JP5273438B2 - Method for calculating solubility of peptide-added biomolecule, peptide tag design method and inclusion body formation prevention method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quantitatively calculating solubility of peptide and protein from an amino-acid sequence in a physicochemically correct method in accordance with the thermodynamic law, in advance (prior to synthesis or expression) and at exponentially higher calculation accuracy than by a conventional method. <P>SOLUTION: A method for calculating the solubility S of peptide additive biomolecule comprising an arbitrary amino-acid sequence at the end of a biomolecule from a thermodynamic theory. Gibbs free energy &Delta;G<SB>Aggr</SB>of association of the peptide is defined by a quadratic function of the number of residues N of amino acid X added to the biomolecule expressed by an equation (1); &Delta;G<SP>X</SP><SB>Aggr</SB>=&Delta;G<SB>Aggr0</SB>+&Delta;G<SP>X</SP><SB>Aggr1</SB>N+&Delta;G<SP>X</SP><SB>Aggr2</SB>N<SP>2</SP>, and the Gibbs free energy &Delta;G<SB>Aggr</SB>of association of the peptide is used to calculate the solubility S of the peptide additive biomolecule by an equation(2): S=exp(&Delta;G<SB>Aggr</SB>/RT). <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

The present invention relates to a method for calculating the solubility of biomolecules from amino acid sequences based on thermodynamic models of biomolecule aggregation.
Furthermore, the present invention relates to a method for designing a peptide tag having a desired solubility using the above calculation method and a method for preventing the formation of inclusion bodies (inactive aggregates accumulated in cells).

In the post-genomic era, research on elucidating the structure and function of proteins based on genetic information has attracted attention in order to elucidate the mechanism of biological phenomena and the causes of diseases. Proteins in the living body have a thermodynamically stable fold state and express a specific function when they take their natural structure. Therefore, synthesizing the target protein while maintaining the original three-dimensional structure (natural structure) outside the living body is a very important issue not only in proteomic research but also in industrial use and pharmaceutical application fields.
Proteins are denatured by environmental changes such as heat, pH, salt concentration, and the presence of a reducing agent, not only reducing or deactivating activity, but also greatly reducing solubility. This is because the bonds supporting the higher-order structure in the molecule are broken and the fold state is broken, and in the denatured protein, the highly hydrophobic part that was inside the molecule is exposed to the outside, In order to come into contact with the solvent (water), the proteins tend to aggregate and precipitate. Even in the natural state, proteins tend to aggregate and precipitate when there are many hydrophobic residues on the molecular surface or near the isoelectric point.

Such an aggregation phenomenon occurs when the concentration of molecules exceeds a certain solubility limit concentration (solubility). As a method for suppressing protein aggregation, a technique for improving solubility by manipulating solvent conditions such as pH, temperature and salt concentration is generally used. For example, in protein structure analysis by NMR, which requires a high concentration analysis sample, the solubility of the protein is improved by adding a low concentration of a surfactant such as CHAPS.
However, this method is based on the theory that protein solubility is determined by the hydrophobicity / hydrophilicity of the molecule, and depends heavily on the intuition and experience of researchers. Optimization of solvent conditions requires effort and time. Is needed. In addition, there is a problem that the functional three-dimensional structure of the protein is not maintained under controlled solvent conditions.

Patent Document 1 describes a method of measuring protein solubility from a solubility curve based on observation of interference fringes of a protein solution around a crystal when the concentration of a precipitant in the protein solution is increased.
However, this method also semi-empirically “predicts” the solubility, and it is necessary to determine the optimum solvent conditions from a large amount of screening with low accuracy and using the temperature, pH, and concentration of various additives as parameters. Efficient.
In addition, Non-Patent Document 1 describes a method for predicting aggregation tendency of amino acid sequences of proteins from theoretical calculation. However, as parameters used in the calculation formula, actual values of solubility of peptides are obtained from literature and experiments, and the accuracy is low, and the versatility is low for industrial use.

On the other hand, the present inventors have developed a method for dramatically improving the solubility of a target protein to which the tag is added by attaching a tag consisting of a short hydrophilic amino acid to the target protein (Patent Document 2). reference). According to this method, the solubility can be improved without affecting the functional expression or the three-dimensional structure of the target protein.
Although this method can be used under a wide range of solvent conditions and is an epoch-making method with excellent versatility, in principle it is only a mathematical expression of an empirical rule. For this reason, establishment of a methodology for strictly defining the protein dissolution phenomenon is desired.

Now that genetic engineering has been developed, various recombinant proteins obtained from mass synthesis methods are used in a wide range of fields such as pharmaceuticals, foods, and diagnosis. Among these, Escherichia coli is widely used as a host for mass synthesis because it grows quickly and can be easily cultured.
However, in a recombinant protein expression system using E. coli, most of the produced target protein becomes insoluble and forms an inactive inclusion body (inclusion body). For this reason, it is necessary to further refold (rewind) the obtained protein after making the three-dimensional structure completely broken with a denaturant or the like.
Conventionally, in order to obtain the target protein in a solubilized state, a method for expressing a highly soluble protein (solubility enhancing tag) such as MBP (maltose binding protein) as a fusion protein with the target protein, protein concentration, and type of additive Various methods such as an optimization method, a dialysis method, and a dilution method are used in appropriate combination.
However, these methods are individually addressed for each target protein, and the yield may be low. In addition, in the method using the above-described solubility-improving tag, the molecular weight of the protein used as the tag is 10 kD or more, and there is a problem that the addition of the tag affects the function or structure of the target protein. Removal is necessary, which is costly and troublesome.
WO2004 / 90515 JP 2006-188507 A "Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins", Serrano et al., Nat Biotechnol., Vol.22, p1302-1306, 2004 "A theory of linear and helical aggregation of macromolecules.", Oosawa, F., and M. Kasai., J. Mol. Biol., Vol.4, p10-21, 1962

As described above, there is currently no thermodynamic model that theoretically “correctly” represents aggregation of peptides and proteins (generally, biomolecules), and there is no method for adjusting the solubility systematically with high accuracy. Therefore, the actual situation is that qualitative solubility prediction is performed by an empirical method of accumulating experimental data for each target protein. For this reason, in comprehensive analysis of a vast population such as proteome research and peptide synthesis, it is possible to predict quantitative solubility more efficiently, and development of a highly versatile solubilization technique is expected.
Therefore, the present invention establishes a method for predicting the solubility of a target protein under an arbitrary solution condition from a theoretical calculation based on amino acid sequence information before actually synthesizing it, and designing a peptide tag for improving solubility by this theoretical calculation. Thus, there is provided a method for improving the solubility of a protein by providing the tag without changing the function, the three-dimensional structure, the thermal stability, or the like. Another object of the present invention is to provide a method for preventing the formation of inclusion bodies in an expression system using Escherichia coli as a host using the tag.

As a result of intensive studies to solve the “solubility” of peptides and proteins from a new viewpoint,
(1) Focusing on the phenomenon in which biomolecules aggregate when the solubility (solubility limit concentration) is exceeded, the ease of this aggregation (ie, “dissolution (limit concentration)”) is determined by the molecular association model of Oosawa & Kasai (non- based on Patent Document 2), further one molecule associates with the association of biological molecules (Figure 1) Gibbs associated to the free energy (hereinafter, referred to as association free energy.) can be represented by .DELTA.G _Aggr,
(2) The association free energy ΔG _Aggr of a peptide having an arbitrary amino acid sequence is calculated by the sum of the association free energy ΔG ^x _Aggr of each amino acid species X in all types of amino acids (all amino acid species) constituting the peptide. And ΔG ^x _Aggr is approximately represented by a quadratic function of the number of amino acid species (residues) contained in the peptide,
(3) The theoretical value of the solubility of the biomolecule to which the peptide calculated from the amino acid sequence based on the thermodynamic theory is almost the same as the actually measured value, and the combination of the type and number of amino acids constituting the peptide is the same , The solubility is almost the same regardless of the amino acid sequence pattern, so that the amino acid sequence that can obtain the desired solubility by calculation can be designed (identified),
(4) The formation of aggregates including inclusion bodies can be prevented by adding to the target protein a peptide tag consisting of an amino acid sequence that provides high solubility designed in (3).
As a result, the present invention has been completed.

That is, in the present invention,
The first invention is a method for calculating the solubility S of a peptide-added biomolecule in which a peptide having an arbitrary amino acid sequence is added to the end of a biomolecule (amino acid, peptide, protein, etc.) based on thermodynamic theory. The Gibbs free energy ΔG _Aggr of the peptide association is the sum of ΔG ^X _Aggr defined by a quadratic function of the number N of residues of amino acid species X constituting the amino acid sequence represented by the following formula (1): The gist is a method of calculating and calculating the solubility S of the peptide-added biomolecule by the following formula (2) using the Gibbs free energy ΔG _Aggr of the peptide association.

ΔG ^X _Aggr = ΔG _Aggr0 + ΔG ^X _Aggr1 N + ΔG ^X _Aggr2 N ² Formula (1)
(In the above formula (1), X is the amino acid species added to the biomolecule, ΔG ^X _Aggr is the Gibbs free energy of association associated with the addition of the amino acid species X to the biomolecule (ie, the amino acid species X (Association free energy) (J / mol), ΔG _Aggr0 is the Gibbs free energy (J / mol) of the association of the biomolecule, ΔG ^X _Aggr1 is the first term of the Gibbs free energy of association of the amino acid species X (J / mol), ΔG ^X _Aggr2 is the second term (J / mol) of the Gibbs free energy of association of the amino acid species X, and N is the number of residues of the amino acid species X contained in the added peptide.

S = exp (ΔG _Aggr / RT) Equation (2)
(In the above formula (2), S is the solubility of the peptide-added biomolecule, R is the gas constant, T is the absolute temperature, ΔG _Aggr is the free energy of association of each amino acid species X with respect to all amino acid species X contained in the peptide _Represents the sum of ΔG ^X _Aggr (ΣΔG ^X _Aggr (J / mol)).)

Here, a variant in which one amino acid species x of the amino acid species X is added to the end of a standard biomolecule (standard biomolecule) is prepared, and the variant is dissolved in an arbitrary solvent. The concentration of the soluble fraction when added above the limit concentration is measured, and from the change in solubility when the number of residues n of the amino acid species x is changed, the Gibbs association of the amino acid species x is determined by regression analysis. The free energies ΔG ^x _Aggr1 and ΔG ^x _Aggr2 can be calculated by the following equation (3).

s = exp ((ΔG ^x _Aggr1 n + ΔG ^x _Aggr2 n ² ) / RT) Equation (3)
(In the above formula (3), s is the ratio between the solubility of the mutant and the solubility of the standard biomolecule, R is the gas constant, T is the absolute temperature, and x is a specific type added to the standard biomolecule. Amino acid species, n is the number of residues of the amino acid species x, ΔG ^x _Aggr1 is the first term (J / mol) of the Gibbs free energy of association of the amino acid species x, and ΔG ^x _Aggr2 is the association of the amino acid species x Represents the second order term (J / mol) of Gibbs free energy.)

When the amino acid sequence of the peptide is selected from the combination patterns of the type of amino acid species X and the number N of residues thereof, the second invention provides peptide-added biomolecules in each combination pattern by the solubility calculation method described above. The gist is a method of designing the amino acid sequence of the peptide by calculating the solubility S of the peptide, selecting a combination pattern in which the solubility S has a desired value, and selecting the combination pattern.
The gist of the third invention is a method of preventing inclusion body formation of the target protein by expressing the target protein as a fusion protein with the peptide tag designed by the above-described method in the fungus body or in a test tube.

In a protein having a natural structure, some amino acids are buried in the protein and are not in contact with the solvent (water), and therefore, the influence of the amino acid on the inside is small as a factor that determines the solubility of the protein. Therefore, in the present invention, when theoretically determining the solubility of a protein based on the molecular association model of Oosawa & Kasai, the characteristics of all amino acids constituting the protein are not considered but the terminal side exposed on the protein surface is exposed. The association free energy of several amino acid residues is used. At this time, there is provided a method in which several amino acid residues on the terminal side are regarded as a peptide added to a protein, and the solubility of this peptide is determined by theoretical calculation. In this way, paying attention only to the amino acid on the terminal side, the solubility is determined from a micro value indicating the likelihood of the association of the amino acid on the terminal side and a macro value indicating the likelihood of the association between the proteins. Thus, the solubility of the peptide can be obtained with high accuracy by theoretical calculation without being limited by the target protein and solvent conditions.
Here, the target to which the peptide tag is attached is not limited to a protein, and can be a biomolecule including a peptide (in the present invention, one amino acid is defined by being expanded to “peptide”), and its state ( There are no particular restrictions on the modified state or the natural state), the type and number of amino acids constituting the amino acid, combinations thereof, and the like.
The peptide added to such a biomolecule is not particularly limited as long as it has a low molecular weight and does not form a three-dimensional structure. The number of amino acid residues is preferably 30 residues or less, and more preferably 20 residues or less. Particularly in the case of 3 to 15 residues, an excellent effect is obtained in that the solubility is significantly improved and the influence on the target biomolecule is small. The molecular weight is preferably up to about 30,000. When the number of amino acid residues and the molecular weight are within the above ranges, no correction parameter is required in the approximate expression represented by the above formula (1), and the biomolecule is used for analytical research or medical application. In some cases, it is excellent in that the peptide can be used as it is without being removed.

Energy (association free energy, ΔG _Aggr ) is required when the monomer molecules are bonded and aggregated. The value varies depending on the amino acid species constituting the molecule, but by using two parameters (association free energy ΔG ^X _Aggr1 and ΔG ^X _Aggr2 ) for each amino acid, the sum of the association free energies ΔG ^X _Aggr of the constituent amino acids is obtained. By calculating, the solubility can be obtained.

The above ΔG ^X _Aggr1 and ΔG ^X _Aggr2 can be experimentally determined under any solution condition.
As this determination method, a solubility curve when the number of amino acid residues added to a standard biomolecule (standard sample) is changed can be obtained by an ordinary method, and a regression analysis method can be obtained from this result. . Specifically, it can be determined by the following procedure.
First, one to several predetermined amino acid species x are added to the end of a standard sample to create a variant of the biomolecule having a different number of amino acid residues (Kato. A. et al., Biopolymers., Vol. 85, No. 1, p12-18, 2006).
Examples of the mutant include those added so that the number of amino acid residues is 1, 3, 5, and 7, for example.
Next, the obtained mutant is added to the solvent under any condition until the apparent dissolution slightly exceeds the limit (the solution becomes slightly cloudy), and the resulting solution is brought into an equilibrium state. Centrifuge until separated into supernatant and precipitate. Subsequently, the precipitate is removed and a supernatant (soluble fraction) is obtained.
The solvent used here is not particularly limited as long as it is an aqueous solution system, and any commonly used solvent can be used. The buffer solution may be appropriately selected according to the pH of the solvent used, and examples thereof include glycine buffer solution, acetate buffer solution, phosphate buffer solution, Tris buffer solution and the like. The concentration may be appropriately selected, but is preferably about 50 mM in order to minimize the influence on the solubility.
Moreover, what is necessary is just to select suitably other solvent conditions, such as temperature, pH, and salt concentration, for example, temperature 4 degreeC, pH 7.0, etc. are mentioned.
The centrifugation condition is not particularly limited as long as it is a condition normally used in the biochemical field. For example, 20,000 × g and 1 hour at 4 ° C. can be used. At this time, it is necessary to confirm that the equilibrium state has been reached. For example, after storing the obtained soluble fraction under measurement conditions, the concentration may be measured again, for example, every 6, 12, or 24 hours to confirm that the concentration does not change. Moreover, it is necessary to confirm the reversibility of aggregation. For example, reversibility can be confirmed by adding the buffer used for the measurement to the insoluble fraction and transferring the insoluble fraction to the soluble fraction.
Then, the solution concentration of this soluble fraction is measured with a spectrophotometer, this is defined as solubility, and a solubility curve with the number of amino acid residues as a variable is obtained.
Then, from the change in solubility when the number of amino acid residues is changed, the association free energies ΔG ^x _Aggr1 and ΔG ^x _Aggr2 of the amino acids are determined by the above equation (3) by regression analysis.
The standard sample is not particularly limited, but includes tryptophan or tyrosine that is easy to express and purify, and capable of spectroscopically measuring the sample concentration, and is completely in the natural state or completely denatured under the measurement conditions. What is in a state should just be. Further, the solubility is preferably several hundreds μM to 1 mM. Furthermore, the molecular weight is preferably 2,000 or more, more preferably about 10,000. Specifically, BPTI-21 and BPTI-22 (both BPTI at positions 21 and 22), which are mutants of the bovine pancreatic trypsin inhibitor BPTI (Bovine Pancreatic Trypsin Inhibitor), or Examples thereof include a peptide fragment composed of an amino acid sequence at positions 40 to 58 of BPTI-22, which is tubularly formed by SS bond, and a peptide fragment artificially designed based on a Cys (GlyAlaAlaSerAlaAla) ₄ CysGlyGly sequence.

As described above, by _obtaining two parameters ΔG ^X _Aggr1 and ΔG ^X _Aggr2 under arbitrary solvent conditions for all 20 types of amino acids, the parameters are values specific to each amino acid. The free energy of association (ΔG _Aggr ) of various peptides having the above can be determined freely, and further the free energy of association ΔG _Aggr of this peptide (the sum ΣΔG of the free energy of association ΔG ^X _Aggr of the amino acid species X contained in the peptide) ^X _Aggr ) can determine the solubility of the biomolecule to which the peptide is added according to the above equation (2) based on thermodynamic theory.
Such a parameter has low pH dependence and can be used in the above formula (2) without correction in a relatively wide pH range, but changes greatly with respect to the solvent conditions of temperature and salt concentration, and is appropriately adjusted. Correction is required.
In addition, parameters of amino acids having relatively similar physicochemical properties (leucine and isoleucine, asparagine and glutamic acid, etc.) tend to show relatively the same value. Therefore, in order to reduce development costs, it is also effective to estimate the approximate solubility of a sample to be obtained by determining representative amino acid parameters and substituting them as parameters for other amino acids.
Thus, the method of calculating the solubility of peptide-added biomolecules from amino acid sequences is highly versatile and effective as a method for solving problems caused by solubility in advanced research and development fields and medical application fields related to proteins. Is

The second invention is a method of designing (identifying) a peptide tag having a desired solubility by using the above formulas (1) to (3).
For example, as described later, a peptide tag consisting of an amino acid sequence composed of a combination of arginine or lysine having a high value of two parameters ΔG ^X _Aggr1 and ΔG ^X _Aggr2 provides high solubility.
FIG. 3 shows an improved state of the NMR spectrum by adding a tag that leads to high solubility. FIG. 3 (A) is an HSQC spectrum of “BPTI-22 (3.8 mM)”, and at this concentration (3.8 mM), the photograph sample is cloudy and has a lot of precipitates, so the shape of the cross peak is not good. Since it is uniform and broad, it is understood that analysis is impossible. On the other hand, FIG. 3 (B) shows the same measurement time (20 minutes) of a mutant (“BPTI-22-C5R (3.8 mM)”) in which 5 arginines (R) were added to the C-terminus of BPTI-22. The HSQC spectrum in FIG. 5 shows that although the sample has the same molecular weight, precipitation is reduced and solubility is improved, so that good spectral quality can be obtained.

  As the number of amino acid residues of such a peptide tag, if the molecular weight of the tag itself is too large, it may affect the functional expression, purification, structural analysis, etc. of the target protein. Preferably it is 3-15 residues.

The third invention is a method for synthesizing a protein that prevents the formation of inclusion bodies by attaching a peptide tag designed by the method according to the first or second invention to a target protein.
The target protein is not particularly limited with respect to the state (denatured or natural) and the type, number, and combination of amino acids constituting it.

The number of amino acid residues of the peptide tag in the third invention is 1 to 20 residues, preferably 3 to 15 residues, and more preferably 5 to 10 residues. When the number of amino acid residues is within the above range, solubility is improved and expression as an inclusion body can be suppressed. If the number of amino acid residues exceeds the above range, the molecular weight of the peptide tag itself is too large, which may affect the functional expression, purification, structural analysis, etc. of the target protein.
The ability to suppress inclusion body formation in the microbial cell is expressed and purified from a fusion protein in which a peptide tag is fused to the end of the target protein. After disrupting the microbial cell, centrifugation is performed, and each concentration in the soluble and insoluble fractions is determined. Is quantified by SDS-PAGE. In addition, the ability to suppress the formation of aggregates in a test tube is obtained by expressing and purifying a fusion protein in which a peptide tag is fused to the end of the target protein, centrifuging and then quantifying the concentration of the soluble fraction spectroscopically. .
Inclusion body formation suppression ability is related to the length of the peptide tag to be added, and the expression level of the inclusion body decreases depending on the number of amino acid residues of the peptide tag used.
Actually, each of the Caspase-Activated DNase (CAD) proteins was created by fusing three types of peptide tags with different amino acid residues to the C-terminus of the peptide fragment consisting of the amino acid sequence at positions 1 to 86, Analysis of the effect on inclusion bodies revealed that inclusion bodies were suppressed by adding a peptide tag that allowed for improved solubility.
For example, when a peptide tag consisting of 9-residue amino acids is added, inclusion body formation can be prevented by 90% or more. This suggests that inclusion body formation correlates with protein solubility. Therefore, by using the solubility-improving peptide tag designed according to the present invention, a highly versatile inclusion body formation suppression technique can be provided.

Such a peptide tag may be added to either the N-terminus or C-terminus of the target protein, or may be added to both the N-terminus and C-terminus, but depending on the three-dimensional structure of the target protein. May give a peptide tag to both the N-terminus and C-terminus, thereby causing an interaction between them and affecting the functional expression and three-dimensional structure of the target protein. In such a case, a peptide tag may be added to either the N-terminus or C-terminus.
Moreover, you may provide a linker to this peptide tag as needed. Examples of the linker include glycine and / or proline, which is about 1 to 5 residues, preferably about 2 to 5 residues. Attaching a linker to the peptide tag is to make the tag itself flexible and to prevent interaction between them by placing a distance between the target protein and the designed peptide tag. This is for the purpose.

  The method for obtaining the fusion protein by attaching the peptide tag to the target protein is not particularly limited. For example, cell-free (in vitro) synthesis or E. coli by gene recombination using a gene that expresses the fusion protein. Examples thereof include a method for transforming a host such as yeast and yeast and culturing the obtained host, and a method for chemical synthesis using a synthesizer.

The first invention of the present application uses a thermodynamic model of aggregation of biomolecules, unlike a conventional method for calculating solubility based on a hydrophobic / hydrophilic model, and the relationship between the value of free energy associated with one molecule and the solubility of the biomolecule. The physicochemical theoretical calculation of the solubility according to the laws of thermodynamics is made possible, and it is possible to predict the solubility quantitatively with high accuracy before protein expression or peptide synthesis.
Therefore,
1. When dealing with peptides and proteins with low solubility or when it is necessary to dissolve proteins at high concentrations (functional analysis at high concentrations or structural analysis by NMR), or the high rate of creation and purification of protein chips and microbioreactors In various cases where solubility is a problem, such as crystallization, a target can be efficiently selected in advance when conducting a comprehensive analysis on a huge population of peptides and proteins.
2. In the calculation of the solubility of the target peptide or target protein, it can be used under solvent conditions such as any pH, temperature, salt concentration, etc., and solubility prediction excellent in versatility can be performed. As a result, for example, application to the development of a solubility calculation system using amino acid sequences, solvent conditions, and protein three-dimensional structure as input data can be expected as shown in FIG.

Moreover, according to the second invention, a peptide tag having a desired solubility can be designed by using the method for obtaining the solubility.
Therefore,
3. When a peptide or protein with low solubility is to be analyzed, by adding a peptide tag that can dramatically improve its solubility without changing the function, structure, stability, etc. of the analysis target, etc. It enables efficient analysis and can reduce the analysis cost.
4). In peptide synthesis, the solubility of the target peptide can be designed in advance.

Furthermore, the third invention is based on the knowledge focusing on the strong correlation between the tendency of protein aggregation and inclusion body formation in E. coli, and the tag designed as described above is added to the target protein. Thus, inclusion bodies can be prevented from forming in E. coli.
Therefore,
5. Solubilized protein can be produced, and the yield can be improved.
6). Several types of peptide tags can improve the solubility of most proteins, and can be expected to be applied to the development of highly versatile inclusion body-preventing expression vectors.

Example 1
[Synthesis of fusion protein]
In this Example 1, mutant BPTI-22 (58aa, molecular weight 5,880) having 22 Ala in the poorly soluble protein bovine pancreatic trypsin inhibitor (BPTI) was used as the target protein.
A pMMHaBPTI-22 expression vector (see J. Mol. Biol., Vol. 298, p493-501, 2000) was used for protein expression. This expression vector has a His tag sequence added to the N-terminus of the trpΔLE leader sequence using a reagent kit (trade name: QuikChange II XLsite-Directed Mutagenesis Kit) manufactured by Stratagene.
Escherichia coli JM109 (DE3) pLysS was transformed with this expression vector, and the resulting colonies were directly inoculated into 100 ml of LB medium containing 50 μg / ml ampicillin and 34 μg / ml chloramphenicol, and 6 ° C. at 6 ° C. Cultured with shaking for hours. Thereafter, the cells were inoculated into 2 L of LB medium and cultured with shaking at 37 ° C. overnight. The resulting culture was transferred to a 500 ml centrifuge tube and centrifuged at 5,000 × g for 20 minutes at 4 ° C. to collect the cells. The collected cells were suspended in 100 mM Tris-HCl buffer (pH 8.7) and washed again by centrifugation at 7,000 × g and 4 ° C. for 20 minutes.
The washed cells are collected in 2 ml of the culture solution in a 50 ml centrifuge tube, and about 10 times the amount of buffer A (50 mM Tris-HCl (pH 8.7), 150 mM NaCl). ). While the suspension was ice-cooled, ultrasonic crushing was performed until the suspension became translucent.
After crushing, centrifugation was performed at 7,000 × g and 4 ° C. for 20 minutes to remove the supernatant, and an insoluble fraction was obtained.
The insoluble fraction was again suspended in buffer B (50 mM Tris-HCl (pH 8.7), 1% by volume NP-40, 1% by weight Deoxycholic acid, 1 mM EDTA), and sonicated. This operation was repeated 3 times until the liquid became white.
Since the target protein forms inclusion bodies in E. coli, an insoluble fraction was obtained after centrifugation.
Subsequently, the insoluble fraction (protein forming the inclusion body) is solubilized with 6MGuHCl (pH 8.7), and stirred for 10 hours in order to cause S—S binding at Cys5 and Cys55 in the BPTI mutant molecule. Oxidized with air. Thereafter, dialysis was carried out with 50 mM Tris-HCl buffer (pH 8.7), and a precipitate appeared when 6 MGuHCl was removed. This is because the target protein also precipitates because the tag protein having the trpΔLE leader sequence added to the target protein precipitates at pH 8.7.
The precipitate was collected by centrifugation at 7,000 × g and 4 ° C. for 20 minutes, 10 times the amount of 70% formal acid was added to the weight of the precipitate, and the CNBr reaction (10 mg / ml CNBr / 70% formal acid), and the tag protein was cleaved from the target protein.
Furthermore, after speed vac for about 2 hours, it dialyzed overnight with mill-Q. Then, dialysis was performed overnight with 10 mM phosphate buffer (pH 6.0). After dialysis, centrifugation was performed at 7,000 × g and 4 ° C. for 20 minutes, the precipitate (tag protein) was removed, and the fusion protein (GGnR sequence (n is the N-terminal or C-terminal of BPTI-22) was removed from the soluble fraction. A fusion protein to which a peptide tag having arginine (R) residues and n = 1, 3, 5) was added was obtained.
Acetic acid was added to this solution and purified by HPLC. The reverse phase column (YMC-Pack PROTEIN-RP S, 5 μm) used for purification was equilibrated with 25% acetonitrile, and the protein was adsorbed onto the column. After adsorption, a linear gradient (25-60%, acetonitrile increase rate: 1% / min) was applied to elute the protein adsorbed on the column.
Then, the absorbance at 220 nm of the eluate was measured online. At this time, the flow rate was 8.0 ml / min.
The fraction in which the peak appeared in the absorbance was identified using ESI-TOFMS “JMS-T100X” (manufactured by JEOL Ltd.), the purified preparation was lyophilized and stored at −80 ° C.

[ _{Determination of} parameters ΔG ^X _Aggr1 and ΔG ^X _Aggr2 ]
The fusion protein thus obtained (a fusion protein to which a peptide tag having a GGnR sequence at the C terminus was added) was dissolved in 100 mM acetate buffer (pH 4.7), and the resulting precipitate was 20,000 × g, Removed by centrifugation at 4 ° C. for 1 hour. Proteins were basically handled on ice in all experimental operations.
The absorbance at 280 nm was measured with a spectrophotometer to determine the solubility limit concentration at 20 ° C. and pH 7.0, and this was used as the solubility. The relationship between “number of added amino acid residues” and “solubility” was experimentally determined. The result is shown in FIG.
In FIG. 4, the horizontal axis represents the number of added amino acid residues, the vertical axis represents the solubility, and the solubility of the standard peptide (nothing added) was set to “1”.
Subsequently, two parameters ΔG ^X _Aggr1 and ΔG ^X _Aggr2 (J / mol) were obtained from this solubility curve (ie, from the “solubility” obtained experimentally) by regression analysis with respect to the number N of added amino acids.
As a result, ΔG ^R _Aggr1 of arginine (R) was determined to be 4.72 J / mol, and ΔG ^R _Aggr2 was determined to be 143.65 J / mol.
Similarly to arginine, when two parameters ΔG ^X _Aggr1 and ΔG ^X _Aggr2 (J / mol) were determined for lysine (K), ΔG ^K _Aggr1 was 4.72 J / mol and ΔG ^K _Aggr2 was 130. It became 47 J / mol.

[Calculation of solubility of peptide tag fusion protein]
In the experiment, the solubility of the BPTI-22 protein without the peptide tag and the fusion protein in which the arginine tag (RRRGG, RRRRRGG (“GG” is given as a linker residue)) was added to the N-terminus of BPTI-22 was measured. It was measured.
That is, 100 mM acetate buffer (pH 4.7) was added to each protein until it became cloudy (aggregated) (about 100 μl), and these protein solutions 20,000 × g were centrifuged at 4 ° C. for 1 hour. And about the soluble fraction of this protein solution, the light absorbency in 280 nm was measured with the spectrophotometer, the protein concentration was calculated | required, and this measured value was made into solubility.
As a result, the solubility of the fusion protein to which the peptide tag is added is compared with the solubility of the BPTI-22 protein (1.70 mM) when the peptide tag whose amino acid sequence is RRRGG is added (solubility is 2.70 mM). When a peptide tag having an amino acid sequence of RRRRRGG was added (solubility is 6.20 mM), it was confirmed to improve 3.65 times respectively.
On the other hand, ΔG ^R _Aggr1 (4.72 J / mol) and ΔG ^R _Aggr2 (143.65 J / mol) of arginine obtained as described above are substituted into the above formula (1), and ΔG ^R _{Aggr of the} peptide tag is Asked.
Here, since all the peptide tags are composed only of arginine, ΔG ^R _Aggr = ΔG _Aggr and the solubility of the peptide tag is derived from the above equation (2).
The solubility of the protein with the addition of 3 arginines is as follows:
ΔG _Aggr = 4.72 × 3 + 143.65 × 3 ² = 1307.01 J / mol
The solubility ratio of both proteins at 277.15 K (4 ° C.) is S = exp (1307.01 / (8.314 × 277.15)) = 1.76
Therefore, at a temperature of 4 ° C, the solubility of the protein added with 3 arginines was calculated to be 1.76 times better than that without the protein, which was in good agreement with the 1.59 times measured in the above experiment. .

Similarly, the solubility of the protein with the addition of 5 arginines is as follows:
ΔG _Aggr = 4.72 × 5 (= 23.6) + 143.65 × 5 ² (3591.25) = 361.85 J / mol
And the ratio of the solubilities of both proteins at 277.15 K (4 ° C.) is S = exp (361.85 / (8.314 × 277.15)) = 4.80
Thus, at a temperature of 4 ° C, the solubility of the protein with 5 arginines added was calculated to be 4.80 times better than that with no added protein, 3.65 times and 24% measured in the above experiment. The theoretical value and the measured value agreed with each other.
When the solubility value obtained by the calculation as described above was compared with the actual measurement value, they were approximated, and it was verified that the solubility calculation can be performed with high accuracy by the calculation method of the present invention.
Moreover, it was confirmed that the solubility of the peptide tag depends on the type and number of amino acids constituting the tag depending on variables, and is not greatly influenced by the sequence of these amino acids.

Next, the fusion protein solution 3.8 mM to which the peptide tag was added and the BPTI-22 solution without the peptide tag were used as analysis samples, and 15N-HSQC spectra were measured by NMR method.
The result is shown in FIG. Here, the same measurement time (20 minutes) between a mutant in which 5 arginines (R) are added to the C-terminus of BPTI-22 (“BPTI-22-C5R (3.8 mM)”) and BPTI-22 The HSQC spectra at were compared. As is clear from FIG. 3, in the above fusion protein, the spectral quality is evaluated by S / N ratio, cross-peak line width and form, and the measurement time is obtained in about 1/8 of the untagged sample. NMR spectra were obtained. Therefore, it has been clarified that the addition of the peptide tag can greatly improve the solubility without changing the function, structure and stability of the protein.

Example 2
In this Example 2, 3, 5 lysine residues were added to the N-terminus of mutant BPTI-22 having 22 Ala in the poorly soluble protein bovine pancreatic trypsin inhibitor (BPTI) as the target protein. And compared with the calculation results.
In the experiment, the solubility of a BPTI-22 protein without a peptide tag and a fusion protein in which a lysine tag (KKKGG, KKKKKGG) was added to the N-terminus of BPTI-22 was measured in the same manner as in Example 1.
That is, 100 mM acetate buffer (pH 4.7) was added to each protein until it became cloudy (aggregated) (about 100 μl), and these protein solutions 20,000 × g were centrifuged at 4 ° C. for 1 hour. And about the soluble fraction of this protein solution, the light absorbency in 280 nm was measured with the spectrophotometer, the protein concentration was calculated | required, and this measured value was made into solubility.
As a result, the solubility of the fusion protein to which the peptide tag is added is compared with the solubility of the BPTI-22 protein (1.70 mM) when a peptide tag whose amino acid sequence is KKKGG is added (solubility is 2.66 mM). When a peptide tag having an amino acid sequence of KKKKKGG was added (solubility was 5.37 mM), it was confirmed to improve 3.16 times experimentally.
On the other hand, ΔG ^K _Aggr1 (4.72 J / mol) and ΔG ^K _Aggr2 (130.47 J / mol) of lysine obtained as described above are substituted into the above formula (1), and ΔG ^K _{Aggr of the} peptide tag is Asked.
Here, since all of the peptide tags are composed only of lysine, ΔG ^K _Aggr = ΔG _Aggr and the solubility of the peptide tag is derived from the above equation (2).
The solubility of the protein with three lysine additions is as follows:
ΔG _Aggr = 4.72 × 3 + 130.47 × 3 ² = 1188.39 J / mol
And the ratio of the solubilities of both proteins at 277.15 K (4 ° C.) is S = exp (1188.39 / (8.314 × 277.15)) = 1.67
Therefore, at a temperature of 4 ° C, the solubility of the protein added with 3 lysines was calculated to be 1.67 times higher than that without the protein, which was in good agreement with the 1.56 times measured in the above experiment. .

Similarly, the solubility of 5 proteins added with lysine is as follows:
ΔG _Aggr = 4.72 × 5 (= 23.6) + 130.47 × 5 ² (326.75) = 328.35 J / mol
And the ratio of the solubilities of both proteins at 277.15 K (4 ° C.) is S = exp (328.35 / (8.314 × 277.15)) = 4.16
Thus, at a temperature of 4 ° C, the solubility of the protein with 5 lysine added was calculated to be 4.16 times better than that without the protein added, 3.16 times and 24% measured in the above experiment. It matched in the range of error.
When the solubility value obtained by the calculation as described above was compared with the actual measurement value, they were approximated, and it was verified that the solubility calculation can be performed with high accuracy by the calculation method of the present invention.

Example 3
[Combination pattern of amino acid X types and the number of residues N]
First, using a pAED vector, <1> the target protein, <2> a fusion protein in which a peptide tag is added to the end of the target protein, <3> a sample in which a His tag is further added to the fusion protein, <4> the above object Four constructs, a sample with a His tag at the end of the protein, were expressed in E. coli.
Here, BPT having a molecular weight of 6 kD to 30 kD was used as the target protein. As the peptide tag, a peptide consisting of a GGRn sequence (n is the number of residues of arginine (R), n = 1, 3, 5) was used.
The sequence was redesigned so that each construct had the greatest improvement in solubility with respect to the target protein.
The solubility of the protein with one arginine added is as follows:
ΔG ^X _Aggr = 4.72 × 1 + 143.65 × 1 ² = 2317.28 J / mol
Therefore, at a temperature of 300K, the solubility of a protein with one arginine added is calculated to be 1.06 times higher than that without a protein.
In addition, the solubility of the protein added with 3 arginines is as follows:
ΔG ^X _Aggr = 4.72 × 3 + 143.65 × 3 ² = 1307.01 J / mol
Therefore, at a temperature of 300 K, the solubility of the protein with 3 arginines added is calculated to be 1.71 times higher than that without the protein.
Similarly, the solubility of the protein with the addition of 5 arginines is as follows:
ΔG ^X _Aggr = 4.72 × 5 + 143.65 × 5 ² = 361.85 J / mol
Therefore, at a temperature of 300K, the solubility of the protein with 5 arginines added is calculated to be 4.26 times higher than that without.
From the above calculation results, the solubility was maximized when 5 amino acid residues were added.
From this result, it was found that the amino acid sequence of the peptide tag that maximizes the solubility of each construct in the target protein was GG5R.

[Preventing inclusion body formation with solubility enhancement tags]
Example 4
In Example 4, a peptide fragment CAD _1-86 C34S having an amino acid sequence of positions 1 to 86 of a mutant in which Cys at position 34 of a basic protein CAD (Caspase-Activated Dnase) is substituted with Ser as a target protein. Mutants were used.
First, fusion proteins in which 3, 6 and 9 arginine residues were added as peptide tags to the C-terminal side of the CAD _1-86 C34S mutant were prepared. C0R (addition number 0) and C3R (addition number 3), respectively. , C6R (addition number 6) and C9R (addition number 9).

That is, when Escherichia coli BL21 (DE3) pLysS was used as a host and the culture temperature was set to 25 ° C. and 37 ° C. were examined. IPTG was added in the range of OD590 nm = 0.6 to 0.7 to induce expression. Thereafter, the cells were cultured for 4 hours, and after measuring the OD, each sample was adjusted so that the OD value at 200 μl was 8.0.
After purifying these samples and disrupting the cells, they were centrifuged at 15,000 × g for 15 minutes to recover the supernatant (soluble fraction) and the precipitate (insoluble fraction), respectively. (PH 8.0) 200 μl was added and quantitative analysis was performed by SDG-PAGE to examine the influence on inclusion body formation. The result is shown in FIG.
From FIG. 5, under the culture conditions of 25 ° C., the amount of protein in the insoluble fraction (ppt) decreases as the number of arginine residues added (the tag length increases) (ie, the soluble fraction ( The amount of protein in sup) is increased), and as the amino acid to be added becomes longer, the solubility is improved, and it was found that there is an effect in preventing inclusion body formation. Note that arginine-based tagging does not exhibit a dissolution effect at 37 ° C. and has an effect at 25 ° C. The CAD _1-86 C34S mutant cannot form a structure at high temperatures and tends to aggregate. At ℃, it is thought that inclusion forms can be prevented by forming a structure and adding a tag. For other unstable proteins, the tag effect can be improved by lowering the culture temperature at the same time as adding the tag. It seems that it can be used to the maximum.
Thus, it was revealed that a peptide tag with improved solubility based on arginine is effective in preventing inclusion bodies formed by basic proteins.

Example 5
In Example 5, acidic protein N-intein (127aa, molecular weight 14388.2) was used as the target protein.
In the same manner as in Example 4, fusion proteins were prepared by adding 3,6,9 aspartic acids as peptide tags to the C-terminus of the N-intein protein, and C0R (addition number 0) and C3R (addition number 3), respectively. ), C6R (addition number 6), and C9R (addition number 9).
BL21 (DE3) pLysS was used as an expression host, and the culture temperature was 37 ° C. Expression was induced by adding IPTG in the range of OD590 nm = 0.6 to 0.7. Thereafter, the cells were cultured for 4 hours, and the influence on inclusion body formation was examined in the same manner as in Example 4. The result is shown in FIG.
As shown in FIG. 6, although there is almost no difference in the expression level between all the cells (all), the soluble fraction (sup) is greatly different, and the number of aspartic acid residues increases and the soluble fraction increases. The expression level in minutes increased, and almost all proteins were expressed as soluble fractions in C9D. That is, it was found that inclusion bodies formed by N-intein can be prevented by adding 9 aspartic acid residues.
From this, it has been clarified that addition of an aspartic acid-based tag is effective in preventing inclusion bodies formed by acidic proteins.
Moreover, from Example 4 and Example 5, it became clear that the inclusion body formation prevention effect of the solubility improvement peptide tag can be obtained with both basic and acidic proteins.
Furthermore, the isoelectric point (pI) of the CAD _1-86 C34S mutant and the pKa of arginine are 9.77 and 12.5, respectively, while the isoelectric point (pI) of N-intein and the pKa of aspartic acid are respectively Since it is 4.36 and 3.9 to 4.0, it is expected that it is effective to design a peptide tag with improved solubility for preventing inclusion body formation based on the isoelectric point of the target protein. It was done.

The present invention is suggested to be applied to the following cases, for example.
・ When you want to use a high concentration of enzyme or protein in a bioreactor or protein chip.
-In an inkjet printer, etc., when it is desired to prevent organic matter from precipitating when the solubility becomes low during ink use.
・ When you want to predict the tendency of formation of inclusion bodies of peptides (proteins).
・ When you want to solubilize low-solubility globular proteins, membrane proteins, etc., such as peptides, hormones, enzymes, and denatured proteins.

It is a schematic diagram of the thermodynamic model showing aggregation of a biomolecule. It is a figure which shows the example of the input screen of a solubility calculation system. The comparison of “solution state (photograph)” and “NMR spectrum” by addition of a solubility-enhancing peptide tag is shown, (A) is the state of BPTI-22, and (B) is the state of BPTI-22-C5R. It is the solubility curve calculated | required experimentally about arginine. It is a photograph of SDG-PAGE showing the influence of arginine-based tagging on inclusion body formation of CAD _1-86 C34S mutant and the culture temperature dependency, (A) is when the culture temperature is 37 ° C., and (B ) When 25 ° C. It is a photograph of SDG-PAGE which shows the influence which aspartic acid type tag addition has on the inclusion body formation of N-intein protein.

Explanation of symbols

ΔG _Aggr meeting free energy

Claims (5)

A method of calculating the solubility S of a peptide-added biomolecule in which a peptide having an arbitrary amino acid sequence is added to the end of the biomolecule based on a thermodynamic theory, wherein the Gibbs free energy ΔG _Aggr of the peptide association is Calculated as the sum of ΔG ^X _Aggr defined by the quadratic function of the number N of residues of amino acid species X constituting the amino acid sequence represented by formula (1), and the Gibbs free energy ΔG _Aggr of the peptide association is A solubility calculation method comprising using the following formula (2) to calculate the solubility S of the peptide-added biomolecule.

ΔG ^X _Aggr = ΔG _Aggr0 + ΔG ^X _Aggr1 N + ΔG ^X _Aggr2 N ² formula (1)
(In the above formula (1), X is an amino acid species added to the biomolecule, ΔG ^X _Aggr is the Gibbs free energy (J / mol) of association associated with addition of the amino acid species X to the biomolecule, ΔG _Aggr0 Is the Gibbs free energy (J / mol) of the association of the biomolecule, ΔG ^X _Aggr1 is the first term (J / mol) of the Gibbs free energy of association of the amino acid species X, and ΔG ^X _Aggr2 is the amino acid species X (Second term of Gibbs free energy of association (J / mol), N represents the number of residues of amino acid species X contained in the added peptide)

S = exp (ΔG _Aggr / RT) Equation (2)
(In the above formula (2), S is the solubility of the peptide-added biomolecule, R is the gas constant, T is the absolute temperature, ΔG _Aggr is the free energy of association of each amino acid species X with respect to all amino acid species X contained in the peptide _Represents the sum of ΔG ^X _Aggr (ΣΔG ^X _Aggr (J / mol)).)
A variant in which one amino acid species x of the above amino acid species X is added to the end of a standard biomolecule, and the variant is added to an arbitrary solvent at a concentration higher than the solubility limit concentration. The solution concentration of the soluble fraction is measured, and from the change in solubility when the number of residues n of the amino acid species x is changed, the Gibbs free energy ΔG ^x _Aggr1 , ΔG ^x of the association of the amino acid species x is determined by regression analysis. The solubility calculation method according to claim 1, wherein _Aggr2 is calculated by the following formula (3).

s = exp ((ΔG ^x _Aggr1 n + ΔG ^x _Aggr2 n ² ) / RT) Equation (3)
(In the above formula (3), s is the ratio between the solubility of the mutant and the solubility of the standard biomolecule, R is the gas constant, T is the absolute temperature, and x is a specific type added to the standard biomolecule. Amino acid species, n is the number of residues of the amino acid species x, ΔG ^x _Aggr1 is the first term (J / mol) of the Gibbs free energy of association of the amino acid species x, and ΔG ^x _Aggr2 is the association of the amino acid species x Represents the second order term (J / mol) of Gibbs free energy.)
  When the amino acid sequence of the peptide is selected from the combination pattern of the type of amino acid species X and the number N of residues thereof, peptide bioaddition in each combination pattern is performed by the solubility calculation method according to claim 1 or 2. A method for designing a peptide tag, wherein the solubility S of a molecule is calculated, a combination pattern in which the solubility S is a desired value is selected, and the amino acid sequence of the peptide is designed.   A method for preventing formation of inclusion bodies of the target protein by expressing the target protein as a fusion protein with a peptide tag designed by the method according to claim 3 in a microbial cell or in a test tube.   The method for preventing inclusion body formation according to claim 4, wherein the peptide tag is an amino acid having 1 to 20 residues.