CN119331857B - Novel recombinant protein plamminogen-CTM and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a novel recombinant protein Plasminogen-CTM and application thereof. The invention firstly provides a novel recombinant protein plamminogen which is based on natural plamminogen protein, and a plurality of recombinant protein mutants are obtained after multi-site mutation is carried out on amino acid sites 742-810 of the natural plamminogen protein in a combined mode. The novel recombinant protein plasmin provided by the invention has enzyme activity without activation of an activator, so that the recombinant protein plasmin can be an effective means for replacing natural plasmin directly separated from blood plasma.

Description

Novel recombinant protein plamminogen-CTM and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a novel recombinant protein Plasminogen-CTM and application thereof.

Background

Plasmin (plasmin), also known as plasmin (Plasmatrypsinogen), is a precursor substance of plasmin (plasmin) and does not have the ability to cleave fibrin. In normal organisms plasmin is present in the zymogen state and is only active by the action of a plasmin activator. Plasmin is a proteolytic enzyme capable of specifically hydrolyzing fibrin gel, and is an important component in fibrinolytic systems. The two systems of in vivo coagulation and fibrinolysis are interdependent and closely connected. Once the body produces coagulation reaction, the fibrinolytic system is almost simultaneously activated, so that redundant thrombus in the body is removed, and the level of fibrinogen in the body is reduced through negative feedback effect, thereby avoiding excessive aggregation of fibrin. The lack of plasminogen can lead to fibrin accumulation, which in turn leads to the development of lesions that impair normal tissue and organ function.

The most common method for obtaining plasmin is currently the plasma separation and obtaining method. However, the method has the problems of high plasmin separation cost, limited raw material sources, high price of plasmin finished products and the like. Although plasmin obtained by a gene mutation method, a synthetic biological means and the like can overcome the problems of high preparation cost and limited raw material sources of plasmin to a certain extent, the plasmin synthesized at present has the problems of low activity or no activity and the like. For example, a recombinant human plasminogen protein having a length of about 248 amino acids, and a method for preparing and using the same are disclosed in the patent with publication number CN118308334 a. However, the recombinant human plasminogen prepared in this protocol is an inactive zymogen form and requires the addition of an activator (e.g., urokinase) to activate to obtain active plasmin.

In view of the foregoing, there is a need to provide new methods or strategies to ameliorate the deficiencies of the prior art.

Disclosure of Invention

In view of the above, the present invention aims to provide a plurality of variants and applications of novel recombinant protein Plasminogen, and the specific technical scheme is as follows.

Novel recombinant protein plaminogen, which is based on natural plaminogen protein, and is any one of mutants after following mutation at 742-810 amino acid positions of the natural plaminogen protein:

(1) The first mutant comprises 772 th Y mutation to F, 775 th Q mutation to L, 778 th T mutation to V, 788 th N mutation to L, 793 rd Y mutation to F, 801 th T mutation to V, 809 th N mutation to L and 810 th N mutation to L (SEQ ID NO. 7)

(2) The second mutant is composed of 757 th Q mutant into L, 772 th Y mutant into F, 775 th Q mutant into L, 778 th T mutant into V, 788 th N mutant into L, 793 rd Y mutant into F, 801 th T mutant into V, 809 th N mutant into L and 810 th N mutant into L (SEQ ID NO. 8)

(3) Third mutant, 742T mutant V, 753T mutant V, 757Q mutant L, 772Y mutant F, 775Q mutant L, 778T mutant V, 788N mutant L, 793Y mutant F, 801T mutant V, 809N mutant L and 810N mutant L (SEQ ID NO. 9), or

(4) Fourth mutant 753T mutation to V, 757Q mutation to L, 772Y mutation to F, 775Q mutation to L, 778T mutation to V, 788N mutation to L, 793Y mutation to F, 801T mutation to V, 809N mutation to L and 810N mutation to L (SEQ ID NO. 1).

As a preferred technical means, the novel recombinant protein plaminogen is based on a natural plaminogen protein, and the following mutation is carried out on amino acid positions 753-810 of the natural plaminogen protein:

The mutation of T at 753 to V, Q at 757 to L, Y at 772 to F, Q at 775 to L, T at 778 to V, N at 788 to L, Y at 793 to F, T at 801 to V, N at 809 to L and N at 810 to L.

As a preferable technical means, the amino acid sequence of the novel recombinant protein plamminogen is shown as SEQ ID NO. 1.

Further, the amino acid sequence of the novel recombinant protein plamminogen also comprises a sequence shown in any one of SEQ ID NO. 7-9.

A recombinant expression vector comprising any one of the novel recombinant proteins Plasminogen described above.

Further, the types of the vectors include plasmid vectors, phage vectors, or animal and plant virus vectors.

A recombinant bacterium expressing any of the novel recombinant proteins Plasminogen described above.

Further, the recombinant bacteria include escherichia coli, bacillus subtilis, pichia pastoris or saccharomyces cerevisiae.

The use of any of the novel recombinant proteins plamminogen described above in the manufacture of a medicament for dissolving fibrin clots.

The use of any of the novel recombinant proteins plamminogen described above in the manufacture of a medicament for preventing the formation of a fibrin clot.

Further, the dosage forms of the medicament include tablets, injections and/or sprays.

The use of any of the novel recombinant proteins plamminogen described above in the preparation of an in vitro fibrinolysis reagent.

The beneficial technical effects are as follows:

The invention obtains a plurality of recombinant plasma protein variants with enzyme activity by carrying out mutation modification on a plurality of sites of C-terminal (C-terminal) of the natural plasma protein in a combined mode, wherein the recombinant plasma protein variants with optimal effect have the enzyme activity which is equal to that of the natural plasmin, can effectively promote the dissolution of fibrin clots, and in addition, the crystal structure of the optimal mutant is similar to that of the natural protein, and the total hydrophilcity and hydrophilcity performance of the optimal mutant is also similar to that of the natural plasma protein, so that the recombinant plasma protein variants have wide clinical application value and market development value.

Although the enzyme activity of the other mutants is weaker than that of the optimal mutant and the natural plasmin, the other mutants can have the enzyme activity without additionally adding an activator for activation, so that the mutant can also be an alternative scheme for replacing the natural plasmin directly separated from the blood plasma.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 is a diagram of electrophoresis bands of a plurality of recombinant proteins prepared according to the present invention;

FIG. 2 is a crystal structure of Plasminogen-CTM predicted by Alphafold;

FIG. 3 is a crystal structure of a native plaminogen protein predicted by Alphafold;

FIG. 4 shows the results of hydrophilic-hydrophobic analysis of recombinant protein Plasminogen-CTM prepared by the invention;

FIG. 5 is a diagram of the electrophoresis bands of the unmutated recombinant protein plasmigen;

FIG. 6 shows the results of the enzyme activity measurement of recombinant protein plasmin-CTM and other recombinant proteins or plasmin prepared by the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Herein, "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.

As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Materials and reagents:

SD (Sprague Dawley) rats were purchased from the university of south China laboratory animal center, male rats, 4 weeks old, order number B241009410.

Coli BL21 (DE 3) competent cells were purchased from Beijing Soy Bao technology Co.

Plasmin (derived from human plasma) was purchased from Shanghai Michelia Biochemical technologies Co., ltd., cat# P920011.

The pET-22b (+) plasmid is a commercially available E.coli expression vector, purchased from Beijing, biotech Co., ltd. The vector tags were N-pelB and C-His, and the vector resistance was AMPICILLIN (ampicillin).

Restriction enzymes NdeI and XhoI were purchased from NEB (Beijing) Inc. His-tag protein purification resin (Nickel column), purchased from Shanghai-Haimai bioengineering Co., ltd., cat# LM-616.IPTG, ampicillin, DMSO (dimethyl sulfoxide) were all purchased from beijing solebao technologies. DMEM medium was purchased from GIBCO. CCK8 reagent is purchased from Chongqing Bao optical technology Co., ltd and is used according to the operation of the reagent instruction.

Culture medium:

The LB medium contains 5g yeast extract, 10 g tryptone, 10 g sodium chloride per liter, and the pH is adjusted to 7.0.

The preparation method comprises dissolving 5g yeast extract, 10 g tryptone, 10 g sodium chloride in 950 mL double distilled water, adjusting pH to 7.0 with sodium hydroxide solution, and fixing volume to 1L with double distilled water. If a solid medium is formulated, agar is added at 1.5 g/100 mL. Autoclaving at 121 ℃ 30min.

The experimental reagents which are not specifically described in the present invention are all conventional reagents in the art and can be prepared according to the conventional methods in the art or purchased from related reagent suppliers, and the experimental methods which are not specifically described are all conventional methods in the art, and reference is made to related experimental manuals, such as molecular cloning experimental manuals or the specifications of related reagent manufacturers.

Example 1

1. The sequence design and expression purification of recombinant protein Plasminogen-CTM.

The embodiment provides a novel recombinant protein, which is obtained by carrying out site-directed mutagenesis transformation on natural Plasminogen protein, and is named as recombinant protein Plasminogen-CTM, the amino acid sequence of which is shown as SEQ ID NO.1, the total length of which is 820 amino acids, and the nucleotide sequence of which is shown as SEQ ID NO.2, and the total length of which is 2460 bp. When the gene is synthesized, ndeI restriction sites (CATATG) and XhoI restriction sites (CTCGAG) are added to the 5 'and 3' ends of the gene, respectively. The gene sequence with the enzyme cutting site is shown in SEQ ID NO. 3. Sequencing and verifying the synthesized genes, wherein the genes with correct sequences are used for subsequent vector construction. The specific sequences involved are shown in Table 1.

TABLE 1

2. Vector construction

The pET-22b (+) plasmid is preferred as an expression vector in this example. The pET-22b (+) plasmid and the target gene (SEQ ID NO. 1) were subjected to a double cleavage reaction using restriction enzymes NdeI and XhoI, respectively, and then the target gene was ligated into the pET-22b (+) vector by ligation. It will be appreciated that the present embodiment may also be implemented using phage vectors, animal and plant viral vectors, or the like.

3. Recombinant bacterial transformation

(1) Competent cells of E.coli BL21 (DE 3) (Beijing Soy Bao) were removed from the-80℃refrigerator and ice-bath 5 min.

(2) After glycerol of BL21 (DE 3) competent cells is preserved and melted, competent cells are added into the connection product, the gun head is sucked and put for 3 times and is uniformly mixed, and the ice bath is kept stand for 30 min.

(3) The wall of the tube was quickly wiped with absorbent paper and then heat-shocked at 42 ℃ to 90 s, immediately ice-bath 2 min.

(4) 800. Mu.L of LB liquid medium was added under aseptic conditions, and 45 min was cultured at 37℃and 150 rpm.

(5) The cells were collected by centrifugation at 8000 rpm and min, a part of the supernatant was discarded, E.coli was resuspended in about 100. Mu.L of the remaining supernatant, and then spread evenly on LB solid medium containing 100. Mu.g/mL ampicillin, placed in a 37℃incubator, and cultured upside down for 12 h.

(6) The monoclonal is selected and inoculated into liquid LB medium containing 100 mug/mL ampicillin, and positive clone identification is carried out after culturing at 37 ℃ for 12-16 h.

Coli is preferred as the recombinant bacterium in this example. It will be appreciated that this embodiment may also be implemented using bacillus subtilis, bi Chi yeast or saccharomyces cerevisiae strains, etc.

4. Protein expression and purification

(1) Inoculating, namely preparing a liquid LB culture medium, sterilizing, placing the sterilized liquid LB culture medium in an ultra-clean workbench, cooling to room temperature, adding ampicillin into the LB culture medium in the ultra-clean workbench, uniformly mixing the ampicillin to ensure that the final concentration of the ampicillin is 100 mug/mL, inoculating escherichia coli (positive clone) containing target gene plasmids into the LB culture medium according to the amount of 200 mug/L, placing the LB culture medium in a shaking table, and culturing for 8-10 h according to the conditions that the rotating speed is 170 rpm and the temperature is 37 ℃.

(2) After 8-10 h of shaking culture, 2 mL bacterial liquid is taken out, the OD600 value of the bacterial liquid is measured by a spectrophotometer, when the OD600 value of the bacterial liquid reaches 0.6-0.8, 200 mu L/mL of IPTG is added into the bacterial liquid, and then 8 h of shaking culture is carried out according to the conditions of 37 ℃ and 170 rpm.

(3) Purifying by adding IPTG and shake culturing 8 h, taking out bacterial liquid, centrifuging 5 min at 4deg.C at 8000 rpm, removing supernatant after centrifuging, and retaining precipitate (the precipitate is Escherichia coli).

(4) Ultrasonic crushing, namely, carrying out ultrasonic crushing on escherichia coli, centrifuging, obtaining a precipitate which contains target protein, washing the precipitate obtained by centrifuging by using a washing solution I (50 mmol/L Tris-HCl, 1mol/L urea and 10 mL/L Triton X-100), then washing by using a washing solution II (50 mmol/L Tris-HCl,2 mol/L urea and 5 mL/L Triton X-100), and then dissolving by using an inclusion body dissolving solution (50 mmol/L Tris-HCl,8 mol/L urea and 100 mmol/L NaCl).

(5) Finally, carrying out gradient renaturation on the inclusion body solution, wherein the method comprises the steps of filling the inclusion body solution into a dialysis bag (Beijing Soy Bao technology Co., ltd., product number: YA 1071), and then sequentially placing the dialysis bag into urea solutions of 6M, 4M, 2M, 1M and 0.5M for gradient renaturation, wherein renaturation is carried out at the low temperature of 4 ℃ under the condition of 2-4 h of each urea concentration. Purifying the solution after renaturation by His-tag protein purification resin (nickel column, shanghai-associated Michael) to obtain recombinant protein Plasminogen-CTM (histidine tag is added in the design of the gene sequence of the target protein). After purification, it was identified whether the purified target protein was successfully obtained by running polyacrylamide-gel electrophoresis (SDS-PAGE). The results showed that the target protein of about 91.870 kDa was obtained (see FIG. 1).

The high-order structures of the recombinant protein plamminogen-CTM and the natural plamminogen protein prepared in the example are predicted, and the results are shown in FIG. 2 and FIG. 3. FIG. 2 shows the protein crystal structure of the novel recombinant protein Plasmogen-CTM, FIG. 3 shows the crystal structure of native Plasmogen, and the results show that Plasmogen-CTM is similar to the three-dimensional structure of native Plasmogen with no significant changes.

Example 2

This example provides a novel recombinant protein plamminogen-CTM hydrophilic-hydrophobic property verification.

Further, hydrophilicity and hydrophobicity prediction was performed on the recombinant protein Plasminogen-CTM prepared in example 1, and the results are shown in FIG. 4. The abscissa represents the amino acid number of the protein, the ordinate represents the hydrophilicity and hydrophobicity, the larger the ordinate represents the hydrophobicity, and if negative, the hydrophilicity.

The results show that the total hydrophilic-hydrophobic properties of the novel recombinant protein plaminogen-CTM are similar to those of the natural plaminogen, and there is no obvious difference.

Example 3

This example provides recombinant plamminogen proteins and their hydrophilicity and hydrophobicity assays.

First, a recombinant approach was used to synthesize a native plaminogen protein, called recombinant plaminogen protein (unmutated hydrophilic and hydrophobic amino acids). The method steps of prokaryotic expression vector construction, recombinant bacterial transformation, protein expression and purification of the recombinant Plasminogen protein are the same as in example 1. The amino acid sequence SEQ ID NO.4, the nucleotide sequence SEQ ID NO.5 and the nucleotide sequence SEQ ID NO.6 with enzyme cleavage sites of the unmutated recombinant protein Plasminogen constructed in the example are shown in Table 2.

TABLE 2

After purification, it was identified whether the purified target protein was successfully obtained by running polyacrylamide-gel electrophoresis (SDS-PAGE), see FIG. 5.

The results show that the protein of interest (unmutated recombinant protein plamminogen) of about 90 kDa was obtained, in agreement with its theoretical prediction (91.94 kDa). Table 3 shows basic information (theoretical values) of plamminogen-CTM and the unmutated recombinant protein plamminogen, as follows.

TABLE 3 Table 3

As can be seen from Table 3, the hydrophilicity of the plamminogen-CTM was similar to that of the unmutated recombinant protein plamminogen.

Example 4

This example provides mutants of other recombinant plaminogen proteins, see in particular table 4.

TABLE 4 Table 4

The method steps of prokaryotic expression vector construction, transformation, protein expression and purification of mutants of other recombinant plamminogen proteins provided in this example are the same as in example 1. After purification, it was examined by running polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether the purified target protein was successfully obtained, and the results are shown in FIG. 1.

As can be seen from the SDS-PAGE result of FIG. 1, after modification of hydrophilic amino acid mutation, recombinant plaminogen proteins after different site mutations can be effectively expressed, and isoelectric points and hydrophobicity values of the recombinant plaminogen proteins are shown in Table 5.

TABLE 5

As can be seen from Table 5, the molecular weights, isoelectric points and average hydrophilicity values of Plaminogen-8, plaminogen-9 and Plaminogen-11 were similar, with no large difference.

The amino acid sequences of mutants Plaminogen-8, plaminogen-9 and Plaminogen-11 of the recombinant Plaminogen proteins according to this example are shown in the following Table.

TABLE 6

Example 5

This example provides an enzyme activity assay for a plurality of recombinant plaminogen protein mutants prepared in example 4.

Preparation of protease solution recombinant protein mutants Plasminogen-8, plasminogen-9, plasminogen-CTM, plasminogen-11 and natural cellulose 50 μg were precisely weighed in sequence and dissolved in 500 mL hydrogen phosphate dibasic-citric acid buffer (operated in a constant temperature water bath) of 0.02M and pH 6.0 at 40 ℃ to obtain solutions for standby.

Enzyme activity determination 3% fibrin solution 10 mL was added to a large tube, buffer 3 mL was added to equilibrate to about 5min in a 60 ℃ water bath, each recombinant protein mutant solution 0.5 mL was added to each experimental group, mixed well and the time was recorded. 2 min, taking out the reaction solution by a dropper, dripping the reaction solution into a color mixing hole which is filled with dilute iodine solution (0.75 mL) in advance, and recording the time as T (min) when the color of the liquid in the hole is changed from purple to brownish red and the color is the same as the standard color, namely reaching the end point of the reaction. Each experimental group is used for carrying out 3 parallel experiments, and the average enzyme activity of the recombinant protein of each experimental group is calculated in IU/mg.

As shown in FIG. 6, the average enzyme activities of plasmin-CTM were comparable to that of native plasmin, while the average enzyme activities of plasmin-8, plasmin-9 and plasmin-11 were weaker, and the specific results are shown in Table 7.

TABLE 7

The above results demonstrate that unmutated plasmin (i.e., the native plasmin protein) has little enzymatic activity, and the novel recombinant protein plasmin-CTM exhibits the highest enzymatic activity, which is not significantly different from that of native plasmin, indicating that the novel recombinant protein plasmin-CTM has no need for an activator and also has enzymatic activity, and that the enzymatic activity is high, and has potential to be an effective means for replacing native plasmin isolated directly from plasma. On the other hand, the recombinant proteins Plasmogen-8, plasmogen-9 or Plasmogen-11 have enzyme activities although their enzyme activities are weaker compared to Plasmogen-CTM and native plasmin, but do not require additional activators to activate them. Among them, plasmin-11, which has relatively minimal enzyme activity, can also have an enzyme activity value up to about 30% of that of native plasmin, so plasmin-8, plasmin-9 and plasmin-11 can also be alternatives to native plasmin that is isolated directly from plasma.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.

Claims (7)

1. The novel recombinant protein plaminogen is characterized in that the novel recombinant protein plaminogen is based on natural plaminogen protein, and any one of mutants after the following mutation is carried out on amino acid positions 742-810 of the natural plaminogen protein:

(1) A first mutant, wherein the 772 th Y mutation is F, the 775 th Q mutation is L, the 778 th T mutation is V, the 788 th N mutation is L, the 793 rd Y mutation is F, the 801 st T mutation is V, the 809 th N mutation is L and the 810 th N mutation is L, the amino acid sequence of the first mutant is shown as SEQ ID NO.7, or

(2) A second mutant, wherein the 757 th Q mutation is L, the 772 th Y mutation is F, the 775 th Q mutation is L, the 778 th T mutation is V, the 788 th N mutation is L, the 793 rd Y mutation is F, the 801 th T mutation is V, the 809 th N mutation is L and the 810 th N mutation is L, the amino acid sequence of the second mutant is shown as SEQ ID NO.8, or

(3) Third mutant, wherein the 742 th T mutation is V, the 753 th T mutation is V, the 757 th Q mutation is L, the 772 th Y mutation is F, the 778 th Q mutation is L, the 778 th T mutation is V, the 788 th N mutation is L, the 793 rd Y mutation is F, the 801 st T mutation is V, the 809 th N mutation is L and the 810 th N mutation is L, the amino acid sequence of the third mutant is shown as SEQ ID NO.9, or

(4) And a fourth mutant, wherein the 753 th T mutation is V, the 757 th Q mutation is L, the 772 th Y mutation is F, the 775 th Q mutation is L, the 778 th T mutation is V, the 788 th N mutation is L, the 793 rd Y mutation is F, the 801 th T mutation is V, the 809 th N mutation is L and the 810 th N mutation is L, and the amino acid sequence of the fourth mutant is shown as SEQ ID NO. 1.

2. A recombinant expression vector, wherein a target gene is connected to the recombinant expression vector, and the target gene is a novel recombinant protein plamminogen gene of any mutant of claim 1.

3. A recombinant bacterium, characterized in that it expresses a novel recombinant protein plamminogen of any one of the mutants according to claim 1.

4. Use of a novel recombinant protein plamminogen of any of the mutants according to claim 1 for the preparation of a medicament for dissolving fibrin clots.

5. Use of a novel recombinant protein plamminogen of any of the mutants according to claim 1 for the preparation of a medicament for preventing fibrin clot formation.

6. The use according to claim 4 or 5, wherein the medicament is in the form of a tablet, injection or spray.

7. Use of the novel recombinant protein plamminogen of any one of the mutants according to claim 1 for the preparation of an in vitro fibrinolysis clot reagent.