Patent Application
20110071281
The present invention relates to a method of protein electrolysis, a protein obtained by the above-mentioned protein electrolysis method, and a use of said method for protein modification.
Since its introduction in 1975, two-dimensional electrophoresis (2-DE) has been widely used for the analysis of protein samples from pure protein preparation to crude tissue extracts. The first dimensional electrophoresis, i.e. isoelectric focusing (IEF), has been improved by using an immobilized pH gradient (IPG) strip. To achieve optimal resolution and visualization, interfering compounds, such as salts, nucleotides, polysaccharides, lipids and particulate materials, need to be removed from the samples prior to IEF. It is generally believed that the presence of high salts would increase conductivity, thereby hindering focusing in IEF. Moreover, rapid transport of water and ions would trigger electroosmosis, which results in protein aggregation and artifacts.
However, electrolytic reactions always accompany the electrophoresis operated at a voltage significantly higher than the redox potential of water, including anodic oxidation generating oxygen gas and protons and cathodic reduction generating hydrogen gas and hydroxide ions. The presence of electrolytes would definitely increase current and therefore increases the rate of electrolysis and power output. Consequently, the pH of the solution at the anode decreases and that at the cathode increases. In addition, bubbles are formed at both electrodes. Accordingly, IEF is usually performed at low voltage for a prolonged time (for instance, at 30 V for 4 hours rather than at 500 V for 1 hour) to reduce the undesired effects caused by high salts. Unfortunately, conventional IEF systems (such as Ettan IPGphore system or Protein IEF system) are closed buffer systems so that the stalled ions would increase the rate of electrolysis.
According to current researches, carbamylation (Righetti et al., 2006; McCarthy et al., 2003), formation of oligomers and β-elimination (i.e. desulfuration) of cysteine resides can only be observed when the samples are inappropriately processed. Proper reduction and alkylation prior to the IEF process can prevent the scrambled disulfide bonds formation and β-elimination of cysteine resides during IEF process. However, protein modifications happened in the high salt IEF process is never described in the previous studies.
It is known in the art that electrolytic reduction only occurs at the surface of cathode. Surprisingly, the Inventors find that electrolytic reduction also occurs at a distance from cathode while the electrolysis is performed in the presence of high salts. The present invention uses said high-salt electrolysis to electrolytically reduce a protein and consequently result in the modification of said protein.
Thus, the present invention provides a method of protein electrolysis, comprising the following steps:
In a preferable embodiment of present invention, said electrolytic reduction comprises the following steps: applying said protein sample into a cathodic chamber of an electrolyzer and performing electrolysis.
In a preferable embodiment of present invention, said salt is selected from chloride, bromide, acetate, sulfate or carbonate of an alkali metal or an alkaline earth metal; more preferably, said alkali metal is Li, Na, or K; said alkaline earth metal is Mg or Ca; more preferably, said salt is selected from LiCl, NaCl, NaOAc, Na2SO4, CaCl2, KCl or MgCl2; even more preferably, from LiCl, NaCl, NaOAc or Na2SO4; most preferably, is NaCl.
In a preferable embodiment of present invention, said salt has a concentration of 20 mM to 1000 mM; more preferably, 50 mM to 500 mM; even more preferably, 100 mM to 300 mM; most preferably, 100 mM.
In a preferable embodiment of present invention, said protein sample further comprises an acid; more preferably, said acid is acetic acid, phosphoric acid, citric acid or glycine; even more preferably, is acetic acid.
In a preferable embodiment of present invention, said acid has a concentration of 1˜2%.
In a preferable embodiment of present invention, said step (b) is reducing acid amino residues of said protein sample; more preferably, said step (b) is modifying carboxyl groups of said protein sample into aldehyde groups or alcohol groups.
The present invention also provides a modified protein, which is obtained by the aforesaid method of protein electrolysis. More preferably, said modified protein is a pseudo-protein or a pseudo-peptide.
The present invention also provides a use of the aforesaid method of protein electrolysis for protein modification, which is used for reducing acid amino residues of a protein; more preferably, for modifying carboxyl groups of a protein into aldehyde groups or alcohol groups.
To sum up, the present invention provides a method for protein modification by electrolytic reduction, a modified protein obtained by the method, and a use of said electrolytic reduction method for protein modification. Comparing to traditional chemical methods using reductants, the method of present invention has advantages of low cost, reduced contamination and easily-controllable reaction.
Except for the definition hereinafter, all the scientific terminologies should be explained as their original means, which is understood by those ordinary skilled in the art. If any argument is caused, the definitions in this specification shall be used as the major explanations.
The term “salt” herein is referred as a salt selected from alkali metal salts or alkaline earth metal salts; preferably, chloride, bromide, acetate, sulfate or carbonate thereof; more preferably, any of the aforesaid salts of alkali metal Li, Na, or K; any of the aforesaid salts of alkaline earth metal Mg or Ca; more preferably, a salt selected from LiCl, NaCl, NaOAc, Na2SO4, CaCl2, KCl or MgCl2; even more preferably, from LiCl, NaCl, NaOAc or Na2SO4; most preferably, NaCl. As the protein electrolysis of present invention is achieved by accumulation of free metal ions around cathode and closely related to the electrophoretic mobility of the metal ions, any salt of an alkali metal or an alkaline earth metal is suitable for present invention.
The term “high concentration” herein indicates that the alkali metal salt or alkaline earth metal salt has a concentration of 20 mM or above; preferably, 20 mM to 1000 mM; more preferably, 50 mM to 500 mM; even more preferably, 100 mM to 300 mM; most preferably, 100 mM. Protein modification can be performed on any protein sample by using the alkali metal salt or alkaline earth metal salt having the above-mentioned concentration. The above-mentioned concentration is merely for convenience of regular operation, and those ordinarily skilled in the art can perform protein modification under a salt concentration out of the above-mentioned range by changing other operating conditions accordingly.
The term “acid” herein is referred as any organic or inorganic acid; more preferably, acetic acid, phosphoric acid, citric acid or glycine; even more preferably, acetic acid. The acid is presented to adjust the pH of the electrolytic buffer and to keep the protein in the cathodic chamber. It does not participate in reducing reaction. Therefore, any acid that is able to decrease the pH of electrolytic buffer is suitable for the present invention.
Regarding the two-dimensional electrophoresis in the following examples, the first dimensional isoelectric focusing was performed by using Ettan IPGphor Isoelectric Focusing System (GE Healthcare). First, an IPG gradient strip with pH of 4˜8, 4˜10 or 4˜11 was prepared on GelBond® PAG Film using model 475 Delivery System (Bio-RAD) for gradient making. The length of said strip was 7 cm. Said strip was 4% polyacrylamine gel, wherein the ratio of bisacrylamide to acrylamide was adjusted to 0.045. The concentrations of acrylamido buffer (the 0.2 M stock solution thereof was purchased from Fluka) used for the preparation of said gradient strip were calculated by Doctor pH software (Hoefer). According to the routine condition used in the first dimensional isoelectric focusing, the protein sample was loaded into an IPG strip and the IPG strip was rehydrated for 4˜10 hour. After that, the isoelectric focusing electrophoresis was conducted.
Before the second dimensional electrophoresis, SDS-PAGE, the IPG strips were transferred into a tray, and soaked in a buffer containing 75 mM Tris-HCl (pH 7.8), 0.1% SDS and 0.002% bromophenol blue for 10 minutes, twice. The second dimensional SDS-PAGE electrophoresis was performed by Tris-Tricine SDS-PAGE in accordance with the method disclosed by Schagger etc. (1987). In other words, the SDS-PAGE was performed in 7.5% or 10% polyacrylamide gel while the ratio of bisacrylamide to acrylamide thereof was adjusted to 0.03.
The following examples are just the best exemplary embodiments, and they are not intended to limit the scope of present invention. Those ordinarily skilled in the art can make appropriate changes and amendments according to the disclosure of present invention without departing from the spirits of present invention.
Day 1 zebrafish embryos were collected and stored at −70° C. For protein sample preparation, one gram of zebrafish embryos were homogenized in 10 ml of cold TE buffer (20 mM Tris-HCL, pH 8.0, 5 mM EDTA) on ice and the homogenate was centrifuged at 27,000×g at 4° C. for 30 minutes. The supernatant was subjected to 90% ammonium sulfate precipitation and the pellet was dissolved in TE buffer. This step was repeated once again and the protein solution was then desalted into the rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer, pH 3˜10) (GE healthcare) by Millipore centricon YM-10 (Millipore) as a salt-free protein sample.
For high-salt electrophoresis, the 100× stock solution of a salt (such as NaCl, Na2SO4 and (NH4)2SO4) was added into said protein sample and said rehydration buffer to achieve the desired final concentration. After rehydrating the IPG strip for 4˜10 hours, the routine IEF (i.e. the first dimensional IEF electrophoresis proceeded for 8,000˜12,000 voltage-hours) can be performed and then the second dimensional electrophoresis is proceeded by using 7.5% polyacrylamide gel. Alternatively, the protein samples in high salts can be subjected to electrophoresis at the maximal voltage of 500 V for 200 voltage-hours as well as in-gel dialysis and re-focusing IEF, and then the second SDS-PAGE electrophoresis is proceeded.
According to
In order to closely observe the proteins having high pI value produced in high-salt two-dimensional electrophoresis, a purified bovine RNase A (Sigma) was used for the following experiments.
In the light of foregoing, proteins having a higher pI value are produced in the absence of protein denaturing agent, such as urea. Therefore, urea does not play a role of protein modification, and the high-salt 2-DE does make an irreversible pI drift of bovine RNase A.
In this experiment, 0.5% IPG buffer was used to replace the rehydration buffer for RNase A protein sample preparation (2 μg/125 μl). The first dimensional IEF electrophoresis was performed at the maximal voltage of 500 V for 0, 0.5, 1 or 1.5 hours by using a IPG strip with pH gradient of 4˜11. Also, in-gel dialysis and re-focusing IEF were conducted in accordance with example 1; the second dimensional SDS-PAGE electrophoresis was performed by using 7.5% polyacrylamide gel, and then silver staining was performed.
According to the results shown in
Electrolytic reaction plays a key role in the formation of the above-mentioned artifacts including cathodic drift, and that is because electrolysis of water will be enhanced during high salt electrophoresis. Proton accumulation at the anode and hydroxide ion accumulation at the cathode not only cause protonation and deprotonation of proteins at the two electrodes, respectively, but also vanish the pH gradient within the IPG strips near the electrodes. Subsequently, the isoelectric focusing effect is exempted.
In order to determine the influence of pH value on the artifacts, the pH values of the IPG strips were monitored. After IEF, the strips were rinsed by distilled water and then immersed in a solution of phenol red (Merck). Phenol exhibits a color transition from yellow, orange to purple over the pH range 6.8 to 8.4. In the IPG strips used in high salt IEF, the bright yellow band representing acidity and the purple band representing alkalinity moved toward the center of the strips (data not shown).
As shown in
The electrolytic reducing power generated during electrolysis was further examined.
First, IPG strips were rehydrated for 4 hours by 6 mM, 12 mM, 25 mM or 50 mM NaCl or salt-free distilled water. The aforementioned blank strips were subjected to IEF at the maximal voltage of 500 V for 600 or 8,000 voltage-hours. After that, the strips were rinsed in distilled water and stained in 0.5 mg/ml phenol red for 10 sec to reveal the acidification and alkalization of the strips themselves. In addition, IPG strips were rehydrated for 4 hours by 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, purchased from Sigma) aqueous solution comprising 6 mM, 12 mM, 25 mM or 50 mM NaCl or the salt-free MTT aqueous solution, and then subjected to IEF at the maximal of 500 V for 600 voltage-hours.
MTT is likely to be reduced to insoluble purple formazan. In the salt-free electrophoresis, there is little MTT being reduced. Relatively, after 600 voltage-hours IEF in the presence of 6 mM or 12 mM NaCl, formazan is produced at sites near cathode; and, after 600 voltage-hours IEF in the presence of 25 and 50 mM NaCl, formazan is localized to 2 to 3 cm away from the cathode (data not shown).
The foregoing results indicate that high concentrations of NaCl interfere with the electrophoresis of MTT, and the cathodic reduction reactions occur away from the cathode. Therefore, localization of MTT at the cathode seems not required for its reduction. Moreover, the extent of MTT reduction is the highest in the presence of high concentration of LiCl, NaCl or CaCl2, and it is the lowest in the presence of high (NH4)2SO4, with the descending sequence of LiCl=NaCl>CaCl2>KCl>MgCl2>>(NH4)2SO4 (data not shown).
Furthermore, application of moist filter paper and anion exchanger paper (size 10×3 mm2) between the electrodes and IPG strip does not affect the MTT reduction. However, the use of cation exchanger paper significantly decreases the MTT reduction at sites near the cathode. This indicates that freely movable cations are necessary for spreading of reducing paper for MTT reduction.
The following experiments were performed by using a home-made horizontal electrolyzer with platinum electrodes. The electrolyzer had a anodic chamber (2.5×3.5×2.0 cm) and a cathodic chamber (2.5×3.5×2.0 cm) that salt-bridged each other with a slice of PVDF membrane (1.8×8.0 cm). Said PVDF membrane had been rinsed sequentially by methanol, water and 1% acetic acid/0.1 M salt in advance. The experiments of this example also can be performed by a commercial electrolyzer.
In the experiments of this example, 5 mg/ml bovine insulin (Sigma) was prepared in 1% acetic acid comprising 0.1 M NaCl or LiCl. The presence of acetic acid can minimize the migration of proteins toward the anodic chamber due to protonation.
3 ml of said bovine insulin sample was loaded into the cathodic chamber while 5 ml of 1% acetic acid comprising 0.1 M NaCl was loaded into the anodic chamber. A small amount of the electrolyzed samples were removed at different time intervals and subjected to SDS-PAGE by using 10% polyacrylamide gel and then staining with GelCode blue dye (purchased from Thermo Scientific) to determine disulfide bond reduction. As results, the reduction of bovine insulin into A and B chains has been detected after electrolyzing for 1.5 hours. After 2 hours electrolysis, and most bovine insulin has been reduced into A and B chains (data not shown). Moreover, the extent of bovine insulin reduction is the highest in the presence of high concentration of LiCl or NaCl, and it is the lowest in the presence of high concentration of (NH4)2SO4, with the descending sequence of LiCl=NaCl>CaCl2>KCl>MgCl2>>(NH4)2SO4 (data not shown).
In the light of foregoing, it is clear that alkali and alkaline earth metal ions as electrolytes are able to reduce MTT and protein disulfide bonds, wherein the order of activity among these ions on the reduction of MTT and protein disulfide bonds and the protein alkalization induced by aforesaid electrolysis are identical, and the efficiency of reduction is proportional to ion concentration. Altogether, freely movable metal ions accumulation at sites near the cathode is required for the generation of reducing power.
Moreover, the extent of MTT and protein disulfide bonds reduction is the highest in the presence of high concentration of LiCl or NaCl, and it is the lowest in the presence of high concentration of (NH4)2SO4, with the descending order of LiCl=NaCl>CaCl2>KCl>MgCl2>>(NH4)2SO4. The reactivity order is more correlated with the electrophoretic mobility than with the reactivity of these metals with water. Thus, these alkali metal and alkaline earth metal ions are supposed to be reduced to metals at cathode first, and the metals reduce the migrating metal ions, MTT or protein in the gel subsequently. That is a “relay” of reduction. And this is why the reduction occurs not only at cathode, but also at a distance away from the cathode, and as the spreading of reductive power is correlated with the concentration and mobility of cation ions.
As shown in
In order to examine if specific amino acid residues affect the pI of a given protein, four hypothetical bovine RNase A mutants were obtained from the replacement mutation of the protein sequence of wild type RNase A (WT), as shown in
The results indicate that the reduction of carboxyl groups and amide groups of amino acid residues can convert a given protein to a more basic form. The results are consistent with the experimental data shown in
According to the results of aforesaid examples 1˜3, the high-salt 2-DE of present invention is able to reduce a carboxylic acid into the corresponding aldehyde with a carbonyl group (C═O) and even further into the corresponding alcohol. Thus, Schiff base or hydrazone formation, which results from the interaction between aldehyde intermediates and primary amines or hydrazides, can be employed to detect if any aldehyde group of a given protein is formed during a high-salt IEF.
To provide additional evidences for reduction of carboxylic acids in the high-salt electrolytic condition of the present invention, a mass spectrometric analysis was accessed to determine the molecular weight of the electroreduced RNase A.
5 μg of un-electroreduced RNase A (RNase A) and electroreduced RNase A (ER-RNase A) were subjected to SDS-PAGE electrophoresis. As shown in
Theoretically, the apparent molecular mass would decrease by 16 or 14 Da if a given carboxylic acid is reduced to an aldehyde or an alcohol, respectively. Alternatively, the apparent molecular mass would decrease by 14 Da if a given amide is reduced to a primary amine. Other possible reactions are reduction of disulfide bonds and formation of Schiff base.
[M+H]+/Z of un-electroreduced RNase A peak is 13682.5 while that of electroreduced RNase A peak among a broad range of mass spectra is 13566.6 (data not shown); and the difference of the their molecular weights is 115.9 Da. The molecular weight difference can be resulted from mixed reduction of carboxylic acids, amides, disulfide bonds and formation of Schiff bases (please refer to the following formula),
i.e. formation of 2 aldehyde groups (2×16 Da) and 6 amine or alcohol groups (6×14 Da). In addition, higher-molecular-weight forms also appear. The increase in molecular weight cannot be explained by dimerization via Schiff base formation.
Therefore, the high salt electrolysis of present invention can be applied to produce a pseudo-protein or a pseudo-peptide with aldehyde groups, which may be used for conjugation with amine-containing molecules and protein cross-linking, for instance, for conjugation with the amine-containing haptens for immunization.
| Number | Date | Country | Kind |
|---|---|---|---|
| 98131483 | Sep 2009 | TW | national |
