JP5765700B2 - Soluble control tag design apparatus and method and program thereof - Google Patents

Description

  The present invention relates to a technique for controlling solubilization or insolubilization of proteins.

  Protein production is an important issue in fields such as biochemistry, structural science, pharmacy, and industry. In order to obtain a protein successfully by genetic recombination, it is necessary to overcome the three steps of expression, solubility and purification. Until now, living cells have often been used in protein expression systems. Escherichia coli is one of the preferred hosts because it is genetically easy to handle and a large amount of recombinant protein can be obtained. In addition to methods using microorganisms and cultured cells, methods using protein synthesis systems extracted from prokaryotes and eukaryotes have also been proposed. These techniques can express the target protein in large quantities, and further dramatically increase the solubility of the protein and facilitate purification.

  A reliable approach to increase protein solubility is to add highly soluble proteins to the target protein. In general, the added sequence is called a tag. Several proteins that function as solubilizing tags have been reported in the literature. For example, glutathione-S-transferase (GST) in Non-Patent Document 1, maltose binding protein (MBP) in Non-Patent Document 2, thioredoxin (Trx) in Non-Patent Document 3, N-use substance (NusA) has been reported. These proteins are well known empirically as having high solubility.

  Similar to solubilization tags, affinity tags have also been developed to facilitate the purification of recombinant proteins. Both MBP and GST have solubilization tags and functions as affinity tags. GST binds strongly to glutathione resin, and MBT binds strongly to amylase resin.

Nygren, P.A.et.al, `` Engineering proteins to facilitate bioprocessing '' Trends Biotechnol. 12 (1994), 184-188 Nallamsetty, S. and Waugh, D.S.``Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners '' Protein Expr. Purif. 45 (2006), 175-182 LaVallie, E.R.et.al, `` A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm '' Biotechnology (NY) 11 (1993), 187-193 Davis, G.D.et.al, `` New fusion protein systems designed to give soluble expression in Escherichia coli '' Biotechnol. Bioeng 65 (1999) 382-388

  Despite being a useful tool for protein solubility and purification, tags are not useful for all proteins. Researchers must express various recombinant proteins with different tags attached and compare their solubility to find the optimal tag. In addition, it is necessary to remove the tag for biochemical research and therapeutic protein testing. This is because the large tag size affects both the structure and function of the target protein. These problems are particularly impeding high efficiency cloning and expression projects.

  The present invention proposes a method of designing a tag that controls the solubility of a protein based on the idea that the amino acid sequence of the terminal region affects the solubility of the protein.

  The present invention designs a soluble control tag based on data read from a database that stores amino acid sequences of proteins that have been experimentally confirmed to be soluble or insoluble. Specifically, the amino acid sequences at the N-terminus of the soluble protein and the insoluble protein are read from the database, and the read amino acid sequence is analyzed to obtain the soluble control tag.

  Thus, by analyzing the amino acid sequences found at the N-terminus of soluble proteins and insoluble proteins, soluble control tags can be appropriately designed based on actual data. That is, the soluble control tag varies depending on conditions such as an expression system, but a tag that meets the conditions can be designed by obtaining the soluble control tag based on actual expression system data.

The soluble control tag design apparatus of the present invention is an apparatus for designing a soluble control tag based on data read from a database storing amino acid sequences of soluble protein and insoluble protein, and the residue length of the soluble control tag to be obtained. An input unit for inputting L, an arithmetic unit for obtaining a soluble control tag based on data read from the database, and an output unit for outputting the soluble control tag obtained by the arithmetic unit,
The computing unit is
(1) generating an amino acid similarity group sequence in which all combinations of amino acid similarity groups are defined which are sequences of amino acid similarity groups of L residue length;
(2) reading an amino acid sequence having an L residue length contained in the N-terminal K residues (K ≧ L) of the soluble protein and the insoluble protein from the database;
(3) counting the number of appearances of each amino acid similarity group sequence at the N-terminus of each of the soluble protein and the insoluble protein based on the read-out amino acid sequence;
(4) When designing a solubilization tag that enhances solubility, the appearance frequency of each amino acid similarity group sequence at the N-terminus of the soluble protein; when designing an insolubilization tag that enhances insolubility, each amino acid similarity at the N-terminus of the insoluble protein Calculating the frequency of occurrence of the group sequence, and obtaining an amino acid similarity group sequence having an appearance frequency higher than a predetermined threshold as a frequent amino acid similarity group sequence;
(5) clustering a plurality of frequent amino acid similarity group sequences into a plurality of clusters;
(6) A soluble protein when designing a solubilizing tag; an amino acid sequence of L residue length read from the N-terminal K residue of an insoluble protein when designing an insolubilizing tag; and the frequent amino acid similarity group sequence If the amino acid sequence corresponds to the frequent amino acid similarity group sequence, the number of occurrences of each type of amino acid at each location in the amino acid sequence is counted based on the amino acids contained in the amino acid sequence. A step of sequentially performing a process of uploading the read amino acid sequence;
(7) summing up the number of appearances for each type of amino acid at each location in the amino acid sequence determined by the frequent amino acid similarity group sequence in the same cluster;
(8) obtaining an amino acid sequence comprising a combination of amino acids having the highest number of appearances at each location as a solubilization tag or an insolubilization tag;
Execute.

  Thus, by calculating the number of appearances of the amino acid similarity group sequence contained at the N-terminus of the soluble protein or insoluble protein, the amount of calculation can be reduced as compared with the case of determining the number of appearances of the amino acid sequence. In addition, when compared with the same length of residues, the total number of amino acid similarity group sequences is much smaller than the total number of amino acid sequences. Therefore, even when there are few protein data stored in the amino acid sequence database, a certain tendency can be found in the number of appearances.

In the step (4), when a soluble tag is obtained, an amino acid similarity group sequence in which an S value and a p value calculated by the following formulas are S> 0.9 and p <1 × 10 ⁻⁵ is a frequent amino acid similarity group. It may be obtained as an array.

  The step (5) converts the frequent amino acid similarity group sequence into a 20-dimensional coordinate value in which the amino acid included in the frequent amino acid similarity group is “1” and the amino acid not included is “0”. Clustering may be performed by generating a dendrogram based on the Euclidean distance of the similar group arrangement and cutting the dendrogram at a predetermined height.

The soluble control tag design method of the present invention is a method of designing a solubilized tag that increases the solubility of a protein or an insolubilized tag that increases insolubility as a soluble control tag,
(1) generating an amino acid similarity group sequence in which all combinations of amino acid similarity groups are defined which are sequences of amino acid similarity groups of L residue length;
(2) reading an amino acid sequence having an L residue length contained in the N-terminal K residues (K ≧ L) of the soluble protein and the insoluble protein from a database storing the amino acid sequences of the soluble protein and the insoluble protein;
(3) counting the number of appearances of each amino acid similarity group sequence at the N-terminus of each of the soluble protein and the insoluble protein based on the read-out amino acid sequence;
(4) When designing a solubilization tag, the frequency of appearance of each amino acid similarity group sequence at the N-terminus of the soluble protein, and when designing an insolubilization tag, the frequency of occurrence of each amino acid similarity group sequence at the N-terminus of the insoluble protein. Calculating an amino acid similarity group sequence having an appearance frequency higher than a predetermined threshold as a frequent amino acid similarity group sequence;
(5) clustering a plurality of frequent amino acid similarity group sequences into a plurality of clusters;
(6) A soluble protein when designing a solubilizing tag; an amino acid sequence of L residue length read from the N-terminal K residue of an insoluble protein when designing an insolubilizing tag; and the frequent amino acid similarity group sequence If the amino acid sequence corresponds to the frequent amino acid similarity group sequence, the number of occurrences of each type of amino acid at each location in the amino acid sequence is counted based on the amino acids contained in the amino acid sequence. A step of sequentially performing a process of uploading the read amino acid sequence;
(7) summing up the number of appearances for each type of amino acid at each location in the amino acid sequence determined by the frequent amino acid similarity group sequence in the same cluster;
(8) obtaining an amino acid sequence comprising a combination of amino acids having the highest number of appearances at each location as a solubilization tag or an insolubilization tag;
Is provided.

The program of the present invention is a program for designing a solubilization tag that enhances the solubility of a protein or an insolubilization tag that enhances insolubility as a solubility control tag.
(1) The number of occurrences of each amino acid similarity group sequence that defines all combinations of amino acid similarity groups, each of which appears at the N-terminus of each of the soluble protein and the insoluble protein. Securing an area to be
(2) reading an amino acid sequence having an L residue length contained in the N-terminal K residues (K ≧ L) of the soluble protein and the insoluble protein from a database storing the amino acid sequences of the soluble protein and the insoluble protein;
(3) counting the number of appearances of each amino acid similarity group sequence at the N-terminus of each of the soluble protein and the insoluble protein based on the read-out amino acid sequence;
(4) When designing a solubilization tag, the frequency of appearance of each amino acid similarity group sequence at the N-terminus of the soluble protein, and when designing an insolubilization tag, the frequency of occurrence of each amino acid similarity group sequence at the N-terminus of the insoluble protein. Calculating an amino acid similarity group sequence having an appearance frequency higher than a predetermined threshold as a frequent amino acid similarity group sequence;
(5) clustering a plurality of frequent amino acid similarity group sequences into a plurality of clusters;
(6) A soluble protein when designing a solubilizing tag, and an amino acid sequence of L residue length read from the N-terminal K residue of the insoluble protein when designing an insolubilizing tag and the frequent amino acid similarity group sequence If the amino acid sequence corresponds to the frequent amino acid similarity group sequence, the number of occurrences of each type of amino acid at each location in the amino acid sequence is counted up based on the amino acids contained in the amino acid sequence. Sequentially performing processing for the read amino acid sequence;
(7) summing up the number of appearances for each type of amino acid at each location in the amino acid sequence determined by the frequent amino acid similarity group sequence in the same cluster;
(8) obtaining an amino acid sequence comprising a combination of amino acids having the highest number of appearances at each location as a solubilization tag or an insolubilization tag;
(9) outputting a solubilized tag or an insolubilized tag;
Is executed.

  According to the present invention, by analyzing the amino acid sequence having an L residue length at the N-terminus of soluble proteins and insoluble proteins, it is possible to design an appropriate soluble control tag that meets the conditions of the expression system and the like.

It is a figure which shows the structure of the soluble control tag design apparatus of embodiment. It is a flowchart which shows operation | movement of the soluble control tag design apparatus of embodiment. It is a figure which shows the amino acid contained in an amino acid similarity group. It is a figure which shows the amino acid similarity group arrangement | sequence of 7 residue length. It is a figure which shows the example of N terminal 20 residues of soluble protein and insoluble protein. It is a figure which shows the example which reads the amino acid sequence of 7 residue length from N terminal 20 residues of protein. (A) It is a figure which shows the amino acid similarity group corresponding to an amino acid sequence. (B) It is a figure which shows the amino acid similarity group sequence corresponding to an amino acid sequence. (C) It is a figure which shows the example which counted the appearance frequency of the amino acid similarity group sequence | arrangement. It is a figure which shows the example which digitized the amino acid similarity group. It is a figure which shows the example which digitized the amino acid similarity group sequence. It is a figure which shows the example which clustered the frequent amino acid similarity group arrangement | sequence. It is a figure which shows the example of the amino acid sequence corresponding to a frequent amino acid similarity group sequence. It is a figure which shows the example which calculated | required the frequency | count of appearance for every kind of amino acid in each place of an amino acid sequence.

  Hereinafter, the soluble control tag designing apparatus and method of the present invention will be described with reference to the drawings. In this embodiment, the soluble control tag is particularly intended for short amino acid sequences of several to 100 residues.

  FIG. 1 is a diagram illustrating a configuration of a soluble control tag designing apparatus 10 according to an embodiment. The soluble control tag designing apparatus 10 includes an input unit 12 for inputting a residue length L of a soluble control tag to be designed, and soluble protein and insoluble protein data stored in a protein database (hereinafter referred to as “protein DB”) 14. Is used to design a soluble control tag having an L residue length, and an output unit 18 that outputs data of the designed soluble control tag. A RAM 20 and a ROM 22 are connected to the CPU 16. When designing the solubilization tag, the CPU 16 writes data necessary for calculation processing into the RAM 20 and reads out data from the RAM 20. The CPU 16 designs the solubilization tag by reading and executing the program 24 stored in the ROM 22. This program 24 is also included in the scope of the present invention.

  The protein DB 14 stores amino acid sequence data of a protein that is confirmed to be a soluble protein or an insoluble protein based on an experiment expressed in a predetermined system. In the present embodiment, a configuration in which the soluble control tag design apparatus 10 includes the protein DB 14 is taken as an example, but the protein DB 14 may be outside the soluble control tag design apparatus 10. In this case, the soluble control tag designing apparatus 10 includes a communication unit for communicating with an external database, and reads the data of the protein DB 14 via the communication unit.

  The soluble control tag designing apparatus 10 is configured by, for example, a personal computer. The input unit 12 includes, for example, a keyboard, a mouse, a CD-ROM reader, and the like. The output unit 18 includes, for example, a monitor, a printer, a CD-ROM writing device, and the like.

  Next, a process for designing a soluble control tag by the soluble control tag designing apparatus 10 will be described. In the following description, a case where a solubilization tag is designed as a solubility control tag is taken up, but an insolubilization tag can be designed by the same method.

(Overview)
FIG. 2 is a flowchart showing the operation of solubilization tag design by the solubility control tag design apparatus 10. In this specification, the outline of the solubilization tag design will be described first, and then the details of each process will be described.

  As shown in FIG. 3, amino acids can be divided into 10 amino acid similarity groups based on properties such as hydrophobicity and polarity. In the present embodiment, the amino acid similarity group includes group x including all amino acids, and the number of amino acid similarity groups is 11.

  The soluble control tag design apparatus 10 of the present embodiment uses the concept of a sequence composed of a combination of amino acid similarity groups (this is called “amino acid similarity group sequence”). The amino acid similarity group sequence that is often found in (1) is obtained (this is called “frequent amino acid similarity group sequence”). The above corresponds to steps S10 to S16 of the flowchart shown in FIG.

  Next, the soluble control tag designing apparatus 10 searches the amino acid sequence corresponding to the frequent amino acid similarity group sequence from the amino acid sequences included in the N-terminus of the soluble protein, and combines all the searched amino acid sequences. Determine the amino acid sequence as a soluble tag. This corresponds to steps S18 to S22 of the flowchart shown in FIG.

(Detailed explanation of each process)
Next, each process of solubilization tag determination is demonstrated in detail. In the following description, a case where a solubilizing tag having a 7-residue length is obtained by analyzing the amino acid sequence of the N-terminal 20 residues will be described as an example.

  First, the soluble control tag designing apparatus 10 receives an input of the residue length of the solubilized tag to be designed. In the present embodiment, data of 7 residue length is input. It is not always necessary to input the residue length every time, and the soluble control tag designing apparatus 10 may store the input residue length as a set value. Further, at this stage, an input of how many residues at the N-terminal are to be analyzed may be accepted. In this embodiment, since the N-terminal 20-residue length is analyzed, data of 20-residue length is input.

As shown in FIG. 4, the soluble control tag designing apparatus 10 generates a combination of all amino acid similarity groups having a length of 7 residues (S10). Amino acid analogs groups, as shown in FIG. 3, since there 11, 7 in the case of residues in length, there are 11 ^seven combinations. However, when the amino acid similarity group (group x) including all amino acids is located at the beginning and the end of the sequence, the amino acid sequence is the same as the 6-residue amino acid sequence. Finally the other group x, to produce a 10 ² × 11 ⁵ amino acid similarity group sequence. In addition, the soluble control tag designing apparatus 10 counts the number of times each amino acid similarity group sequence appears at each N-terminal 20 residue of the soluble protein or insoluble protein, as will be described below. Allocate an area for storing

  Next, the soluble control tag designing apparatus 10 reads out the amino acid sequence having a length of 7 residues from the N-terminal 20 residues of each of the soluble protein and the insoluble protein, and determines the number of appearances of the amino acid similarity group sequence corresponding to the read-out amino acid sequence. Count (S12). Details will be described below.

  FIG. 5 is a diagram showing examples of N-terminal 20 residues of soluble and insoluble proteins. FIG. 6 is a diagram showing an example in which an amino acid sequence having a length of 7 residues from the N-terminal 20 residues is extracted. There are 20−7 + 1 = 14 methods for extracting a 7-residue sequence from 20 N-terminal residues. That is, the first to seventh amino acid sequence at the N-terminal, the second to eighth amino acid sequence at the N-terminal, and the amino acid sequence from the 14th to 20th at the N-terminal. Next, an amino acid similarity group sequence corresponding to the extracted amino acid sequence is obtained, and the number of appearances of the amino acid similarity group sequence is counted up.

  FIG. 7A shows an example of an amino acid sequence extracted from the N-terminus. Below the amino acid sequence, an amino acid similarity group corresponding to each amino acid is shown. For example, the amino acid similarity group corresponding to “A” (alanine) is “a” (hydrophobic), “e” (minimal side chain), “x” (all) (however, at the top of the sequence) (Except for “x”), the amino acid similarity group corresponding to “E” (glutamic acid) includes “b” (polarity), “i” (negative charge), “j” (chargeability), “x” ( All).

  FIG. 7B shows an amino acid similarity group sequence corresponding to the amino acid sequence shown in FIG. In the amino acid sequence shown in FIG. 7 (a), there are 2, 3, 4, 3, 3, 4, and 4 amino acid similarity groups corresponding to each amino acid in the sequence. It corresponds to 3456 amino acid similarity group sequences. The soluble control tag designing device 10 sequentially obtains amino acid similarity group sequences corresponding to the read amino acid sequences, and counts up the number of appearances.

FIG. 7 (c), the N-terminus of the soluble protein, in each of the N-terminus of the insoluble protein is a diagram showing an example of a respective number of occurrences of the 10 ² × 11 ⁵ pieces of the total amino acid similarity group sequence. Hereinafter, in this specification, the number of occurrences of a predetermined amino acid similarity group sequence appearing at the N-terminus of a soluble protein is “Mp”, and the number of occurrences of a predetermined amino acid similarity group sequence appearing at the N-terminus of an insoluble protein is “Mn”. And Further, the total number of all amino acid similarity group sequences appearing at the N-terminus of the soluble protein is defined as the segment number “Np”, and the total of all amino acid similarity group sequences appearing at the N-terminus of the insoluble protein is defined as the segment number “Nn”.

Next, the soluble control tag designing apparatus 10 calculates the appearance frequency of each amino acid similarity group sequence in the solubilized protein (S14). In the present embodiment, the appearance frequency is represented by an S value and a p value represented by the following expression. The S value is a value indicating how specific the sequence appears in the data set obtained from the soluble protein, and the p value is a value indicating how rare the sequence is.

Subsequently, the soluble control tag design apparatus 10 obtains an amino acid similarity group sequence (this is referred to as a “frequent amino acid similarity group sequence”) whose appearance frequency is higher than a predetermined threshold (S16). Specifically, an amino acid similarity group sequence in which S value and p value satisfy both S> 0.9 and p <1 × 10 ⁻⁵ is extracted as a frequent amino acid similarity group sequence.

  Next, the soluble control tag design apparatus 10 clusters the obtained plurality of frequent amino acid similarity group sequences (S18). The distance between frequent amino acid similarity group sequences is defined as follows. First, a frequent amino acid similarity group is digitized according to the type of amino acid contained in the amino acid similarity group, and then an amino acid similarity group sequence comprising a combination of amino acid similarity groups is digitized. Next, a specific example is shown.

FIG. 8 is a diagram showing an example in which the amino acid similarity group is digitized. As shown in FIG. 8, the amino acid similarity group can be represented by a 20-dimensional coordinate value by setting the amino acid included in the amino acid similarity group to “1” and not including “0”. For example, group a (hydrophobic) is (1,1,1,1,1,1,1,1,1,1,1,1,0,0,0,0,0,0,1,0). ) Since the amino acid similarity group sequence is composed of a combination of seven amino acid similarity groups, it is represented by coordinates of 20 × 7 = 140 dimensions.
FIG. 9 is a diagram showing an example of quantifying frequent amino acid similarity group sequences. In the figure, the underlined number indicates the beginning of the numerical value representing each amino acid similarity group.

  Thus, the Euclidean distance between the frequent amino acid similarity group sequences can be obtained by digitizing the frequent amino acid similarity group sequences. The soluble control tag design apparatus 10 generates a dendrogram of frequent amino acid similarity group sequences using the Euclidean distance, and performs clustering by cutting the dendrogram at an appropriate height. Specifically, the distance between the clusters is calculated by the longest distance method (the longest distance among the individual distances included in the cluster is used as the cluster distance), and the nearest cluster (initially a member of the cluster) is calculated. Is a frequent amino acid similarity group sequence), and a dendrogram is generated by repeating the process of sequentially integrating the sequences. The clustering method itself using such a dendrogram is known.

  FIG. 10 is a diagram illustrating a dendrogram generated by the soluble control tag designing apparatus 10. In this example, 10 clusters are generated.

Next, the soluble control tag designing apparatus 10 searches the amino acid sequence corresponding to the frequent amino acid similarity group sequence from the amino acid sequence of the N-terminal 20 residues of the soluble protein.
FIG. 11 is a diagram showing examples of amino acid sequences corresponding to frequent amino acid similarity groups. In this example, “IHVGLDT”, “CKREMPA”, and the like are searched for as amino acid sequences corresponding to the frequent amino acid similarity group sequence “abxxaca”. The soluble control tag designing apparatus 10 counts the number of appearances of each type of amino acid at each location in the sequence based on the searched amino acid sequence, and stores it in the RAM 20.

  Similarly, the soluble control tag designing apparatus 10 stores the number of appearances for each type of amino acid for each location in the sequence for other frequent amino acid similarity group sequences included in the same cluster. Then, as shown in FIG. 11, the soluble control tag designing apparatus 10 adds up the number of appearances of each type of amino acid at each location in the sequence obtained using all the frequent amino acid similarity group sequences included in the same cluster. (S20). Based on the number of appearances of each type of amino acid at each location in the sequence determined in this way, the solubilized tag is determined by combining the most frequently occurring amino acids at each location (S22).

  FIG. 12 is a diagram visually showing the number of appearances of amino acids at each place in the sequence. The horizontal axis indicates each location of the array, which is first, second,..., Seventh from the left. The vertical axis shows the frequency of the appearing amino acids, and the more frequently appearing amino acids are shown in a larger font in the upper part. In this example, “E” is the most common in the first, second and fourth sequences, “L” is the most common in the seventh sequence, and in other cases, there is a significant difference in the number of appearing amino acids. Since there was no, “x” (all) is set. In this case, “EEExExL” is determined as the solubilization tag. Whether there is a significant difference can be determined by a threshold value. For example, it can be determined that there is a significant difference when there is a difference of 10% or more in the number of appearances of the most frequently seen amino acid and the second most frequently occurring amino acid.

Heretofore, the configuration of the soluble control tag designing apparatus 10 of the present embodiment and the operation of solubilizing tag design have been described. When designing an insolubilizing tag, in step S16 for extracting a frequent amino acid similarity group sequence, an amino acid similarity group sequence that frequently appears in the insoluble protein is obtained using the following formula, and an amino acid sequence corresponding to the frequent amino acid similarity group is obtained. In step S20 for searching for an insoluble protein, the search may be performed from the N-terminus of the insoluble protein.

In the present embodiment, since an amino acid similarity group sequence that frequently appears at the N-terminus of a soluble protein and an insoluble protein is first obtained, the amount of calculation can be reduced as compared with the case of using an amino acid sequence. Further, by counting the number of appearances of the amino acid similarity group sequence having a smaller total number than the amino acid sequence, the tendency of the number of appearances can be grasped even when the data is small. In the case of 7 residues in length, there are 20 ⁷ amino acid sequences, and there are about 80 times as many sequences as the amino acid similarity group sequence, so the number of appearances assigned to each amino acid sequence is about 1/80, In particular, every sequence has a small number of occurrences, making it difficult to grasp the characteristics of soluble or insoluble proteins. On the other hand, by using the amino acid similarity group sequence, a characteristic amino acid similarity group sequence can be obtained even when there is relatively little data.

  The soluble control tag designing apparatus of the present invention has been described in detail with reference to the embodiment, but the present invention is not limited to the above-described embodiment.

  In the above-described embodiment, an example in which a soluble control tag is designed using an amino acid similarity group sequence has been described. However, when there is a large amount of data in the protein DB, an amino acid sequence that is uniquely included in a soluble protein or an insoluble protein It is good also as searching directly.

  In the above-described embodiment, an example in which clustering is performed using a tree diagram has been described. However, a clustering method is not limited to a method using a tree diagram, and a known method such as a k-average method is employed. be able to. Further, when the number of frequent amino acid similarity group sequences is not large, clustering is not necessarily performed.

  In the above-described embodiment, the frequent amino acid similarity group sequence is obtained by using the S value and the p value. However, the threshold for determining whether or not to frequently appear may be determined by another method.

  By adding the solubilization tag and the insolubilization tag designed by the soluble control tag designing apparatus of the present invention, the influence on the solubility of the protein was evaluated.

(Generation of soluble control tags)
Using a database of proteins expressed in a wheat germ cell-free system, 16 types of solubilization tags and 12 types of insolubilization tags shown in Table 1 below were designed.

Nine types of genes to which soluble control tags are added were selected according to the following criteria.
(1) Matches with RefSeq and has no transmembrane domain.
(2) According to the data of HGPD (Human Gene and Protein Database), the molecular weight is about 50 kDa, and the degree of solubilization is different.
Nine genes are shown in Table 2 below.

The gene sequence corresponding to the solubilization tag and insolubilization tag shown in Table 1 is added to the N-terminal of the gene shown in Table 2 upstream of the gene sequence encoding the protein, and the protein is expressed in a wheat germ cell-free system and solubilized. Table 3 shows the results of examining how the solubility of the protein was changed as compared with the case where no tag or insolubilizing tag was added.

  As shown in Table 3, the solubilization tag and the insolubilization tag designed by the soluble control tag design apparatus of the present invention affected the solubilization / insolubilization of proteins.

  In the present invention, a soluble control tag is obtained based on actual data, so that a tag suitable for the condition can be designed, which is useful for protein production.

10 Soluble Control Tag Design Device 12 Input Unit 14 Protein Database 16 CPU
18 Output unit 20 RAM
22 ROM
24 programs

Claims (9)

An apparatus for designing a soluble control tag based on data read from a database storing amino acid sequences of soluble protein and insoluble protein,
An input unit for inputting the residue length L of the soluble control tag to be obtained;
An arithmetic unit for obtaining a soluble control tag based on data read from the database;
An output unit for outputting the solubility control tag obtained by the calculation unit;
With
The computing unit is
(1) generating an amino acid similarity group sequence in which all combinations of amino acid similarity groups are defined for amino acids having L residue length, the sequences of amino acid similarity groups classified based on the properties of amino acids;
(2) reading an amino acid sequence having an L residue length contained in the N-terminal K residues (K ≧ L) of the soluble protein and the insoluble protein from the database;
(3) counting the number of appearances of each amino acid similarity group sequence at the N-terminus of each of the soluble protein and the insoluble protein based on the read-out amino acid sequence;
(4) When designing a solubilization tag that enhances solubility, the appearance frequency of each amino acid similarity group sequence at the N-terminus of the soluble protein; when designing an insolubilization tag that enhances insolubility, each amino acid similarity at the N-terminus of the insoluble protein Calculating an appearance frequency of the group sequence and obtaining an amino acid similarity group sequence having an appearance frequency higher than a predetermined threshold as a frequent amino acid similarity group sequence; and (5) clustering a plurality of frequent amino acid similarity group sequences into a plurality of clusters. A step of classification;
(6) A soluble protein when designing a solubilizing tag; an amino acid sequence of L residue length read from the N-terminal K residue of an insoluble protein when designing an insolubilizing tag; and the frequent amino acid similarity group sequence If the amino acid sequence corresponds to the frequent amino acid similarity group sequence, the number of occurrences of each type of amino acid at each location in the amino acid sequence is counted based on the amino acids contained in the amino acid sequence. A step of sequentially performing a process of uploading the read amino acid sequence;
(7) summing up the number of appearances for each type of amino acid at each location in the amino acid sequence determined by the frequent amino acid similarity group sequence in the same cluster;
(8) obtaining an amino acid sequence comprising a combination of amino acids having the highest number of appearances at each location as a solubilization tag or an insolubilization tag;
A soluble control tag design device that performs. In the step (4), when a soluble tag is obtained, an amino acid similarity group sequence in which an S value and a p value calculated by the following formulas are S> 0.9 and p <1 × 10 ⁻⁵ is a frequent amino acid similarity group. The soluble control tag design apparatus according to claim 1, which is obtained as an array.

The step (5)
The frequent amino acid similarity group sequence is converted into a 20-dimensional coordinate value in which the amino acid contained in the frequent amino acid similarity group is “1” and the non-included amino acid is “0”.
Generate a dendrogram based on the Euclidean distance of the frequent amino acid similarity group sequence,
Clustering is performed by cutting the dendrogram at a predetermined height.
The soluble control tag design apparatus according to claim 1 or 2. A method for designing a solubilization tag that increases the solubility of a protein or an insolubility tag that increases insolubility as a solubility control tag,
(1) generating an amino acid similarity group sequence in which all combinations of amino acid similarity groups are defined for amino acids having L residue length, the sequences of amino acid similarity groups classified based on the properties of amino acids;
(2) reading an amino acid sequence having an L residue length contained in the N-terminal K residues (K ≧ L) of the soluble protein and the insoluble protein from a database storing the amino acid sequences of the soluble protein and the insoluble protein;
(3) counting the number of appearances of each amino acid similarity group sequence at the N-terminus of each of the soluble protein and the insoluble protein based on the read-out amino acid sequence;
(4) When designing a solubilization tag, the frequency of appearance of each amino acid similarity group sequence at the N-terminus of the soluble protein, and when designing an insolubilization tag, the frequency of occurrence of each amino acid similarity group sequence at the N-terminus of the insoluble protein. Calculating an amino acid similarity group sequence having an appearance frequency higher than a predetermined threshold as a frequent amino acid similarity group sequence;
(5) clustering a plurality of frequent amino acid similarity group sequences into a plurality of clusters;
(6) A soluble protein when designing a solubilizing tag, and an amino acid sequence of L residue length read from the N-terminal K residue of the insoluble protein when designing an insolubilizing tag and the frequent amino acid similarity group sequence If the amino acid sequence corresponds to the frequent amino acid similarity group sequence, the number of occurrences of each type of amino acid at each location in the amino acid sequence is counted up based on the amino acids contained in the amino acid sequence. Sequentially performing processing for the read amino acid sequence;
(7) summing up the number of appearances for each type of amino acid at each location in the amino acid sequence determined by the frequent amino acid similarity group sequence in the same cluster;
(8) obtaining an amino acid sequence comprising a combination of amino acids having the highest number of appearances at each location as a solubilization tag or an insolubilization tag;
A soluble control tag design method comprising: In the step (4), when a soluble tag is obtained, an amino acid similarity group sequence in which an S value and a p value calculated by the following formulas are S> 0.9 and p <1 × 10 ⁻⁵ is a frequent amino acid similarity group. The soluble control tag design method according to claim 4, which is obtained as an array.

The step (5)
The frequent amino acid similarity group sequence is converted into a 20-dimensional coordinate value in which the amino acid contained in the frequent amino acid similarity group is “1” and the non-included amino acid is “0”.
Generate a dendrogram based on the Euclidean distance of the frequent amino acid similarity group sequence,
Clustering is performed by cutting the dendrogram at a predetermined height.
The soluble control tag design method according to claim 4 or 5. A program for designing a solubilization tag that increases the solubility of a protein or an insolubilization tag that increases insolubility as a solubility control tag,
(1) Amino acid similarity group sequences that are classified based on the properties of amino acids and that define all combinations of amino acid similarity groups for amino acids with L residue length are soluble in amino acid similarity group sequences. Ensuring an area for storing the number of occurrences at each N-terminus of the protein and insoluble protein;
(2) reading an amino acid sequence having an L residue length contained in the N-terminal K residues (K ≧ L) of the soluble protein and the insoluble protein from a database storing the amino acid sequences of the soluble protein and the insoluble protein;
(3) counting the number of appearances of each amino acid similarity group sequence at the N-terminus of each of the soluble protein and the insoluble protein based on the read-out amino acid sequence;
(4) When designing a solubilization tag, the frequency of appearance of each amino acid similarity group sequence at the N-terminus of the soluble protein, and when designing an insolubilization tag, the frequency of occurrence of each amino acid similarity group sequence at the N-terminus of the insoluble protein. Calculating an amino acid similarity group sequence having an appearance frequency higher than a predetermined threshold as a frequent amino acid similarity group sequence;
(5) clustering a plurality of frequent amino acid similarity group sequences into a plurality of clusters;
(6) A soluble protein when designing a solubilizing tag, and an amino acid sequence of L residue length read from the N-terminal K residue of the insoluble protein when designing an insolubilizing tag and the frequent amino acid similarity group sequence If the amino acid sequence corresponds to the frequent amino acid similarity group sequence, the number of occurrences of each type of amino acid at each location in the amino acid sequence is counted up based on the amino acids contained in the amino acid sequence. Sequentially performing processing for the read amino acid sequence;
(7) summing up the number of appearances for each type of amino acid at each location in the amino acid sequence determined by the frequent amino acid similarity group sequence in the same cluster;
(8) obtaining an amino acid sequence comprising a combination of amino acids having the highest number of appearances at each location as a solubilization tag or an insolubilization tag;
(9) outputting a solubilized tag or an insolubilized tag;
A program that executes In the step (4), when a soluble tag is obtained, an amino acid similarity group sequence in which an S value and a p value calculated by the following formulas are S> 0.9 and p <1 × 10 ⁻⁵ is a frequent amino acid similarity group. The program according to claim 7, which is obtained as an array.

The step (5)
The frequent amino acid similarity group sequence is converted into a 20-dimensional coordinate value in which the amino acid contained in the frequent amino acid similarity group is “1” and the non-included amino acid is “0”.
Generate a dendrogram based on the Euclidean distance of the frequent amino acid similarity group sequence,
Clustering is performed by cutting the dendrogram at a predetermined height.
The program according to claim 7 or 8.

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