until a final concentration 75% was reached. For iCGP, 0.5 g was completely redissolved in 0.1 N HCl at a concentration of 20 g/L. A concentration of 5 N NaOH was added drop-wise to the mixture till the desired pH value was reached. Similar to the procedures of ethanol fractionation of soluble form, the precipitate was collected by centrifugation, and the supernatant was further titrated with NaOH
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to the next desired pH value. The procedures were carried out repeatedly till pH reached 7. All the solid of each fraction was weighed after lyophilization.
Determination of amino acid compositions
The amino acid compositions of each fractionated CGP was analyzed by a method using post derivation with PITC as previously described (Tseng et al., 2012). Briefly, CGP was hydrolyzed at 108 oC in 6 N HCl, the hydrolyzed amino acids were then reacted with PITC, and analyzed with a Hitachi HPLC L-2100 system equipped with an Inertsil ODS-2 C-18 column (GL Sciences, Shinjuku, Tokyo, Japan) and a UV/VIS detector. Calibration curves were established using amino acid standards.
Measurement of molecular weight
The molecular weight distribution of sCGP was determined by gel permeation chromatography (GPC) equipped with Ultrahydrogel columns (Waters, Milford, MA) and a refractive index detector. Each fraction of sCGP was dissolved in water at a concentration of 0.5 mg/mL for analysis, and pullulan was used as calibration standards.
Measurement of solubility
Five mg of iCGP or sCGP was dissolved in 5 mL 0.1 M buffer solution at different pHs (KCl-HCl: pH 2; citrate-phosphate: pH 3, 4, 5.5; phosphate: pH 6, 8; PBS: pH 7.4; borate: pH 9, 10, 11; KCl-NaOH: pH 12, 13). After vortexing for 5 min at room temperature, the sample
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was centrifuged at 1000 xg for 3 min. When no precipitate was found, another 5 mg was added, and the procedures of vortexing and centrifugation were repeated until the appearance of precipitates.
Transmission Electron Microscopy Analysis
One mg of iCGP was dissolved in 0.5% phosphotungstic acid which pH was adjusted to 3 with 1 N NaOH. The solution was sonicated for 10 min, and then 20 μL was applied onto the copper mesh. After 30 s, the excessive liquid was wiped off. After three repeats, the mesh was stored at 40 oC incubator until observation under a transmission electron microscope (JEOL JEM-1200EX II) the next day.
Measurement of thermal response
The thermal responses were carried out for 10 mg/mL of sCGP in phosphate saline buffer (PBS, pH=7.4) and 2.5 mg/mL of iCGP in 0.1M citrate-phosphate buffer (pH =3.0), respectively. The changes in turbidity were monitored with either a heating rate or a cooling rate of 1 oC/min by a spectrophotometer (UV-750, Jasco, Tokyo, Japan) equipped with a stirring unit, a piezo heating unit, and a cooling water bath. The temperature at which the solution exhibits 50% relative turbidity is defined as UCST (upper critical solution temperature).
Results
Effect of cultivation temperature on CGP synthesis
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A previous study showed the selection of an E. coli host and an IPTG concentration for CGP preparation, but lacks an analysis of how cultivation temperature affects the iCGP/sCGP ratio (Tseng et al., 2012). In this study, the recombinant CGP purified from E. coli exhibited different ratios of soluble and insoluble forms at different culture temperatures as shown in Fig. 1A. The iCGP/sCGP ratio increased to 0.45 at a high temperature of 37 oC whereas sCGP was found dominant with an iCGP/sCGP ratio of 0.13 at a low temperature of 17 oC. In the temperature range of 22 – 32 oC, the ratio of iCGP/sCGP stayed around 0.35. Cultivation temperature also affected the volumetric yield of CGP production. At 17 oC the yield was as low as 0.32 g/L, then increased to around 1.1 g/L at 22 oC, and peaked at 1.5 g/L at 27 oC. Further increases in temperature caused the yield to drop slowly to 0.84 g/L at 32 oC, and then to 0.61 g/L at 37 oC.
A portion of the bacteria at each temperature was treated with the BugBuster reagent which could disrupt the bacteria in a mild way to release the contents of cytoplasm but not strong enough to solubilize the inclusion bodies. Both the supernatant lysates and debris pellets after the treatment were analyzed by SDS-PAGE (Fig. 1B). Most of the CGP was found to exist in the cell debris, indicating that CGP could not be solubilized by the buffer. However, the expressed CGP synthetase had different distributions between the supernatant and the pellet. At lower cultivation temperatures, more amounts of the soluble enzyme, CGP synthetase, were noted, consistent with other observations of E. coli culture at low cultivation temperatures that
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could slow down the formation of inclusion body (Vera et al., 2007 ). Because most enzymes in the inclusion body are misfolded and tend to lose biological functions due to their incorrect configurations, the CGP synthetase in the inclusion body of the culture at high temperatures might be unable to participate CGP production. Thus, less amounts of enzyme remained soluble in the cytoplasm for the active function, leading to the decreases in CGP yield. On the other hand, at a lower temperature, the enzyme seems to have a loose substrate specificity which allows the incorporation of more lysine instead of arginine into the side chain during synthesis. As a result, a higher ratio of soluble form was produced with a high content of lysine. When the temperature increased, the substrate specificity seemingly becomes tighter, and more arginine and less lysine are incorporated. Thus, a higher ratio of insoluble form was observed.
Fractionation of CGP
After purification, the iCGP was further fractionated at different pH values of 3, 4, 5, 6, and 7 except that obtained at temperature 17 oC which had insufficient amounts for processing. As shown in Fig 2A, only two major fractions at pH of 4 and 5 weighed significantly among all the fractions, and the amounts of other fractions totaled less than 10%. Interestingly, the portions of the two major fractions appeared to almost the same percentage at different cultivation temperatures, and the fraction at pH 5 is about twice the amount at pH 4.
Meanwhile, various ethanol concentrations were used to fractionate the sCGP. In the range between 15% and 75% ethanol, the portions of soluble form displayed a skewed distribution
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toward low ethanol concentrations (Fig. 2B). Some fair and approximate amounts of sCGP, more than 20% of the total, were, respectively, fractionated into 15%, 30%, and 45% ethanol for the cultivation temperatures from 17 to 32 oC except that at 37 oC under which condition almost 50% of soluble form was exclusively fractionated into 15% ethanol. For all cultivation temperatures, a slightly higher amount of sCGP appeared at a low ethanol concentration, the amounts of fractionated sCGP slightly decreased as the ethanol concentration increased, and less than 5% of the total amount was fractionated into 75% ethanol.
Amino acid analysis of the fractionated CGP
Further analysis of amino acid compositions revealed that the ratio of arginine to lysine differs in each fractionated CGP as shown in Fig. 3. For iCGP, a higher content of arginine was present in the fraction that formed precipitates at pH 4 than that at pH 5. Before fractionation proceeded, the purified iCGP was first redissolved at pH 1, and then pH was raised stepwise to 7. The collected precipitates at pH 4 and pH 5 were completely soluble below pH 3, and precipitation occurred when pH increased to 4, indicating that the fraction at pH 4 was soluble at pH 3 but not at pH 4, and similarly that the fraction at pH 5 was soluble at pH 4 but not at pH 5. Namely, a high content of arginine required a more acidic condition for dissolution (Fig. 3A). As for ethanol fractionation of sCGP, the environment of a low ethanol concentration was similar to that of aqueous solution of pH 7 under which condition the iCGP was prepared from the cell disruption. Comparably high contents of arginine were also detected in those fractions
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that form precipitates at low ethanol concentrations (Fig. 3B), suggesting that sCGP fractionated at a lower ethanol concentration had a lower solubility in water. The Arg/Lys ratio then follows a decreasing trend with the increasing ethanol concentrations for all fractions. It was noted that the fractions by the same ethanol concentration have a similar Arg/Lys ratio irrespective of different cultivation temperatures.
When the fractions of sCGP were subjected to the analysis of molecular weight distribution with GPC, it was found that the one with a higher arginine content, as fractionated at a lower ethanol concentration, also appeared to have a higher molecular weight as shown in Fig. 4A. A positive correlation was further observed for the Arg/Lys ratio and the molecular weight of each fraction, and such correlation was independent of the cultivation temperature (Fig. 4B). The molecular weight of sCGP ranges from 18 to around 30 kD, and a high molecular weight was accompanied with a high Arg/Lys ratio which was fractionated in a low ethanol concentration. The results indicated that both Arg/Lys ratio and molecular weight distribution affect the solubility of CGP.
Solubility of recombinant CGP
In an attempt to examine the effect of pH on solubility, the CGP purified from the bacterial culture at 27 oC was used before fractionation proceeded. In the pH range from 2 to 12, sCGP exhibited a higher solubility than iCGP at each corresponding pH values as shown in Fig. 5A. However, both forms have different pH dependency. The solubility of sCGP decreased with
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increasing pH from 110 mg/mL at pH 2, sharply dropped to 27 mg/mL at pH 3, and then gradually decreased to below 1 mg/mL at pH 12. The iCGP had a lower solubility of around 10 mg/mL at pH 2, then stayed below 1 mg/mL, hardly soluble from pH 4 to 11, but slightly increased to 1.7 mg/mL at pH 12 and 18 mg/mL at pH 13. Such dependence of solubility and pH behaved differently in comparison with a previous study which indicated CGP became soluble at a low pH 2 or a high pH 9 (Lang et al., 1972). A further examination into the solubility of each fraction at pH 3, 7.4, and 11 expectantly revealed that the solubility is a function of Arg/Lys ratio (Fig. 5B). The higher ratio of Arg/Lys the fraction exhibited, the less soluble the CGP became. For the three pH values of 3, 7.4, and 11, a rough linear trend was observed between the solubility and the Arg/Lys ratio for sCGP.
Thermal responses of CGP
As Fig. 6A depicted the thermal response of each fraction of sCGP at physiological pH during heating, an abrupt change in turbidity at a low temperature below 5 oC was observed, and then the solution stayed clear until a higher temperature around 55 - 60 oC was reached. Each fraction of the sCGP showed dual phase transition temperatures in PBS. Below 5 oC, a UCST, the critical temperature at which a solution becomes miscible, was found to associate with the content of arginine, and the fraction with a higher ratio of Arg/Lys had a slightly higher UCST. When the temperature exceeded the second phase change temperature around 55 – 60 o
C, formation of fibril agglomerates began, and the agglomerates settled down after leaving
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undisturbed for a period of time. A subsequent cooling process was performed in an attempt to redissolve the agglomerates (Fig. 6B). Surprisingly no phenomena of hysteresis was observed, and the fibril agglomerates remained even after further incubation at 4 oC or room temperature up to 96 h. However, when the heating temperature was limited to 45 oC, below the second phase change temperature, hysteresis were observed for each fraction of sCGP during the cooling process (Fig. 6C), and the clear solution turned cloudy again when the temperature was dropped below 5 oC, reaching its UCST.
On the other hand, iCGP, which became soluble at acidic conditions, exhibited only one phase change temperature at pH 3 in 0.1M citrate phosphate buffer (Fig. 7A). In contrast to the abrupt phase change of sCGP, the changes in turbidity seemed gradual over a wide temperature range from 15 to 40 oC. When the heating temperature was raised to above the denaturing temperature of the soluble form, no fibril formation appeared, and hysteresis was observed during a subsequent cooling process. Similar to the soluble form which UCST depended on the Arg/Lys ratio, the fractions of pH 4 and 5 showed UCSTs at 31 and 28 oC, respectively, indicating that a higher ratio of Arg/Lys resulted in a higher UCST. When examined under TEM, the insoluble agglomerates of both fractions seemed to be compact aggregates without a hollow center. The fraction of pH 4 showed a size of around 300 nm (Fig. 7B2) whereas the fraction of pH 5 had a larger size of around 400 nm (Fig. 7B4).
Discussion
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This study employed different ethanol concentrations and pHs to fractionate CGP which was prepared at different cultivation temperatures. The fractionation approach employed in this study revealed the role of lysine content in the solubility of CGP and in the distribution of molecular weight of CGP quantitatively as well as in the thermal responses of CGP. The CGP fractionated at the same condition, such as the same ethanol concentration or the same pH, has an approximate lysine content irrespective of the cultivation temperatures, and therefore presents similar properties which can be expressed as a function of Arg/Lys ratio. A previous report showed that the incorporation of lysine plays a key role in the synthesis of sCGP by examining different strains carrying different cphA genes (Wiefel and Steinbüchel, 2014). The results of this study demonstrated that the role of lysine not only in the solubility but also in determining other properties of CGP, such as the thermal responses.
In the past some polypeptides have shown phase transition, mostly LCST, for example elastin like peptides containing repeats of valine-proline-glycine-X-proline where X is a nonproline amino acid (Rodríguez-Cabello et al., 2006), ethyl and butyl modified poly(glycine) (Lahasky et al., 2012), poly(N-substituted α/β-asparagine)s (Tachibana et al., 2003), and polyethylene glycol modified poly(glutamic acid) (Chen et al., 2011). Polypeptides with a UCST property have been rarely reported. In this study both sCGP and iCGP were found to exhibit UCST when the solution was subjected to heating. At the physiological condition, the thermal response of sCGP occurred at a low temperature below 5 oC whereas that of iCGP was
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hardly observed in PBS due to its limited solubility. When the heating process continued to around 55 – 60 oC, sCGP underwent a second phase change similar to LCST (lower critical solution temperature), the critical temperature when the immiscibility of solution occurs. However, hysteresis was not observed during the process of cooling the heated sCGP because of the formation of fibril agglomerates. The irreversible denaturation of sCGP might be due to the dismantling of intramolecular hydrogen bonds. On the other hand, a second phase change did not occur for iCGP when heating temperature was raised to 65 oC, and the hysteresis of UCST was observed during the heating and cooling processes of the iCGP at an acidic condition of pH 3. The phase change of iCGP spanned a wider temperature range as compared with the abrupt change of the sCGP.
The lack of hydrophobic moiety on CGP might be unable to prompt the formation of micelles that generally result from amphipathic molecules. Hydrogen bonding was attributed to the phase transition of UCST of several polymers (Seuring and Agarwal, 2012; Shimada et al., 2013). The CGP agglomerates might form through hydrogen bonding between the arginine side chains. From a viewpoint of molecular structure, CGP could be viewed as a slightly twisted string along with its side chains, arginine and lysine, alternating on the outer. Some intermolecular hydrogen bonding might be bridged with the guanidino group of arginine. When the arginine is substituted by lysine, less nitrogen atoms could diminish the hydrogen bonding, thus increasing the solubility of CGP at a moderate temperature. More lysine substitutions, as
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indicated by low Arg/Lys ratios, further weaken the hydrogen bonding, thus lowering UCST, which was observed in the fractions of soluble and insoluble forms. The iCGP possesses a higher content of arginine and the slow dissociation and annealing of the numerous hydrogen bonds presumably caused the gradual phase changes during either the heating or cooling process as suggested by the observations of UCST spanning from 15 to 40 oC. However, the high content of lysine of sCGP is prone to denaturation, similar to protein denaturation, at a high temperature over 55 oC at which solution began to turn from clear to cloudy similar to the indication of LCST but resulted in an irreversible phase change of fibril formation.
On the other hand, both the amine group of lysine and the guanidino group of arginine are ionized with a pKa of 10.5 and 12.5, respectively. Therefore the more substitution of arginine with lysine could lower the isoelectric point of CGP, presumably increasing the solubility at pH 7. The extent of ionization at low pH might bring water to bridge the hydrogen bonding between CGP molecules, and could be ascribed to the dependence of solubility on pH. Altogether the pH and thermal responses make CGP possess dual pH and temperature sensitive properties.
More lysine molecules were found in the CGP obtained from cultivation at a low temperature which also allowed the production of more soluble CGP synthetase to direct the CGP synthesis. On the other hand, at a high cultivation temperature, less amount of soluble enzyme could produce an approximate amount of CGP, suggesting that the enzyme was less
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active at a low temperature. Thus a high yield of cyanophycin exists around 27 – 30 oC when a sufficient amount of soluble enzyme operates with a moderate activity. The inefficiency of CGP synthetase at low temperature might cause less substrate specificity to allow the use of lysine instead of arginine as substrate, resulting in a low ratio of iCGP/sCGP.
In conclusion, this study showed that using cultivation temperature can modulate the proportions of soluble and insoluble CGP, and correlated the lysine content with the solubility, molecular weight, and thermal response of CGP in a quantitative way. The characteristic of thermal and pH responses can be expected in biomedical applications, such as drug delivery system.
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Aboulmagd, E., Oppermann-Sanio, F.B., Steinbüchel, A., (2000) Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Arch Microbiol 174, 297-306. Allen, M.M., Hutchison, F., Weathers, P.J., (1980) Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308. J Bacteriol 141, 687-693. Chen, C., Wang, Z., Li, Z., (2011) Thermoresponsive polypeptides from pegylated polyL-glutamates. Biomacromolecules 12, 2859-2863. Frey, K.M., Oppermann-Sanio, F.B., Schmidt, H., Steinbüchel, A., (2002) Technicalscale production of cyanophycin with recombinant strains of Escherichia coli. Appl Environ Microbiol 68, 3377-3384. Hai, T., Oppermann-Sanio, F.B., Steinbüchel, A., (1999) Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19. FEMS Microbiol Lett 181, 229-236. Lahasky, S.H., Hu, X., Zhang, D., (2012) Thermoresponsive Poly(α-peptoid)s: Tuning the Cloud Point Temperatures by Composition and Architecture. ACS Macro Lett. 1, 580–584. Lang, N.J., Simon, R.D., Wolk, C.P., (1972) Correspondence of cyanophycin granules with structuredgranules in Anabaena cylindrical. Arch Microbiol 83, 313–320. Mackerras, A.H., De Chazal, N.M., Smith, G.D., (1990) Transient accumulation of cyanophycin in Anabaena cylindrica and Synechocystis 6308. J Gen Microbiol 136, 2057-2065. Obst, M., Steinbüchel, A., (2004) Microbial degradation of poly(amino acid)s. Biomacromolecules 5, 1166-1176. Rodríguez-Cabello, J.C., Reguera, J., Girotti, A., Arias, F.J., Alonso, M., (2006) Genetic engineering of protein-based polymers: The example of elastinlike polymers. Ordered Polymeric Nanostructures at Surfaces. Springer, pp. 119-167. Sallam, A., Kast, A., Przybilla, S., Meiswinkel, T., Steinbüchel, A., (2009) Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization. Appl Environ Microbiol 75, 29-38. Seuring, J., Agarwal, S., (2012) Polymers with upper critical solution temperature in aqueous solution. Macromol Rapid Comm 33, 1898-1920. Shimada, N., Nakayama, M., Kano, A., Maruyama, A., (2013) Design of UCST
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polymers for chilling capture of proteins. Biomacromolecules 14, 1452-1457. Shin, J.H., Lee, S.Y., (2014) Metabolic engineering of microorganisms for the production of L-arginine and its derivatives. Microb Cell Fact 13, 166. Simon, R.D., (1976) The biosynthesis of multi-l-arginy-poly(l-aspartic acid ) in the filamentous cyanobacterium Anabaena cylindrica. Biochim Biophys Acta 422, 407418. Simon, R.D., Lawry, N.H., McLendon, G.L., (1980) Structural characterization of the cyanophycin granule polypeptide of Anabaena cylindrica by circular dichroism and Raman spectroscopy. Biochim Biophys Acta 626, 277-281. Simon, R.D., Weathers, P., (1976) Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in Cyanobacteria. Biochim Biophys Acta 420, 165-176. Solaiman, D.K., Garcia, R.A., Ashby, R.D., Piazza, G.J., Steinbüchel, A., (2011) Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer. N Biotechnol 28, 552-558. Steinle, A., Oppermann-Sanio, F.B., Reichelt, R., Steinbüchel, A., (2008) Synthesis and accumulation of cyanophycin in transgenic strains of Saccharomyces cerevisiae. Appl Environ Microbiol 74, 3410-3418. Steinle, A., Steinbüchel, A., (2010) Establishment of a simple and effective isolation method for cyanophycin from recombinant Saccharomyces cerevisiae. Appl Microbiol Biotechnol 85, 1393-1399. Steinle, A., Witthoff, S., Krause, J.P., Steinbüchel, A., (2010) Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases. Appl Environ Microbiol 76, 1062-1070. Stevens, S.E., Paone, D.A., (1981) Accumulation of Cyanophycin Granules as a Result of Phosphate Limitation in Agmenellum quadruplicatum. Plant Physiology 67, 716719. Tachibana, Y., Kurisawa, M., Uyama, H., Kakuchic, T., Kobayashi, S., (2003) Biodegradable thermoresponsive poly(amino acid)s. Chem. Commun, 106-107. Tseng, W.-C., Fang, T.-Y., Chen, S.-Y., (2016) Cellular biocompatibility of cyanophycin substratum prepared with recombinant Escherichia coli. Biochem Eng J 105, 97-106. Tseng, W.-C., Fang, T.-Y., Cho, C.-Y., Chen, P.-S., Tsai, C.S., (2012) Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene. Biotechnol Prog 28, 358-363. Vera, A., González-Montalbán, N., Arís, A., Villaverde, A., (2007) The conformational
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quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng 96, 1101-1106. Wiefel, L., Steinbüchel, A., (2014) Solubility behavior of cyanophycin depending on lysine content. Appl Environ Microbiol 80, 1091-1096. Ziegler, K., Deutzmann, R., Lockau, W., (2002) Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense. Z Naturforsch C 57, 522-529. Ziegler, K., Diener, A., Herpin, C., Richter, R., Deutzmann, R., Lockau, W., (1998) Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). Eur J Biochem 254, 154-159.
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Figure Legends
Figure 1. Effect of cultivation temperature on CGP yield and expression of CGP synthetase
(A) the volumetric yield of CGP (●) and the ratio of iCGP and sCGP (■) obtained after purification from the recombinant E. coli cultivated at different temperatures. The arrows indicate the readings of the data on the corresponding axes. (mean ± S.D.; n=3 independent cultivation) (B) the SDS-PAGE of the recombinant E. coli treated with the BugBuster reagent. (M: protein markers; L and P: the cell lysate and cell pellet after centrifugation of the treated bacteria, respectively)
Figure 2. Distribution of iCGP and sCGP after fractionation
CGP was purified from the recombinant E. coli cultivated at different temperatures (▬:17 oC; ▬:22 oC; ▬:27 oC; ▬:32 oC; ▬:37 oC). Fractionation of the purified CGP was performed with different pHs for iCGP and with different ethanol concentrations for sCGP. (A) the distributions of the fractions at different pH values, (B) the distributions of the fractions at different ethanol concentrations. (mean ± S.D.; n=3 independent cultivation)
Figure 3. Amino acid compositions of the fractionated CGP
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Each fractionated CGP was subject to amino acid analysis. (A) the ratio Arg/Lys of each fraction of iCGP, (B) the ratio Arg/Lys of each fraction of sCGP. The CGP was obtained from cultivation at different temperatures (▬:17 oC; ▬:22 oC; ▬:27 oC; ▬:32 oC; ▬:37 oC) (mean ± S.D.; n=3 independent cultivation)
Figure 4. Molecular weight distribution of the fractionated sCGP
(A) a typical elution profile of sCGP fractionated at different ethanol concentrations (▬:15% ethanol; ▬:30% ethanol; ▬:45% ethanol; ▬:60% ethanol; ▬:75%), (B) the dependence of the average molecular weight and the Arg/Lys ratio of the fractionated sCGP from different cultivation temperatures (▬:17 oC; ▬:22 oC; ▬:27 oC; ▬:32 oC; ▬:37 oC).
Figure 5. Dependence of solubility on pH
(A) the dependence of pH and the solubility of sCGP (▬) and iCGP (▬) in different buffers, (B) the solubility of CGP as a function of Arg/Lys ratio in three different buffers (▬: citratephosphate at pH 3; ▬: PBS at pH 7.4; ▬: borate buffer at pH 11).
Figure 6. Thermal response of fractionated sCGP
(A) the changes in the turbidity of sCGP fractionated at different ethanol concentrations (▬:15% ethanol; ▬:30% ethanol; ▬:45% ethanol; ▬:60% ethanol; ▬:75%) when the solution was heated at 1 oC/min under stirring. The images showed the solution at 4oC, 37oC, and 65oC, respectively. (B) the changes in turbidity when the solution was subsequently cooled
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at 1 oC/min under stirring after being heated to 67 oC, (C) the changes in turbidity when the solution was subsequently cooled at 1 oC/min under stirring after being heated to 45 oC. The arrows indicate the thermal heating (in red) or cooling (in blue) along with temperature.
Figure 7. Thermal response and TEM image of fractionated iCGP
(A) the changes in the turbidity of iCGP fractionated at different pHs (▬: pH 5; ▬: pH 4) when the solution was heated at 1 oC/min (solid lines) and then cooled at 1 oC/min (dashed lines) under stirring at pH 3. The images showed the solution of iCGP fractionated at pH 4 at 15oC and 50oC, respectively. The arrows indicate the thermal heating (in red) or cooling (in blue) along with temperature. (B) the TEM images of iCGP fractionated at pH 5 (B1 and B2) and at pH 4 (B3 and B4).
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S0168-1656(17)30155-4 http://dx.doi.org/doi:10.1016/j.jbiotec.2017.04.001 BIOTEC 7844
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
25-1-2017 22-3-2017 1-4-2017
Please cite this article as: Tseng, Wen-Chi, Fang, Tsuei-Yun, Hsieh, Yuan-Chieh, Chen, Chien-Yu, Li, Meng-Che, Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2017.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli Wen-Chi Tseng*1, Tsuei-Yun Fang**2, Yuan-Chieh Hsieh1, Chien-Yu Chen 1, Meng-Che Li 1 1
Department of Chemical Engineering, National Taiwan University of Science and
Technology, Taipei, Taiwan; 2Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan *Corresponding author: Wen-Chi Tseng Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan E-Mail: [email protected] Fax: 886-2-2462-2586 Tel: 886-2-2730-1078 ** Corresponding author: Tsuei-Yun Fang Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung 202, Taiwan E-Mail: [email protected] Fax: 886-2-2462-2192 Tel: 886-2-2462-2586 Highlights
Low temperature cultivation was found to favor the production of the soluble form of cyanophycin.
The soluble form of cyanophycin showed upper critical solution temperatures (UCST) below 5 oC in phosphate buffered saline whereas the insoluble form exhibited a UCST around 28-31 oC at pH 3.
The particle size of the insoluble form of CGP was around 300 – 500 nm as revealed by transmission electron microscopy.
Abstract
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Cyanophycin, also known as cyanophycin granule polypeptide (CGP), is a non-ribosomal polypeptide consisting of aspartic acid as a backbone with arginine and lysine as the side chains. CGP has soluble (sCGP) and insoluble (iCGP) forms based on its aqueous solubility. In order to investigate the role of lysine in its physical properties, CGP was prepared with recombinant Escherichia coli cultivated at different temperatures, and the purified sCGP and iCGP were further fractionated with different ethanol concentrations and pHs, respectively. Low temperature cultivation was found to favor the production of more sCGP. The ratio of iCGP/sCGP increased from 0.12 to 0.35 when the cultivation temperature being raised from 17 to 37 oC. After fractionation the same fraction of either sCGP or iCGP contained an approximate content of lysine, and therefore showed expectantly similar physical properties, irrespective of the cultivation temperatures. A high arginine/lysine ratio was found to result in low solubility and high molecular weight. Fractions of sCGP showed upper critical solution temperatures (UCST) below 5 oC in phosphate buffered saline whereas iCGP exhibited a UCST around 28-31 oC at pH 3. The particle size of iCGP was around 300 – 500 nm as revealed by transmission electron microscopy. The thermal responses of CGP present a potential in biomedical applications, such as drug delivery.
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Keywords: cyanophycin; upper critical solution temperature (UCST); biopolymer; polypeptide
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Introduction Cyanophycin, multi-L-arginyl-poly-L-apspartic acid, is a non-ribosomal polypeptide which was originally found as granules of inclusion body in cyanobacteria under nutrient limitations, and also named as cyanophycin granule polypeptide (CGP) (Allen et al., 1980; Mackerras et al., 1990; Stevens and Paone, 1981). Its structure contains a backbone of poly(aspartate) which β-carboxylic group is linked to the amine group of arginine also by a peptide bond (Simon et al., 1980; Simon and Weathers, 1976). Unlike most proteins, the synthesis of CGP is directed by a single enzyme, cyanophycin synthetase, not by ribosomes (Hai et al., 1999; Simon, 1976; Ziegler et al., 1998). CGP can be produced by recombinant strains when the gene coding CGP synthetase is introduced with an expression vector (Aboulmagd et al., 2000; Frey et al., 2002; Tseng et al., 2012; Ziegler et al., 2002; Ziegler et al., 1998).
Some previous studies have proposed the applications of CGP. The peptides from CGP after partial digestion can serve as nutrition supplements (Obst and Steinbüchel, 2004; Sallam et al., 2009; Solaiman et al., 2011), and complete digestion of CGP can be employed for the preparation of arginine (Shin and Lee, 2014). Recently, the film formed by crosslinked CGP has been shown to be a biocompatible material with minimal activation of macrophages, suggesting its potential use in biomedical applications (Tseng et al., 2016).
Compared with the typical CGP isolated from cyanobacteria, the recombinant CGP has a lower molecular weight with a narrower polydispersity, and sometimes possesses a low
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percentage of lysine, around 10% of the total amino acids, instead of arginine as the side chain (Steinle et al., 2008). In addition to lysine, metabolic engineering could also facilitate the incorporation of other amino acids with a structure similar to arginine, such as ornithine and citrulline, by changing the metabolisms with genetic manipulation (Steinle et al., 2008; Steinle et al., 2010). Under certain culture conditions, a high lysine content up to around 25% of total amino acids has been found without the need of metabolic engineering (Tseng et al., 2012). Original CGP with arginine as the side chain is insoluble at the physiological condition, and becomes soluble in an acidic condition of pH as low as 2 or in an alkali condition of pH above 9 (Lang et al., 1972). When the substitution of lysine occurs, the recombinant CGP becomes soluble (Wiefel and Steinbüchel, 2014). Therefore, the recombinant CGP can be classified as soluble (sCGP) and insoluble (iCGP) forms according to the solubility in aqueous solution at physiological pH. On the other hand, a previous study has shown an increase in the yield of CGP during purification by raising the purification temperature, presumably due to the enhanced solubility at a high temperature (Steinle and Steinbüchel, 2010).
The changes in solubility at different pH values and temperatures suggest that CGP might characteristically possess pH and thermal responses. Because the incorporation of lysine will lead to the changes in solubility, understanding the role of lysine can be achieved through investigating a quantitative correlation of lysine contents with both thermal and pH responses of CGP. However, unlike most synthetic polymers which defined structures can be altered by
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polymerization reaction, the lysine levels of CGP might result from random substitutions of arginine under the enzymatic reaction of CGP synthetase, and therefore lack a precise control during the bacteria cultivation. In this study, different pH values and ethanol concentrations were employed to fractionate CGP according to its solubility, and the amino acid contents of each fraction were examined as well as the property of thermal responses. First, CGP was prepared with recombinant Escherichia coli cultured at different temperatures to investigate the effect of temperature on the production ratio of iCGP to sCGP. After purification, both insoluble and soluble forms were fractionated in the presence of different pH values and ethanol concentrations, respectively. Each fraction was subjected to amino acid analysis in order to determine the amino acid compositions, and the solubility was also measured at different pH values. Thermal responses were examined by the changes in turbidity with temperature. The size of CGP agglomerate was observed under transmission electron microscopy.
Materials and Methods
Materials
Isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin, and chloramphenicol was obtained from Ameresco (Solon, OH). Bacto yeast extract and tryptone were from BectonDickinson (Franklin Lakes, NJ). Phenylisothiocyanate (PITC) and amino acid standards were from Pierce (Rockford, IL). Other chemicals were from Sigma-Aldrich (St. Louis, MO) and
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were used as received. Water was deionized by a Milli-Q water purification system (Bedford, MA).
Preparation of CGP
Recombinant CGP was prepared according to the method as previously described (Tseng et al., 2012). Briefly, Escherichia coli BL21-CodonPlus(DE3)-RIL (Agilent/Stratagene, Santa Clara, California) harboring CGP synthetase gene cphA on pET21b was grown at 17, 22, 27, 32, and 37 oC, respectively, in the terrific medium which consisted of 12 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 2.31 g/L KH2PO4 and 12.54 g/L K2HPO4 supplemented with 100 μg/mL ampicillin plus 34 μg/mL chloramphenicol. When the cell density reached 3-4 absorbance at 600 nm, IPTG was added at a final concentration of 1 mM to the culture for the induction of cphA expression. After further cultivation to the period when the cell density remained almost unchanged within 2 h, the cells were harvested, and the CGP was purified by acid hydrolysis as follows. The harvested cells were disrupted in 0.1 N HCl under constant stirring for 12 h at 40 oC followed by centrifugation at 12500 xg for 30 min to remove the cell debris. After the cell lysate was titrated to pH 7, the precipitate obtained by centrifugation is iCGP, and the supernatant was further mixed with ethanol at a final concentration of 70% (v/v) to form precipitates of sCGP. Both forms were lyophilized prior to further processing.
SDS-PAGE analysis of CGP synthetase
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A small portion of the harvested cells cultured at each different temperatures was employed to analyze the expression of CGP synthetase. The cells were disrupted by the BugBuster protein extraction reagent instead of acid hydrolysis. After centrifugation both debris pellet and supernatant lysate were analyzed on SDS-PAGE.
Fractionation of purified CGP
Because the iCGP was harvested at pH 7 and sCGP was precipitated with 70% ethanol, pH values from 2 - 7 and ethanol concentrations from 15% - 75% (v/v) were employed to subdivide the purified sCGP and iCGP mainly based on solubility, respectively. One gram of sCGP was first redissolved in deionized water at a concentration of 5 g/L. Then a calculated amount of 95% ethanol was added drop-wise to the desired final concentrations of 15%, 30%, 45%, 60%, and 75% (v/v). At the conclusion of each desired ethanol concentration, the precipitate was collected after centrifugation at 12500 xg for 30 min. Ethanol was subsequently added to the remaining supernatant till a next desired concentration was reached. Again, the precipitate was separated by centrifugation from the supernatant to which ethanol was added for further fractionation. The fractionation procedures were repeated until a final concentration 75% was reached. For iCGP, 0.5 g was completely redissolved in 0.1 N HCl at a concentration of 20 g/L. A concentration of 5 N NaOH was added drop-wise to the mixture till the desired pH value was reached. Similar to the procedures of ethanol fractionation of soluble form, the precipitate was collected by centrifugation, and the supernatant was further titrated with NaOH
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to the next desired pH value. The procedures were carried out repeatedly till pH reached 7. All the solid of each fraction was weighed after lyophilization.
Determination of amino acid compositions
The amino acid compositions of each fractionated CGP was analyzed by a method using post derivation with PITC as previously described (Tseng et al., 2012). Briefly, CGP was hydrolyzed at 108 oC in 6 N HCl, the hydrolyzed amino acids were then reacted with PITC, and analyzed with a Hitachi HPLC L-2100 system equipped with an Inertsil ODS-2 C-18 column (GL Sciences, Shinjuku, Tokyo, Japan) and a UV/VIS detector. Calibration curves were established using amino acid standards.
Measurement of molecular weight
The molecular weight distribution of sCGP was determined by gel permeation chromatography (GPC) equipped with Ultrahydrogel columns (Waters, Milford, MA) and a refractive index detector. Each fraction of sCGP was dissolved in water at a concentration of 0.5 mg/mL for analysis, and pullulan was used as calibration standards.
Measurement of solubility
Five mg of iCGP or sCGP was dissolved in 5 mL 0.1 M buffer solution at different pHs (KCl-HCl: pH 2; citrate-phosphate: pH 3, 4, 5.5; phosphate: pH 6, 8; PBS: pH 7.4; borate: pH 9, 10, 11; KCl-NaOH: pH 12, 13). After vortexing for 5 min at room temperature, the sample
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was centrifuged at 1000 xg for 3 min. When no precipitate was found, another 5 mg was added, and the procedures of vortexing and centrifugation were repeated until the appearance of precipitates.
Transmission Electron Microscopy Analysis
One mg of iCGP was dissolved in 0.5% phosphotungstic acid which pH was adjusted to 3 with 1 N NaOH. The solution was sonicated for 10 min, and then 20 μL was applied onto the copper mesh. After 30 s, the excessive liquid was wiped off. After three repeats, the mesh was stored at 40 oC incubator until observation under a transmission electron microscope (JEOL JEM-1200EX II) the next day.
Measurement of thermal response
The thermal responses were carried out for 10 mg/mL of sCGP in phosphate saline buffer (PBS, pH=7.4) and 2.5 mg/mL of iCGP in 0.1M citrate-phosphate buffer (pH =3.0), respectively. The changes in turbidity were monitored with either a heating rate or a cooling rate of 1 oC/min by a spectrophotometer (UV-750, Jasco, Tokyo, Japan) equipped with a stirring unit, a piezo heating unit, and a cooling water bath. The temperature at which the solution exhibits 50% relative turbidity is defined as UCST (upper critical solution temperature).
Results
Effect of cultivation temperature on CGP synthesis
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A previous study showed the selection of an E. coli host and an IPTG concentration for CGP preparation, but lacks an analysis of how cultivation temperature affects the iCGP/sCGP ratio (Tseng et al., 2012). In this study, the recombinant CGP purified from E. coli exhibited different ratios of soluble and insoluble forms at different culture temperatures as shown in Fig. 1A. The iCGP/sCGP ratio increased to 0.45 at a high temperature of 37 oC whereas sCGP was found dominant with an iCGP/sCGP ratio of 0.13 at a low temperature of 17 oC. In the temperature range of 22 – 32 oC, the ratio of iCGP/sCGP stayed around 0.35. Cultivation temperature also affected the volumetric yield of CGP production. At 17 oC the yield was as low as 0.32 g/L, then increased to around 1.1 g/L at 22 oC, and peaked at 1.5 g/L at 27 oC. Further increases in temperature caused the yield to drop slowly to 0.84 g/L at 32 oC, and then to 0.61 g/L at 37 oC.
A portion of the bacteria at each temperature was treated with the BugBuster reagent which could disrupt the bacteria in a mild way to release the contents of cytoplasm but not strong enough to solubilize the inclusion bodies. Both the supernatant lysates and debris pellets after the treatment were analyzed by SDS-PAGE (Fig. 1B). Most of the CGP was found to exist in the cell debris, indicating that CGP could not be solubilized by the buffer. However, the expressed CGP synthetase had different distributions between the supernatant and the pellet. At lower cultivation temperatures, more amounts of the soluble enzyme, CGP synthetase, were noted, consistent with other observations of E. coli culture at low cultivation temperatures that
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could slow down the formation of inclusion body (Vera et al., 2007 ). Because most enzymes in the inclusion body are misfolded and tend to lose biological functions due to their incorrect configurations, the CGP synthetase in the inclusion body of the culture at high temperatures might be unable to participate CGP production. Thus, less amounts of enzyme remained soluble in the cytoplasm for the active function, leading to the decreases in CGP yield. On the other hand, at a lower temperature, the enzyme seems to have a loose substrate specificity which allows the incorporation of more lysine instead of arginine into the side chain during synthesis. As a result, a higher ratio of soluble form was produced with a high content of lysine. When the temperature increased, the substrate specificity seemingly becomes tighter, and more arginine and less lysine are incorporated. Thus, a higher ratio of insoluble form was observed.
Fractionation of CGP
After purification, the iCGP was further fractionated at different pH values of 3, 4, 5, 6, and 7 except that obtained at temperature 17 oC which had insufficient amounts for processing. As shown in Fig 2A, only two major fractions at pH of 4 and 5 weighed significantly among all the fractions, and the amounts of other fractions totaled less than 10%. Interestingly, the portions of the two major fractions appeared to almost the same percentage at different cultivation temperatures, and the fraction at pH 5 is about twice the amount at pH 4.
Meanwhile, various ethanol concentrations were used to fractionate the sCGP. In the range between 15% and 75% ethanol, the portions of soluble form displayed a skewed distribution
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toward low ethanol concentrations (Fig. 2B). Some fair and approximate amounts of sCGP, more than 20% of the total, were, respectively, fractionated into 15%, 30%, and 45% ethanol for the cultivation temperatures from 17 to 32 oC except that at 37 oC under which condition almost 50% of soluble form was exclusively fractionated into 15% ethanol. For all cultivation temperatures, a slightly higher amount of sCGP appeared at a low ethanol concentration, the amounts of fractionated sCGP slightly decreased as the ethanol concentration increased, and less than 5% of the total amount was fractionated into 75% ethanol.
Amino acid analysis of the fractionated CGP
Further analysis of amino acid compositions revealed that the ratio of arginine to lysine differs in each fractionated CGP as shown in Fig. 3. For iCGP, a higher content of arginine was present in the fraction that formed precipitates at pH 4 than that at pH 5. Before fractionation proceeded, the purified iCGP was first redissolved at pH 1, and then pH was raised stepwise to 7. The collected precipitates at pH 4 and pH 5 were completely soluble below pH 3, and precipitation occurred when pH increased to 4, indicating that the fraction at pH 4 was soluble at pH 3 but not at pH 4, and similarly that the fraction at pH 5 was soluble at pH 4 but not at pH 5. Namely, a high content of arginine required a more acidic condition for dissolution (Fig. 3A). As for ethanol fractionation of sCGP, the environment of a low ethanol concentration was similar to that of aqueous solution of pH 7 under which condition the iCGP was prepared from the cell disruption. Comparably high contents of arginine were also detected in those fractions
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that form precipitates at low ethanol concentrations (Fig. 3B), suggesting that sCGP fractionated at a lower ethanol concentration had a lower solubility in water. The Arg/Lys ratio then follows a decreasing trend with the increasing ethanol concentrations for all fractions. It was noted that the fractions by the same ethanol concentration have a similar Arg/Lys ratio irrespective of different cultivation temperatures.
When the fractions of sCGP were subjected to the analysis of molecular weight distribution with GPC, it was found that the one with a higher arginine content, as fractionated at a lower ethanol concentration, also appeared to have a higher molecular weight as shown in Fig. 4A. A positive correlation was further observed for the Arg/Lys ratio and the molecular weight of each fraction, and such correlation was independent of the cultivation temperature (Fig. 4B). The molecular weight of sCGP ranges from 18 to around 30 kD, and a high molecular weight was accompanied with a high Arg/Lys ratio which was fractionated in a low ethanol concentration. The results indicated that both Arg/Lys ratio and molecular weight distribution affect the solubility of CGP.
Solubility of recombinant CGP
In an attempt to examine the effect of pH on solubility, the CGP purified from the bacterial culture at 27 oC was used before fractionation proceeded. In the pH range from 2 to 12, sCGP exhibited a higher solubility than iCGP at each corresponding pH values as shown in Fig. 5A. However, both forms have different pH dependency. The solubility of sCGP decreased with
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increasing pH from 110 mg/mL at pH 2, sharply dropped to 27 mg/mL at pH 3, and then gradually decreased to below 1 mg/mL at pH 12. The iCGP had a lower solubility of around 10 mg/mL at pH 2, then stayed below 1 mg/mL, hardly soluble from pH 4 to 11, but slightly increased to 1.7 mg/mL at pH 12 and 18 mg/mL at pH 13. Such dependence of solubility and pH behaved differently in comparison with a previous study which indicated CGP became soluble at a low pH 2 or a high pH 9 (Lang et al., 1972). A further examination into the solubility of each fraction at pH 3, 7.4, and 11 expectantly revealed that the solubility is a function of Arg/Lys ratio (Fig. 5B). The higher ratio of Arg/Lys the fraction exhibited, the less soluble the CGP became. For the three pH values of 3, 7.4, and 11, a rough linear trend was observed between the solubility and the Arg/Lys ratio for sCGP.
Thermal responses of CGP
As Fig. 6A depicted the thermal response of each fraction of sCGP at physiological pH during heating, an abrupt change in turbidity at a low temperature below 5 oC was observed, and then the solution stayed clear until a higher temperature around 55 - 60 oC was reached. Each fraction of the sCGP showed dual phase transition temperatures in PBS. Below 5 oC, a UCST, the critical temperature at which a solution becomes miscible, was found to associate with the content of arginine, and the fraction with a higher ratio of Arg/Lys had a slightly higher UCST. When the temperature exceeded the second phase change temperature around 55 – 60 o
C, formation of fibril agglomerates began, and the agglomerates settled down after leaving
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undisturbed for a period of time. A subsequent cooling process was performed in an attempt to redissolve the agglomerates (Fig. 6B). Surprisingly no phenomena of hysteresis was observed, and the fibril agglomerates remained even after further incubation at 4 oC or room temperature up to 96 h. However, when the heating temperature was limited to 45 oC, below the second phase change temperature, hysteresis were observed for each fraction of sCGP during the cooling process (Fig. 6C), and the clear solution turned cloudy again when the temperature was dropped below 5 oC, reaching its UCST.
On the other hand, iCGP, which became soluble at acidic conditions, exhibited only one phase change temperature at pH 3 in 0.1M citrate phosphate buffer (Fig. 7A). In contrast to the abrupt phase change of sCGP, the changes in turbidity seemed gradual over a wide temperature range from 15 to 40 oC. When the heating temperature was raised to above the denaturing temperature of the soluble form, no fibril formation appeared, and hysteresis was observed during a subsequent cooling process. Similar to the soluble form which UCST depended on the Arg/Lys ratio, the fractions of pH 4 and 5 showed UCSTs at 31 and 28 oC, respectively, indicating that a higher ratio of Arg/Lys resulted in a higher UCST. When examined under TEM, the insoluble agglomerates of both fractions seemed to be compact aggregates without a hollow center. The fraction of pH 4 showed a size of around 300 nm (Fig. 7B2) whereas the fraction of pH 5 had a larger size of around 400 nm (Fig. 7B4).
Discussion
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This study employed different ethanol concentrations and pHs to fractionate CGP which was prepared at different cultivation temperatures. The fractionation approach employed in this study revealed the role of lysine content in the solubility of CGP and in the distribution of molecular weight of CGP quantitatively as well as in the thermal responses of CGP. The CGP fractionated at the same condition, such as the same ethanol concentration or the same pH, has an approximate lysine content irrespective of the cultivation temperatures, and therefore presents similar properties which can be expressed as a function of Arg/Lys ratio. A previous report showed that the incorporation of lysine plays a key role in the synthesis of sCGP by examining different strains carrying different cphA genes (Wiefel and Steinbüchel, 2014). The results of this study demonstrated that the role of lysine not only in the solubility but also in determining other properties of CGP, such as the thermal responses.
In the past some polypeptides have shown phase transition, mostly LCST, for example elastin like peptides containing repeats of valine-proline-glycine-X-proline where X is a nonproline amino acid (Rodríguez-Cabello et al., 2006), ethyl and butyl modified poly(glycine) (Lahasky et al., 2012), poly(N-substituted α/β-asparagine)s (Tachibana et al., 2003), and polyethylene glycol modified poly(glutamic acid) (Chen et al., 2011). Polypeptides with a UCST property have been rarely reported. In this study both sCGP and iCGP were found to exhibit UCST when the solution was subjected to heating. At the physiological condition, the thermal response of sCGP occurred at a low temperature below 5 oC whereas that of iCGP was
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hardly observed in PBS due to its limited solubility. When the heating process continued to around 55 – 60 oC, sCGP underwent a second phase change similar to LCST (lower critical solution temperature), the critical temperature when the immiscibility of solution occurs. However, hysteresis was not observed during the process of cooling the heated sCGP because of the formation of fibril agglomerates. The irreversible denaturation of sCGP might be due to the dismantling of intramolecular hydrogen bonds. On the other hand, a second phase change did not occur for iCGP when heating temperature was raised to 65 oC, and the hysteresis of UCST was observed during the heating and cooling processes of the iCGP at an acidic condition of pH 3. The phase change of iCGP spanned a wider temperature range as compared with the abrupt change of the sCGP.
The lack of hydrophobic moiety on CGP might be unable to prompt the formation of micelles that generally result from amphipathic molecules. Hydrogen bonding was attributed to the phase transition of UCST of several polymers (Seuring and Agarwal, 2012; Shimada et al., 2013). The CGP agglomerates might form through hydrogen bonding between the arginine side chains. From a viewpoint of molecular structure, CGP could be viewed as a slightly twisted string along with its side chains, arginine and lysine, alternating on the outer. Some intermolecular hydrogen bonding might be bridged with the guanidino group of arginine. When the arginine is substituted by lysine, less nitrogen atoms could diminish the hydrogen bonding, thus increasing the solubility of CGP at a moderate temperature. More lysine substitutions, as
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indicated by low Arg/Lys ratios, further weaken the hydrogen bonding, thus lowering UCST, which was observed in the fractions of soluble and insoluble forms. The iCGP possesses a higher content of arginine and the slow dissociation and annealing of the numerous hydrogen bonds presumably caused the gradual phase changes during either the heating or cooling process as suggested by the observations of UCST spanning from 15 to 40 oC. However, the high content of lysine of sCGP is prone to denaturation, similar to protein denaturation, at a high temperature over 55 oC at which solution began to turn from clear to cloudy similar to the indication of LCST but resulted in an irreversible phase change of fibril formation.
On the other hand, both the amine group of lysine and the guanidino group of arginine are ionized with a pKa of 10.5 and 12.5, respectively. Therefore the more substitution of arginine with lysine could lower the isoelectric point of CGP, presumably increasing the solubility at pH 7. The extent of ionization at low pH might bring water to bridge the hydrogen bonding between CGP molecules, and could be ascribed to the dependence of solubility on pH. Altogether the pH and thermal responses make CGP possess dual pH and temperature sensitive properties.
More lysine molecules were found in the CGP obtained from cultivation at a low temperature which also allowed the production of more soluble CGP synthetase to direct the CGP synthesis. On the other hand, at a high cultivation temperature, less amount of soluble enzyme could produce an approximate amount of CGP, suggesting that the enzyme was less
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active at a low temperature. Thus a high yield of cyanophycin exists around 27 – 30 oC when a sufficient amount of soluble enzyme operates with a moderate activity. The inefficiency of CGP synthetase at low temperature might cause less substrate specificity to allow the use of lysine instead of arginine as substrate, resulting in a low ratio of iCGP/sCGP.
In conclusion, this study showed that using cultivation temperature can modulate the proportions of soluble and insoluble CGP, and correlated the lysine content with the solubility, molecular weight, and thermal response of CGP in a quantitative way. The characteristic of thermal and pH responses can be expected in biomedical applications, such as drug delivery system.
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References
Aboulmagd, E., Oppermann-Sanio, F.B., Steinbüchel, A., (2000) Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Arch Microbiol 174, 297-306. Allen, M.M., Hutchison, F., Weathers, P.J., (1980) Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308. J Bacteriol 141, 687-693. Chen, C., Wang, Z., Li, Z., (2011) Thermoresponsive polypeptides from pegylated polyL-glutamates. Biomacromolecules 12, 2859-2863. Frey, K.M., Oppermann-Sanio, F.B., Schmidt, H., Steinbüchel, A., (2002) Technicalscale production of cyanophycin with recombinant strains of Escherichia coli. Appl Environ Microbiol 68, 3377-3384. Hai, T., Oppermann-Sanio, F.B., Steinbüchel, A., (1999) Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19. FEMS Microbiol Lett 181, 229-236. Lahasky, S.H., Hu, X., Zhang, D., (2012) Thermoresponsive Poly(α-peptoid)s: Tuning the Cloud Point Temperatures by Composition and Architecture. ACS Macro Lett. 1, 580–584. Lang, N.J., Simon, R.D., Wolk, C.P., (1972) Correspondence of cyanophycin granules with structuredgranules in Anabaena cylindrical. Arch Microbiol 83, 313–320. Mackerras, A.H., De Chazal, N.M., Smith, G.D., (1990) Transient accumulation of cyanophycin in Anabaena cylindrica and Synechocystis 6308. J Gen Microbiol 136, 2057-2065. Obst, M., Steinbüchel, A., (2004) Microbial degradation of poly(amino acid)s. Biomacromolecules 5, 1166-1176. Rodríguez-Cabello, J.C., Reguera, J., Girotti, A., Arias, F.J., Alonso, M., (2006) Genetic engineering of protein-based polymers: The example of elastinlike polymers. Ordered Polymeric Nanostructures at Surfaces. Springer, pp. 119-167. Sallam, A., Kast, A., Przybilla, S., Meiswinkel, T., Steinbüchel, A., (2009) Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization. Appl Environ Microbiol 75, 29-38. Seuring, J., Agarwal, S., (2012) Polymers with upper critical solution temperature in aqueous solution. Macromol Rapid Comm 33, 1898-1920. Shimada, N., Nakayama, M., Kano, A., Maruyama, A., (2013) Design of UCST
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Figure Legends
Figure 1. Effect of cultivation temperature on CGP yield and expression of CGP synthetase
(A) the volumetric yield of CGP (●) and the ratio of iCGP and sCGP (■) obtained after purification from the recombinant E. coli cultivated at different temperatures. The arrows indicate the readings of the data on the corresponding axes. (mean ± S.D.; n=3 independent cultivation) (B) the SDS-PAGE of the recombinant E. coli treated with the BugBuster reagent. (M: protein markers; L and P: the cell lysate and cell pellet after centrifugation of the treated bacteria, respectively)
Figure 2. Distribution of iCGP and sCGP after fractionation
CGP was purified from the recombinant E. coli cultivated at different temperatures (▬:17 oC; ▬:22 oC; ▬:27 oC; ▬:32 oC; ▬:37 oC). Fractionation of the purified CGP was performed with different pHs for iCGP and with different ethanol concentrations for sCGP. (A) the distributions of the fractions at different pH values, (B) the distributions of the fractions at different ethanol concentrations. (mean ± S.D.; n=3 independent cultivation)
Figure 3. Amino acid compositions of the fractionated CGP
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Each fractionated CGP was subject to amino acid analysis. (A) the ratio Arg/Lys of each fraction of iCGP, (B) the ratio Arg/Lys of each fraction of sCGP. The CGP was obtained from cultivation at different temperatures (▬:17 oC; ▬:22 oC; ▬:27 oC; ▬:32 oC; ▬:37 oC) (mean ± S.D.; n=3 independent cultivation)
Figure 4. Molecular weight distribution of the fractionated sCGP
(A) a typical elution profile of sCGP fractionated at different ethanol concentrations (▬:15% ethanol; ▬:30% ethanol; ▬:45% ethanol; ▬:60% ethanol; ▬:75%), (B) the dependence of the average molecular weight and the Arg/Lys ratio of the fractionated sCGP from different cultivation temperatures (▬:17 oC; ▬:22 oC; ▬:27 oC; ▬:32 oC; ▬:37 oC).
Figure 5. Dependence of solubility on pH
(A) the dependence of pH and the solubility of sCGP (▬) and iCGP (▬) in different buffers, (B) the solubility of CGP as a function of Arg/Lys ratio in three different buffers (▬: citratephosphate at pH 3; ▬: PBS at pH 7.4; ▬: borate buffer at pH 11).
Figure 6. Thermal response of fractionated sCGP
(A) the changes in the turbidity of sCGP fractionated at different ethanol concentrations (▬:15% ethanol; ▬:30% ethanol; ▬:45% ethanol; ▬:60% ethanol; ▬:75%) when the solution was heated at 1 oC/min under stirring. The images showed the solution at 4oC, 37oC, and 65oC, respectively. (B) the changes in turbidity when the solution was subsequently cooled
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at 1 oC/min under stirring after being heated to 67 oC, (C) the changes in turbidity when the solution was subsequently cooled at 1 oC/min under stirring after being heated to 45 oC. The arrows indicate the thermal heating (in red) or cooling (in blue) along with temperature.
Figure 7. Thermal response and TEM image of fractionated iCGP
(A) the changes in the turbidity of iCGP fractionated at different pHs (▬: pH 5; ▬: pH 4) when the solution was heated at 1 oC/min (solid lines) and then cooled at 1 oC/min (dashed lines) under stirring at pH 3. The images showed the solution of iCGP fractionated at pH 4 at 15oC and 50oC, respectively. The arrows indicate the thermal heating (in red) or cooling (in blue) along with temperature. (B) the TEM images of iCGP fractionated at pH 5 (B1 and B2) and at pH 4 (B3 and B4).
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