KR100746993B1 - The expression and purification method of human protein tyrosine phosphatase using E.coli system - Google Patents

Abstract

The present invention relates to a method for producing human protein tyrosine phosphatase (PTP) using Escherichia coli, specifically, a method for producing human PTP with enzymatic activity in Escherichia coli using various vectors. It is about. Various human PTPs produced by the methods of the present invention can be usefully used for antibody production of PTP or drug search for PTP related diseases.

Protein Tyrosine Dephosphatase (PTP), Escherichia Coli

Description

Expression and purification method of human protein tyrosine phosphatase using E. coli system

Figure 1a is a schematic diagram showing the structure of the vector pET28a-MBP (maltose binding protein) to be inserted His label, tobacco erch virus (TEV), PTP gene,

1B is a schematic diagram showing the structure of the overexpression vector pET28a-MBP-HTs into which the PTP gene is to be inserted;

MCS: multi-cloning site

(His) ₆ : hexa his-tag, MBP: MBP-label

FIG. 1C is a Venn diagram showing the number and types of PTP-produced overexpression vectors successfully produced.

Figure 2a is an example of a gel picture shown by electrophoresis of the pET28a-MBP-PTP fusion vector,

From the left

Lane 1: pET28a-MBP vector; Lane 2: pET28a-MBP-pk4-1; Lane 3: pET28a-MBP-pk4-2;

Lane 4: pET28a-MBP-pk7-1; Lane 5: pET28a-MBP-pk7-2; Lane 6: pET28a-MBP-pk13-1;

Lane 7: pET28a-MBP-pk13-2; Lane 8: pET28a-MBP-pk16-1; Lane 9: pet28a-MBP-pk16-2

Lane 10: pET28a-MBP-pk18-1; Lane 11: pET28a-MBP-pk18-2; Lane 12: pET28a-MBP-p19-1;

Lane 13: pET28a-MBP-p19-1; Lane 14: pET28a-MBP-p24-1; Lane 15: pET28a-MBP-p24-2;

Lane 16: pET28a-MBP-p25-1; Lane 17: pET28a-MBP-p25-2; Lane 18: pet28a-MBP vector

(In the case of lanes 4 and 5, the fusion vector is not properly formed, and it is completed by retesting. The last 1 and 2 of each construct are plasmid vectors obtained from two different colonies.)

Figure 2b is an example of a gel photograph showing the result of confirming that the PTP is inserted by cutting the pET28a-MBP-HTs-PTP vector using a restriction enzyme,

From the left

Lane 1: marker; Lane 2: pET28a-MBP-pk4-1; Lane 3: pET28a-MBP-pk4-2;

Lane 4: pET28a-MBP-pk13-1; Lane 5: pET28a-MBP-pk13-2;

Lane 6: pET28a-MBP-pk16-1; Lane 7: pET28a-MBP-pk16-2;

Lane 8: pET28a-MBP-pk18-1; Lane 9: pET28a-MBP-pk18-2; Lane 10: pET28a-MBP-p19-1;

Lane 11: pET28a-MBP-p19-2; Lane 12: pET28a-MBP-p24-1; Lane 13: pET28a-MBP-p24-2;

Lane 14: pet-28-MBP-p25-1; Lane 15: pet-28-MBP-p25-2; Lane 16: pET-28-MBP vector;

Lane 16: Marker

Restriction enzyme was treated with Ndel / BamHI up to lanes 1-15, and lane 16 was treated with the pET28-MBP vector with XbaI and XhoI restriction enzymes. Plasmid vector obtained from colony)

3 is a flow diagram showing the whole process from expression of human PTP to purification,

4 is a diagram showing the SDS PAGE results of purified human PTP group,

5 is a graph showing the relative enzymatic activity of purified human PTP produced by the method of the present invention.

The present invention relates to a method for producing human protein tyrosine phosphatase (PTP) using Escherichia coli, and more specifically, to produce human PTP with enzymatic activity in Escherichia coli using various vectors. It is about a method.

Phosphorylation-dephosphorylation of protein tyrosine is a very important means in intracellular signaling systems. Protein tyrosine phosphatase (PTP) is an enzyme that hydrolyzes phosphate groups in tyrosine residues in proteins. About 100 PTPs are present in humans, and about 80 to 90 of these have been reported to show enzymatic activity as actual PTPs. (Alonso A. et al. Cell, 117; 699-711, 2004)

It is known that many PTPs are actually associated with disease because disruption of the intracellular signaling system easily leads to disease, and some PTPs have been targeted for drug development (Current Topics in Med. Chem. 3: 739-835, However, even in the case of PTP 1B, which is the target enzyme for the development of diabetes treatments, the research on the development of inhibitors has been carried out with a great deal of effort for more than 10 years, but there have been no reports of successful development of the treatments (Tayler). , SD Current Topics in Med. Chem. 3, 759-782, 2003) There may be several reasons for this, but one of the representative reasons is that when a drug is administered to an animal or human body, it may bind to PTPs other than the target PTP, causing unexpected toxicity. It is thought to be caused. One way to minimize this problem is to conduct PTP specificity studies on all possible PTPs for drug candidates before applying them to animal experiments. Since about 90 PTPs have similar active site structures, There is a difficulty in developing inhibitors specific for PTP.

The method of expressing and purifying the necessary protein using E. coli expression system has the advantage of obtaining a large amount of protein at low cost and effort, but due to the difference in the intracellular environment of mammals and bacteria, In order to obtain proteins, it is necessary to establish specific expression and purification conditions for each protein. However, until now, only some PTPs are expressed and purified in large quantities, and most PTPs have not been reported to be purified. Therefore, it is impossible to study selectivity of the entire PTP. The reason why the PTP is not properly purified is because the human PTPs are not properly expressed and purified under the general expression conditions, as the inventors have obtained only 30 PTPs when the PTP is purified using the general expression conditions. It is considered to be. As a general method, it is difficult to express human PTP in large quantities, and thus, PTP-specific antibody production is delayed, and drug development is also difficult.

Accordingly, the present inventors have completed the present invention by establishing a method of expressing and purifying each PTP using a human E. coli expression system as a result of efforts to produce each PTP in an active state.

Disclosure of the Invention An object of the present invention is to provide a method for producing human protein tyrosine phosphatase (PTP) with activity, which is closely related to various diseases including cancer, arthritis, diabetes, and can be useful for drug development. To provide.

In order to achieve the above object, the present invention

Iii) transforming Escherichia coli by preparing an expression vector of human tyrosine phosphatase (PTP);

Ii) culturing the transformed Escherichia coli of step iii) to induce expression of PTP; And

Iii) purifying PTP from the cultured Escherichia coli of step ii).

In addition, the present invention provides an expression vector pET28a-MBP-His-TEV-PTP prepared in step iii).

In addition, the present invention provides an expression vector pET28a-GST-PTP prepared in step iii)

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of transforming E. coli by preparing an expression vector of human tyrosine phosphatase (PTP);

Ii) culturing the transformed Escherichia coli of step iii) to induce expression of PTP; And

Iii) purifying PTP from the cultured Escherichia coli of step ii).

So far, more than half of protein tyrosine phosphatase (PTP) is a protein that is difficult to purify to the extent that few cases have been purified in large quantities in an active state, and the present inventors expressed and purified from E. coli against 77 PTPs. I figured out how. Specifically, the present inventors prepared an expression vector by inserting 77 PTPs (see Table 1) into pET28a, an E. coli overexpression vector for mass production. Among them, pET28a-GST-PTP and pET28a-MBP-His-TEV-MCS-PTP, ie, pET28a-, based on pET28a and pET16b to express the remaining PTPs except 26 PTPs expressed and purified in a general manner. MBP-HTs-PTP expression vector was constructed. pET28a-GST-PTP is a GST-labeled expression vector to purify PTP with glutathione resin, and pET28a-MBP-HTs-PTP targets PTPs that form insoluble inclusion bodies. It is an expression vector in which maltose binding protein (MBP) is fused to increase folding and solubility. The pET28a-MBP vector is purified by inserting a tobacco erch virus sequence that can be hydrolyzed by protease between the MBP and the PTP insertion site, and then the MBP site is separated from the PTP to recover only pure PTP. It was made.

In E. coli transformants prepared by introducing the recombinant expression vector, and in step ii), in order to induce the expression of PTP in the transformant, the expression of other proteins is induced by inducing the expression of PTP (expression condition a) ) In case of PTP that forms insoluble inclusion bodies and is not properly expressed, follow the same condition as "expression condition A", but increase the concentration of isopropyl-ß-D-thiogalacto pyranosid (IPTG) than "expression condition A" (expression) Conditions b) E. coli is cultured in the medium to which sorbitol and glycine betain are added (expression condition c) or the same condition as “expression condition c”, but IPTG (isopropyl-) in “expression condition c” The method for reducing the concentration of ß-D-thiogalactopyranosid) or inducing expression at low temperature was selected for each PTP (expression condition e) (see Table 1). .

In the general method, the culture medium is cooled to a concentration lower than room temperature, preferably 18 ° C., when the E. coli transformant is cultured to an OD ₆₀₀ value of 0.12 or less, preferably 0.6 to 0.8, more preferably 0.6. And induce expression with an IPTG of 0.5 mM or less.

The IPTG treatment concentration of “expression condition a” is less than 1.0 mM and preferably 0.5 mM. The IPTG treatment concentration of “expression condition I” is at least 1.0 mM and more preferably 1.0 mM. The final sorbitol concentration is 0.1 to 1 M, and the final glycine betaine concentration is less than 1 M and preferably 50 mM in the medium to which sorbitol and glycine betaine are added. The IPTG treatment concentration of “expression condition d” is lower than the final IPTG treatment concentration of “expression condition c” and is preferably 20 μM. The low temperature of "expression condition e" is preferably less than 18 ° C, preferably 4 ° C. or lower, and the final concentration of IPTG treated in "expression condition e" is 0.5 mM or less and preferably 0.1 mM.

In step iii), the cells recovered after induction of expression in step ii) were crushed to purify PTP using a resin suitable for each expression vector. Specifically, since his tag was included in the pET28a vector for pET28a-PTP, Talon® resin (ClonTech) was used, and for GET-PTP, GST resin (purification method 2) was used, and pET28a- MBP-HTs-PTP was purified using an amylose resin (purification method 1) or Talon® resin (purification method 3). The amylose resin binds to the maltose binding protein (MBP) and the talon resin binds to the His label so that both can be used for PTP purification (see Table 1 and FIG. 4).

PTP expressed by the above method was activated using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), which is a dephosphoryase activity test substance. As a result of the test, as shown in Table 3 and Figure 5, it was confirmed that 77 human PTP purified in the active state by the expression and purification method of the present invention.

Human PTP is a protein tyrosine phosphatase receptor type A, Unitprot accession number: P18433, PTPRB (Unitprot accession number: P23467), PTPRC (Unitprot accession number: Q5T5R0), PTPRD (Unitprot accession number: P23468), PTPRE (PTPRA) Unitprot access number: P23469), PTPRF (Unitprot access number: P10586), PTPRG (Unitprot access number: P23470), PTPRH (Unitprot access number: Q2NKN9), PTPRJ (Unitprot access number: Q12913), PTPRK (Unitprot access number: Q15262 ), PTPRM (Unitprot access number: P28827), PTPRN (Unitprot access number: Q16849), PTPRN2 (type N polypeptide 2, Unitprot access number: Q92932), PTPRO (Unitprot access number: Q16827), PTPRR (Unitprot access number: Q15256) ), PTPRS (Unitprot access number: Q13332), PTPRT (Unitprot access number: O14522), PTPRU (Unitprot access number: Q92729), PTPRZ (Unitprot access number: P23471), PTPN1 (protein tyrosine phosphatase, non-receptor type 1, Unitprot access number: P18031), PTPN2, (Unitprot access number: P17706) PTPN3 (Unitprot access number: P26045), PTPN5 (Unitprot access Number: P54829), PTPN6 (Unitprot access number: P29350), PTPN7 (Unitprot access number: P35236), PTPN11 (Unitprot access number: Q06214), PTPN12 (Unitprot access number: Q05209), PTPN13 (Unitprot access number: Q12923), PTPN14 (Unitprot access number: Q15678), PTPN18 (Unitprot access number: Q99952), PTPN21 (Unitprot access number: Q16825), PTPN22 (Unitprot access number: Q9Y2R2), PTPN23 (Unitprot access number: Q9H3S7), DUSP1 (duase specificityphosph 1, Unitprot access number: P28562, DUSP2 (Unitprot access number: Q05923), DUSP4 (Unitprot access number: Q9PW71), DUSP5 (Unitprot access number: Q16690), DUSP6 (Unitprot access number: Q16828), DUSP7 (Unitprot access number : Q16829), DUSP8 (Unitprot access number: Q13202), DUSP9 (Unitprot access number: Q99956), DUSP10 (Unitprot access number: Q9Y6W6), DUSP16 (Unitprot access number: Q9BY84), MK-STYX (Map kinase phosphatase-like protein) , Unitprot access number: Q9Y6J8), DUSP3 (Unitprot access number: P51452), DUSP11 (Unitprot access number: O75319), DUSP12 (Unitprot access number No .: Q9UN16), DUSP13A (Unitprot access number: Q9UII6) DUSP13B (Unitprot access number: Q9QYJ7), DUSP14 (Unitprot access number: O95147), DUSP15 (Unitprot access number: Q9H1R2), DUSP18 (Unitprot access number: Q8NEJ19) (Unitprot access number: Q8WTR2), DUSP21 (Unitprot access number: Q9H596), DUSP22 (Unitprot access number: Q9NRW4), DUSP23 (Unitprot access number: Q9BVJ7), DUSP24 (Unitprot access number: Q9Y6JB), DUSP25 (Unitprot access number: Q9Y216), DUSP26 (Unitprot access number: Q4GOw2), EPM2A (epilepsy progressive myoclonus type 2A, Unitprot access number: O95278), SSH1 (slingshot homolog 1, Unitprot access number: Q8WYLO), SSH2 (Unitprot access number: Q76176), SSH3 (Unitprot accession number: Q8TE77), PTP4A1 (protein tyrosine phosphatase 4a1, Unitprot accession number: Q93096), PTP4A2 (Unitprot accession number: Q12974), PTP4A3 (Unitprot accession number: O75365), CDC14B (CDC14 cell division cycle 14 homolog B, Unitprot accession number: O60729), TPTE (transmembrane phosphoinositide 3-phosphatase and tensin homolog 2, Unitprot Access number: Q4R6N0), TENC1 (tensin like C1 domain containing phosphatase, Unitprot access number: Q2NL80), MTMR1 (myotubularin related protein 1, Unitprot access number: Q13613), MTMR3 (Unitprot access number: Q13615, MTMR7 (Unitprot access number: Q9Y216), MTMR8, ACP1 (acid phosphatase 1 soluble, Unitprot access number: P24666), CDC25A (cell division cycle 25B, Unitprot access number: P30304), CDC25B (Unitprot access number: P30305), CDC25C (Unitprot access number: P30307) PTP selected from the group consisting of (see Table 1).

In addition, the present invention provides an expression vector pET28a-MBP-HTs-PTP prepared in step iii).

We decided to fuse the maltose binding site to the N-terminus of PTP to increase the solubility of PTP forming an insoluble inclusion body in the E. coli expression system. To this end, a recombinant vector was prepared in which the MBP gene was introduced immediately before the multi-cloning site (MCS) of pET28a. In addition, in order to recover only pure PTP without MBP after purification, a portion that can be hydrolyzed by tobacco etch virus (TEV) protease is introduced downstream of the MBP and the MBP in the pET28a-MBP vector. To complement the disadvantage that His labels attached to the N-terminus often fail, the pET28a-MBP-His-TEV vector is introduced by the addition of six histidines between the MBP and TEV proteases. That is, pET28a-MBP-HTs described in SEQ ID NO: 5 was prepared (see FIG. 1B).

In addition, the present invention provides an expression vector pET28a-GST-PTP prepared in step iii).

The present inventors constructed a pET28a-GST-PTP expression vector using a pET28a-GST vector rather than a pET28a-MBP-HTs-PTP vector for PTPs having a low degree of insolubility among PTPs forming an insoluble inclusion body. Labeling with glutathione S-transferase (GST), that is, glutathione S transferase, does not significantly affect the expressed PTP due to the small size of GST. Therefore, in the case of PTP that is not severely insoluble, a PET28a-GST-PTP expression vector was constructed to purify the PTP.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 MBP Fusion Vector Fabrication

<1-1> pET28a-PTP Fusion Vector Construction

The genes corresponding to the 77 PTP activity domains described in Table 1 were inserted into the pET28a vector (Novagen) using restriction enzymes such as Nde I, Nhe I, BamH I, and Xho I, thereby overexpression vector pET28a-PTP Was produced.

<1-2> Construction of pET28a-MBP Fusion Expression Vector

In order to increase the expression, folding and solubility of PTP in an E. coli expression system, maltose binding protein (MBP) was fused to the N-terminus of PTP. To this end, there is a method using a commercially available MBP vector, but the MBP vector is a multi-cloning site (MCS) different from the pET28a vector (Novagen), which is an overexpression vector inserted with PTP in <Example 1> As a result, there was a difficulty in preparing a recombinant vector. Therefore, a recombinant vector in which the MBP gene was introduced immediately before the multiple restriction enzyme site of the pET28a vector was prepared.

MBP DNA for pET28a-MBP vector production was cut out from pIVEX-MBP fusion vector (Roche) using restriction enzymes XbaI and NdeI. Subsequently, pET28a-MBP vector was prepared by conjugating the pET28a vector treated with the same restriction enzyme as MBP DNA cut out using T4 DNA ligase at 16 ° C. for 4 hours (FIG. 1A).

<1-3> Construction of pET28a-MBP-HTs Expression Vector

In order to remove MBP from the purified protein and obtain only pure PTP domain, a portion that can be hydrolyzed by tobacco etch virus (TEV) protease was introduced at the back of MBP.

First, the PCR product obtained by using a forward primer, a reverse primer, and a pProEX HTb vector (Invitrogen) described in SEQ ID NOs: 1 and 2 as a template, and then treated with NcoI / Ndel, was inserted into a pET28a vector to complete a pET28a-TEV vector. It was. Subsequently, PCR was carried out with the PacI restriction enzyme agonist and the forward primer of SEQ ID NO: 3, the reverse primer of SEQ ID NO: 4, and the pET28a-TEV vector as a template, and the product was treated with PacI and Xho I restriction enzyme. Then, pET28a-MBP-TEV vector was prepared by inserting into pET28a-MBP (FIG. 1B). The two kinds of PCR reaction conditions used above were the primers described above using DNA polymerase (Pfu polymerase, Solgent). Denature the pair and the template at 94 ° C. for 5 minutes, and 1 minute at 94 ° C., b. 1 minute at 55 ° C. The procedure of polymerizing the reaction at 72 ° C. for 2 minutes (a ~ da) was repeated 30 times, and finally, the polymerization reaction was completed at 72 ° C. for 10 minutes. In addition, six histidines were introduced between the MBP and TEV proteases to compensate for the weakness of the His label attached to the N-terminus of MBP in the pET28a-MBP vector. Thus, the pET28a-MBP-His-TEV-MCS vector described in SEQ ID NO: 5 was prepared, which was simply named pET28a-MBP-HTs vector (FIG. 1B).

<1-4> Construction of pET28a-MBP-HTs-PTP Recombinant Construct

Except for the 26 PTP genes of Example <1-1> , which can be expressed under general conditions among 77, 63 PTP genes were restricted from the pET28a-PTP overexpression vector (Nde I, Nhe I, BamH I, or Xho). I) was used to cut out the PTP gene. PET28a-MBP-HTs-PTP expression vector was prepared by treating each gene with the corresponding restriction enzyme pET28-PTP and pET28-MBP-HTs vector at 16 ° C. for 4 hours. E. coli DH5a was transformed with the vector and plated in LB agar medium containing 30 mg / mL kanamacin and incubated overnight at 37 ° C. Figure 2a is an example confirming the pET28a-MBP-PTP vector prepared by separating the recombinant plasmid from the transformed E. coli and agarose gel electrophoresis. Figure 2b is an example of double digestion of purified pET28a-MBP-HTs-PTP at 37 ℃ using the restriction enzyme, followed by 1% DNA agarose gel electrophoresis, confirming that the PTP gene is inserted to be.

Example 2 Expression of pET28a-MBP-PTP in Escherichia Coli

<2-1> PTP expression under normal conditions (expression condition a)

E. coli Rosetta DE3 (Novagen) or E. coli Rosetta DE3 (Novagen) were transformed with pET28a-MBP-PTP or pET28a-MBP-HTs-PTP vector prepared in Example It was incubated for 18 hours at 37 ℃ using the included LB solid medium. A single colony (colony) formed at this time was inoculated in LB medium (including 50 mL, 30 mg / mL kanamycin), incubated for 16 hours at 37 degrees Celsius, and then an appropriate amount was taken for LB medium (1 L, 30 mg / mL kanamycin included). ) Was inoculated. Adjust the amount of E. coli culture so that the OD ₆₀₀ is 0.12 or less and continue to incubate at the same temperature, when the OD ₆₀₀ reaches 0.6 ~ 0.8, the culture is cooled to 18 ℃. Subsequently, IPTG (isopropyl-ß-D-thiogalactopyranosid), which is an expression inducing substance, was added to bring the final concentration of IPTG to 0.5 mM, and further cultured at 18 ° C. for 18 hours to induce the expression of MBP fused PTP.

  The cultured cells were centrifuged and the precipitated cells were suspended in 40 mL lysis buffer (PMSF 1 mM, pH7.5 50 mM Tris, 200 mM NaCl) and lysed by an ultrasonic generator. This was centrifuged to load the supernatant into amylose resin and purified by the batch method described below. The protein purification using the batch method is described in detail as follows. After washing and activating amylose resin (1 mL) twice with 10 mL of column buffer (50 mM Tris pH7.5, 200 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol), the supernatant separated above was added The protein is adsorbed onto the resin by shaking slowly at 4 ° C. for 3 hours. After the aqueous solution was filtered off, the resin was washed twice with column (10 mL), and then 10 mL of elution buffer (column buffer containing 500 mM maltose) was added and then slowly shaken for 1 hour. After filtering, the target protein is recovered. The target protein was eluted by repeating it once more with 5 mL of elution buffer and estimating the size of the protein via a 10% SDS-PAGE gel.

During the process, the insoluble inclusion body was formed upon the expression of several MBP-PTP fusion proteins. These PTPs were prepared using LB medium containing 0.5-1 M sorbitol and 50 mM glycine betaine, or at low temperature. Obtained water-soluble by expressing (Table 1)

<2-2> IPTG concentration increase (expression condition b)

Example <2-1> was expressed in the result, and then, increase the concentration of the expression conditions of Example <2-1> that the expression of slow speed PTP targeted IPTG at 0.5 mM to 1.0 mM protein.

<2-3> Protein Expression in Sorbitol Medium (C)

Inoculate in the same manner as in Example <2-1> and take 10 mL of 50 mL LB medium incubated overnight, and then 1 L sorbitol medium (including 30 mg / mL kanamycin, 1 M sorbitol and 50 mM betain in LB medium). Incubate at 37 ° C until OD ₆₀₀ = 0.6. After the incubator was cooled to 18 ° C. for 20 minutes, IPTG was added thereto and incubated at the same temperature for 24 hours. In the sorbitol medium, the growth rate is very slow, so it was grown at 37 ° C. for more than 8 hours, and the OD was measured in the middle to confirm that the OD ₆₀₀ = 0.6. Aggregates and protein purification are the same as those described in Example <2-1> above.

<2-4> Reduction of IPTG Concentration under Expression Conditions (D)

The protein was expressed by adjusting the IPTG concentration to 20 μM under the conditions of Example <2-1> .

<2-5> Protein expression at low temperature (expression condition e)

After inoculation in the same manner as in <2-1> , 10 mL of 50 mL LB medium incubated overnight was added to the L LB medium and incubated at 37 ° C until OD ₆₀₀ = 0.6.

The culture solution was transferred to a shaker at 4 ° C. and shaken to cool the temperature of the culture solution to 4 ° C., and then incubated for 3 days by adding IPTG to a final concentration of 0.1 mM. Aggregates and protein purification are the same as those described in Example <2-1> .

Example 3 Protein Purification

<3-1> Amylose resin (purification method 1)

The cells precipitated by centrifugation were suspended in 25 mL of cell lysis buffer (pH7.5, 50 mM Tris, 200 mM NaCl) and 1 mM PMSF (phenylmethylsulfonyl fluoride) was added to inhibit the activity of proteolytic enzymes. . After lysing the cells by sonication, centrifugation was performed for 30 minutes, and the supernatant was washed with 10 mL of distilled water and 10 mL of cell lysis buffer, and the supernatant was added to 2 mL of amylose resin, which had been activated, and mixed. The protein was bound to the resin by slow shaking at 4 ° C. for 5 hours using a rocker. After removing the supernatant, the cells were added to a cell lysis buffer (10 mL) containing 10 mM maltose, and the resin was washed for 10 minutes. Finally, 10 mL of elution buffer (cell lysis buffer containing 500 mM maltose) was added thereto, shaken for 2 hours, and only the supernatant was recovered to obtain a target protein (FIG. 4).

<3-2> Using glutathione resin (purification method 2)

GST labeled proteins were purified as in Example <4-1> using a 1 × 4 cm glutathione column. Elution buffer containing 20 mM glutathione, pH 7.5, 50 mM Tris, 200 mM NaCl was used to elute the aliquots containing the target protein from the resin (FIG. 4).

<3-3> Use of Talon resin (purification method 3)

The cells precipitated by centrifugation were suspended in 25 mL of cell lysis buffer (pH7.5, 50 mM Tris, 200 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol) and 1 mM PMSF to inhibit proteolysis. Was added. After crushing the cells using ultrasonic waves, centrifuged at 3013 G for 30 minutes, the supernatant was washed with 10 mL of distilled water and cell lysis buffer, and the activated supernatant was mixed with 1 mL of talon resin and the supernatant, followed by shaking using a shaker at 4 ° C. The protein was bound to the resin by shaking slowly for 1 h at. After removing the supernatant, the resin was washed by shaking with 10 mL of cell lysis buffer and 10 mM imidazole buffer for 10 minutes, respectively. Finally, 10 mL of cell lysis buffer containing 100 mM imidazole was added and shaken for 1 hour, followed by filtration to recover the target protein (FIG. 4).

number Protein name gene Overexpression vector Expression condition Purification Method One T4 PTPRA MBP end One 2 T7 PTPRB GST end 2 3 T17 PTPRC MBP All One 4 T26 PTPRD pET28a end 3 5 T37 PTPRE MBP hemp One 6 T48 PTPRF pET28a end 3 7 T6 PTPRG MBP hemp One 8 T8 PTPRH GST end 2 9 T23 PTPRJ GST end 2 10 T39 PTPRK pET28a end 3 11 T5 PTPRM MBP end One 12 T38 PTPRN MBP end One 13 T12 PTPRN2 MBP I One 14 T15 PTPRO MBP I One 15 T10 PTPRR MBP I One 16 T22 PTPRS MBP end One 17 T20 PTPRT MBP la One 18 T31 PTPRU MBP end One 19 T28 PTPRZ MBP end One 20 PTP1B PTPN1 pET28a end 3 21 T25 PTPN2 MBP end One 22 T36 PTPN3 GST end 2 23 T41 PTPN5 pET28a end 3 24 T18 PTPN6 MBP I One 25 pk32 PTPN7 MBP All One 26 pk28 PTPN11 pET28a end 3 27 T1 PTPN12 MBP All One 28 T32 PTPN13 MBP end One 29 T40 PTPN14 MBP end One 30 T3 PTPN18 MBP end One 31 T2 PTPN21 MBP end One 32 T35 PTPN22 MBP end One 33 T30 PTPN23 MBP end One 34 pk4 DUSP1 MBP end One 35 pk5 DUSP2 MBP end One 36 pk7 DUSP4 MBP end One 37 pk8 DUSP5 MBP end One 38 pk9 DUSP6 MBP I One 39 pk10 DUSP7 MBP All One 40 T33 DUSP8 MBP end One 41 pk12 DUSP9 MBP end One 42 pk13 DUSP10 MBP end One 43 T27 DUSP16 MBP I One 44 T53 MK-STYX MBP end One 45 pk6 DUSP3 MBP I One 46 pk14 DUSP11 MBP end One 47 pk15 DUSP12 MBP end One 48 pk33 DUSP13A MBP I One 49 p44 DUSP13B MBP I One 50 pk30 DUSP14 MBP I One 51 p21 DUSP15 MBP end One 52 pk35 DUSP18 MBP I One 53 NE1 DUSP19 MBP end One 54 p19 DUSP21 MBP end One 55 pk18 DUSP22 MBP end One 56 p12 DUSP23 MBP end One 57 pk17 DUSP24 MBP end One 58 p16 DUSP25 MBP end One 59 p20 DUSP26 pET28a end 3 60 pk19 EPM2A MBP end One 61 p18 SSH1 MBP end One 62 NE3 SSH2 MBP end One 63 pk36 SSH3 MBP All One 64 pk3  PTP4A1 pET28a end 3 65 p49 PTP4A2 MBP end One 66 p26 PTP4A3 MBP end One 67 T29 CDC14B MBP end One 68 T9 TPTE MBP end One 69 p24 TENC1 16b-MBP end One 70 T24 MTMR1 16b-MBP hemp One 71 T19 MTMR3 MBP All One 72 pk16 MTMR7 16b-MBP end One 73 p13 MTMR8 MBP la One 74 T46 ACP1 MBP I One 75 pk1 CDC25A pET28a end 3 76 T47 CDC25B GST end 2 77 T45 CDC25C GST end 2

(88 expression purification attempts but only 77 PTPs were successful)

Example 4 Protein Activity Evaluation

DiFMUP commercially available according to the example of Pastula C. et al. Comb Chem High Throughput Screen. 6, 341-6, 2003; Welte S et al., Anal. Biochem. 338, 32-38, 2005. 100 mM) and pNPP (50 mM) were used to measure the enzyme activity of PTP (Table 2 and FIG. 5).

 Test result of enzyme activity of purified human PTP A2. DSP or VH1-like PTP 2.1 MKP T33 DUSP8 O O activation PK9 DUSP6 O O activation pk8 DUSP5 O O activation pk10 DUSP7 O O activation PK4 DUSP1 activation T27 DUSP16 O O activation sub Total 5 5 2.2 Atypical DSP P12 DUSP23 O O activation P16 DUSP25 O O activation P19 DUSP21 O O activation P21 DUSP15 O O activation P25 DUSP13A O O activation P44 DUSP13B O O activation PK6 DUSP3 O O activation PK15 DUSP12 O O activation PK18 DUSP22 O X activation PK19 EPM2A O O activation PK30 DUSP14 O O activation PK33 DUSP13A O O activation PK35 DUSP18 O O activation T53 STYX O O activation NE1 DUSP19 O O activation pk17 DUSP24 O O activation p20 DUSP26 O O activation PK18 DUSP22 O O activation sub Total 18 17 2.3 Slingshot P18 SSH1 O O activation NE3 SSH2 O O activation pk36 SSH3 O O activation sub Total 3 3 2.4 PRL P26 PTP4A3 O O activation P49 PTP4A2 O O activation PK3 PTP4A1 O O activation sub Total 3 3 2.5 CDC14 T29 CDC14B O O activation sub Total One One 2.6 PTEN P24 TENC1 O X Inactive P45 TENSN O X Inactive sub Total 2 0 2.7 pk16 MTMR7 O O activation T19 MTMR3 O O activation p13 / p23 MTMR8 O O activation T24 MTMR1 O O activation sub Total 4 4 B. Group II Cys-Based PTP T46 ACP1 O O activation sub Total One One C. Group III pk1 CDC25A O O activation T45 CDC25C O O activation T47 CDC25B O O activation sub Total 3 3 Total number of PTPs tested 70 The number of active PTPs 65 The number of inactive PTPs 5 Known as inactive in Ref. 4 Active but known as inactive in Ref. One Known as active in Ref. But inactive 2

* PTP code in the table is named by the inventor in accordance with the experimental order

* Ref. Is described by Alonso A. et al. Cell, 117, 699-711, 2004

As described above, a method for purifying human protein phosphorylated tyrosine phosphatase (PTP) using the E. coli expression system of the present invention is currently active, many PTPs including cancer, arthritis, diabetes Since it is known to be closely related to various diseases and new functions are continuously revealed, PTP purified according to the method of the present invention can be used as a new target enzyme for new drug development and can be used for high-efficiency drug search.

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Claims (10)

Iii) transforming Escherichia coli by preparing an expression vector of a human protein tyrosine phosphatase (PTP); Ii) inducing the expression of PTP by culturing the transformed Escherichia coli of step iii). A) incubating Escherichia coli and treating IPTG with a final concentration of 0.3-0.7 mM, B) incubating Escherichia coli and treating IPTG with a final concentration of 0.7-1.5 mM, C. E. coli was cultured in a medium containing 0.1-1 M of sorbitol and 40-60 mM glycine betaine, and IPTG was treated with a final concentration of 0.5-1.0 mM, D) E. coli is cultured in a medium containing 0.1-1 M of sorbitol and 40-60 mM glycine betaine, and IPTG is treated with a final concentration of 10-50 μM, E) In the group consisting of culturing Escherichia coli and treating IPTG with 0.1-0.2 mM at 3-6 ° C, an expression induction treatment condition suitable for generating each PTP with enzymatic activity in Escherichia coli is selected and the conditions selected above. Inducing expression under; And Iii) a method of producing human PTP with activity comprising the step of purifying PTP from the cultured Escherichia coli of step ii). The method of claim 1, wherein the base vector for the production of the expression vector of step iii) is a pET28a or pET16b vector. The method of claim 1, wherein the IPTG final concentration of the expression induction condition 'a' of step ii) is 0.5 mM. The method according to claim 1, wherein the IPTG final concentration of the expression inducing condition “I” of step ii) is 1.0 mM. The method of claim 1, wherein the expression induction condition 'da' of step ii) is 1M, glycine betaine concentration is 50mM, IPTG final concentration is 0.5mM. The method according to claim 1, wherein the expression induction condition 'la' of step ii) has a sorbitol concentration of 1 M, a glycine betaine concentration of 50 mM, and an IPTG final concentration of 20 mM. The method of claim 1, wherein the purification step of step iii) is performed using a resin selected from the group consisting of amylose resin, glutathione resin, and talon resin. The method according to claim 1, wherein the human PTP is a protein tyrosine phosphatase receptor type A (PTPRA), Unitprot accession number: P18433), PTPRB (Unitprot accession number: P23467), PTPRC (Unitprot accession number: Q5T5R0), PTPRD (Unitprot accession number: P23468 ), PTPRE (Unitprot access number: P23469), PTPRF (Unitprot access number: P10586), PTPRG (Unitprot access number: P23470), PTPRH (Unitprot access number: Q2NKN9), PTPRJ (Unitprot access number: Q12913), PTPRK (Unitprot Accession number: Q15262), PTPRM (Unitprot accession number: P28827), PTPRN (Unitprot accession number: Q16849), PTPRN2 (type N polypeptide 2, Unitprot accession number: Q92932), PTPRO (Unitprot accession number: Q16827), PTPRR (Unitprot Access number: Q15256), PTPRS (Unitprot access number: Q13332), PTPRT (Unitprot access number: O14522), PTPRU (Unitprot access number: Q92729), PTPRZ (Unitprot access number: P23471), PTPN1 (protein tyrosine phosphatase, non- receptor type 1, Unitprot accession number: P18031), PTPN2, (Unitprot accession number: P17706) PTPN3 (Unitprot accession number: P26045), PTPN5 (Un itprot access number: P54829), PTPN6 (Unitprot access number: P29350), PTPN7 (Unitprot access number: P35236), PTPN11 (Unitprot access number: Q06214), PTPN12 (Unitprot access number: Q05209), PTPN13 (Unitprot access number: Q12923) ), PTPN14 (Unitprot access number: Q15678), PTPN18 (Unitprot access number: Q99952), PTPN21 (Unitprot access number: Q16825), PTPN22 (Unitprot access number: Q9Y2R2), PTPN23 (Unitprot access number: Q9H3S7), DUSP1 (dual specificity phosphatase 1, Unitprot access number: P28562), DUSP2 (Unitprot access number: Q05923), DUSP4 (Unitprot access number: Q9PW71), DUSP5 (Unitprot access number: Q16690), DUSP6 (Unitprot access number: Q16828), DUSP7 (Unitprot Access no .: Q16829), DUSP8 (Unitprot access no .: Q13202), DUSP9 (Unitprot access no .: Q99956), DUSP10 (Unitprot access no .: Q9Y6W6), DUSP16 (Unitprot access no .: Q9BY84), MK-STYX (Map kinase phosphatase- like protein, Unitprot access number: Q9Y6J8), DUSP3 (Unitprot access number: P51452), DUSP11 (Unitprot access number: O75319), DUSP12 (Unitprot t Access number: Q9UN16), DUSP13A (Unitprot access number: Q9UII6) DUSP13B (Unitprot access number: Q9QYJ7), DUSP14 (Unitprot access number: O95147), DUSP15 (Unitprot access number: Q9H1R2), DUSP18 (Unitprot access number: Q8NEJ0) , DUSP19 (Unitprot access number: Q8WTR2), DUSP21 (Unitprot access number: Q9H596), DUSP22 (Unitprot access number: Q9NRW4), DUSP23 (Unitprot access number: Q9BVJ7), DUSP24 (Unitprot access number: Q9Y6JB), protSP25 Number: Q9Y216), DUSP26 (Unitprot access number: Q4GOw2), EPM2A (epilepsy progressive myoclonus type 2A, Unitprot access number: O95278), SSH1 (slingshot homolog 1, Unitprot access number: Q8WYLO), SSH2 (Unitprot access number: Q76176) , SSH3 (Unitprot access number: Q8TE77), PTP4A1 (protein tyrosine phosphatase 4a1, Unitprot access number: Q93096), PTP4A2 (Unitprot access number: Q12974), PTP4A3 (Unitprot access number: O75365), CDC14B (CDC14 cell division cycle 14 homolog B, Unitprot accession number: O60729), TPTE (transmembrane phosphoinositide 3-phosphatase and tensin homolog 2 , Unitprot access number: Q4R6N0), TENC1 (tensin like C1 domain containing phosphatase, Unitprot access number: Q2NL80), MTMR1 (myotubularin related protein 1, Unitprot access number: Q13613), MTMR3 (Unitprot access number: Q13615, MTMR7 (Unitprot access) Q9Y216), MTMR8, ACP1 (acid phosphatase 1 soluble, Unitprot access number: P24666), CDC25A (cell division cycle 25B, Unitprot access number: P30304), CDC25B (Unitprot access number: P30305), CDC25C (Unitprot access number: P30307). Expression vector pET28a-MBP-HTs having a cleavage map of Figure 1-b for the expression and purification of PTP (protein tyrosine phosphatase) forming an insoluble inclusion body in E. coli expression system. delete

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