JP2006083126A - Reagent kit for crystallizing protein and crystallization screening method utilizing the reagent kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent kit for screening, usable for efficiently finding out the optimum condition for crystallization of a sample protein in both of an initial screening and secondary screening following the initial screening when carrying out the screening of the crystallization of the protein. <P>SOLUTION: The reagent kit for crystallizing the protein is constituted of a reagent kit for the initial screening and a reagent kit for the secondary screening. The reagent kits for the initial and secondary screening are obtained by using either one of a precipitant and a buffer to be mutually combined as a standard chemical of a standard of combination, using the other chemical to be distributed to and combined with the standard chemical as a distribution chemical, grouping several kinds of standard chemicals based on each number of kinds of the several distribution chemicals, each quantitatively combining them one by one, and dividedly injecting them in many wells arranged in a container formed into the kit. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a crystallization reagent kit for crystallizing a desired protein from a protein-containing sample and a crystallization screening method using the reagent kit.

  Although biological science has made tremendous progress, the understanding of the relationship between its superior function and structure of protein molecules is still insufficient. The most common method for analyzing the three-dimensional structure of a protein molecule is the X-ray structure analysis method, but first, a high-quality single crystal of protein is required. However, it is still difficult to grow a high-quality protein single crystal that can be analyzed sufficiently, and it is not uncommon to take months to years. The biggest cause is that protein crystallization is still being carried out by trial and error. Therefore, research from the viewpoint of crystal growth in order to grow a high-quality single crystal of protein is important.

Conventionally, reagents for protein crystallization are commercially available as kits from several overseas companies, but there are only about tens to 100 types for initial screening, and so-called sparse conditions are selected based on past results. A matrix sampling method (see Non-Patent Document 1) or the like is employed.
There are enormous numbers of protein crystallization reagents, such as precipitants, buffers, additives, and combinations of pH and concentration, but the sparse matrix sampling method compares about 100 types of proven conditions in the past. It is a method that can be tried easily. This is because the precipitant used for protein crystallization has various viscosities, and it is difficult to dispense and mix with a robot. Under conditions where a large number of reagents must be prepared manually, It is also a method for obtaining the maximum effect with relatively little effort.
Jancarik, J. and Kim, S.-H. Sparse matrix sampling method: a screening method for crystallization of proteins. J. Appl. Cryst. 24, (1991) 409-411.)

  As for reagents, so-called crystallization screening kits are manufactured and sold by overseas companies such as Hampton Research, Jena Bio Science, Decode, Molecular Dimensions, and Sigma-Aldrich. They are created based on past protein crystallization databases based on data on proteins with specific properties extracted from kits that have proven results.

  Recently, the number of types of crystallization screening kits has increased, and the management and storage of these reagents is easy. Therefore, the kits are first used to crystallize new samples, and the reagents are prepared by themselves when microcrystals are obtained. Therefore, it is common to refine the conditions further. New crystallization robots have also been developed, crystallization is automated, and a wider range of conditions can be tested.

  However, the crystallization method using the conventional screening kit is based on past performance data, so the maximum effect is expected under limited conditions, but there are many similar conditions. It is not suitable for systematic data arrangement and is not suitable for screening near the condition where crystals are obtained. In addition, the preparation of reagents for optimizing the conditions and the processing of a large amount of protein samples require a large amount of work and time, resulting in a complicated arrangement of crystallization results.

The object of the present invention is to provide a protein crystallization reagent kit and a crystallization screening method using the reagent kit for solving the above-mentioned problems. Based on one of a wide variety of precipitants and buffers, A reagent kit that arranges the conditions set for this quantitatively and regularly, and further subdivides the conditions (pH, precipitant concentration, etc.) regularly for each reagent to create optimization conditions, and this reagent It is intended to provide a crystallization screening method using a kit.
In the reagent kit of the present invention, first, any one of the precipitating agent and the buffering agent that are combined with each other is used as a reference chemical solution that is a reference for the combination, and the other chemical solution that is distributed and combined with the reference chemical solution is used as the distributing chemical solution. Initially, multiple types of reference drug solutions are grouped together according to the number of types of multiple types of distribution drug solutions, and each is quantitatively combined sequentially and dispensed into a number of wells arranged in a kit container. For the screening reagent kit and at least one of the reference chemical liquid and the distribution chemical liquid whose combination is specified in the initial screening reagent kit, the mixing ratio or amount, concentration, pH, and mixing ratio or amount of the raw material drug By subtracting one or more quantitative elements in a generally quantitative or regular manner, the reagent conditions can be subdivided and the number of samples can be divided into multiple And a secondary screening reagent kit in which a combination of each of the increased reference drug solution and distributed drug solution is dispensed into a number of wells arranged in a kit container. Yes.

  Secondly, one or two or more additives are added to each of the combinations in which additives are desirable for protein crystallization from among the combinations of the reference drug solution and the distribution drug solution in the initial screening reagent kit. The initial screening reagent kit is characterized by being divided into groups, grouped for each additive, and added by dispensing.

  Thirdly, in the combination of the individual precipitant and buffer in the reagent kit for secondary screening, either or both of the precipitant mixing ratio and concentration are changed substantially quantitatively or regularly in multiple steps. In addition, by changing either one or both of the pH of the buffer and the titration reagent ratio in multiple steps, approximately quantitatively or regularly, the combinations of the reference drug solution and the distribution drug solution are grouped together and the reagent conditions are subdivided. In addition, the number of reagents is increased.

  Fourth, one or two or more types of additives are added to each of the combinations in which additives are desirable for protein crystallization, compared to the combination of the reference drug solution and the distribution drug solution in the secondary screening reagent kit. It is characterized in that it is distributed, grouped for each additive, added and dispensed into a reagent kit for secondary screening.

  Fifth, some or all of the precipitant is 2-Propanol (isopropanol), MPD (2-methyl-2,4-pentanediol), PEG 4,000 (polyethylene glycol 4,000), PEG 20,000 (polyethylene glycol 20,000), Jeffamine M-600 (Jephamine M-600), Na Form (sodium formate), Na Chlor (sodium chloride), Li Sulf (lithium sulfate), Amm Sulf (ammonium sulfate), KNa Tart (sodium tartrate), Na Acet (sodium acetate) , Tri-Na Citr (trisodium citrate), Mg Sulf (magnesium sulfate), Na2 Malon (disodium malonate) and Na Phos (sodium phosphate), K2 Phos (dipotassium phosphate) It is characterized by that.

  Sixth, part or all of the buffering agent is Acetic acid (acetic acid), Na3 citrate (trisodium citrate), MES (2-morpholinoethanesulfonic acid), HEPES (2- [4- (2hydroxyethyl)- 1-piperazinyl] ethanesulfonic acid), Tris (tris (hydroxymethyl) aminomethane), CHES (N-cyclohexyl-2-aminoethanesulfonic acid).

  Seventh, some or all of the additives are selected from Li Chlor, Mg Chlor, Ca Chlor, and 1,4-Dioxane. It is said.

  Eighth, the number of reagents in the initial screening reagent kit is 144 or more.

  Ninth, the number of reagents in the secondary screening reagent kit is 3456 or more.

  The crystallization screening method of the present invention performs protein crystallization screening using the initial screening reagent described in the above 1, 2, 5, 6, 7 or 8, and has a high crystallization rate as a result of the screening. Screening of the protein is performed by selecting a reagent from the secondary screening reagents described in the above 1, 3, 4, 5, 6, 7 or 9 in which the reagent conditions are subdivided.

  Furthermore, the crystallization state of the protein obtained as a result of the crystallization test is image-recognized, and the image-recognized image is compared with a large number of crystallization state evaluation reference images that have been evaluated according to scores and recognized in advance. Thus, the crystallization state of each crystallized protein is determined separately, and the crystallization rate of each screening reagent for the protein is determined by the score.

  According to the present invention configured as described above, since crystallization screening can be performed by giving a plurality of conditions that change regularly or systematically to one type of chemical solution, the optimum conditions for crystallization by the chemical solution are found. In addition, it is possible to easily find the optimization conditions for each type by screening by giving the above conditions to a plurality of types of chemical solutions.

  Further, as a result of screening optimized as described above, there is an advantage that even higher optimization conditions can be found by further subdividing the conditions for a portion with a high crystallization rate and repeating experiments.

Hereinafter, a protein crystallization screening reagent kit of the present invention, a method for producing the same, and a crystallization method using the reagent kit will be sequentially described with reference to the drawings.
1. Overview of Initial Screening Reagent Preparation Table As shown in Reagent Nos. 1 to 144 in the embodiment of Table 1, the reagent kit of the present invention includes 15 types from isopropanol (2-Propanol) to sodium phosphate (Na Phos). And 16 kinds of precipitating agents consisting of dipotassium phosphate (K2 Phos = which also has a function as a precipitating agent) used as buffer titration reagents No. 139 to 144, and Acet (acetic acid), Citr (Citric acid), MES (2-morpholinoethanesulfonic acid), HEPES (2- [4- (2hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), Tris (tris (hydroxymethyl) aminomethane), CHES ( N-cyclohexyl-2-aminoethanesulfonic acid) is used as a standard, and the amount and type of these are arranged regularly and systematically for each amount or type. It is.

  That is, in the initial screening, six kinds of buffers along each pH are combined without additives for the above 16 kinds of precipitants, and MPD (methylpentanediol) and PEG (polyethylene glycol), which have a particularly high empirical crystal production rate. ), Four additives consisting of Li Chlor (lithium chloride), Mg Chlor (magnesium chloride), Ca Chlor (calcium chloride) and Dioxane (dioxane) are combined. Similarly, for Amm.Sulf (ammonium sulfate), only a single additive 1,4-Dioxane is combined for each of the six types of buffers. As a result, 144 kinds of initial screening reagents are arranged in total. As can be understood from the conceptual diagram of screening reagents shown in FIG. 6 (details will be described later), regular and systematic design (arrangement of combinations) of 144 initial screening reagents has been carried out, and protein crystal experiments Systematic judgment is possible.

2. Outline of Secondary Screening Reagent Preparation Table As for the secondary screening reagent prepared in the same manner as in 1 above, 144 types of screening reagents exist for 16 types of precipitants. What this means is that optimization is aimed at by dividing the precipitant concentration into 4 stages and the buffer pH into 6 stages from the conditions when microcrystals are produced by initial screening. From the conceptual diagram of secondary screening shown in FIGS. 8 and 10 (details will be described later), it can be seen that there are 3456 types of secondary screening for 24 conditions per initial screening condition. The condition optimization by the secondary screening is premised on the use when the microcrystal is produced by the experimental result in the initial screening. This detail is based on the crystal optimization process (details will be described later) shown in FIG.

3. FIG. 12 shows an overall view of a Gilson liquid handler used for preparing the reagent kit of the present invention. In this example, two syringes 2 are provided on a box-shaped body case 1. , 3 is provided with a dispensing unit 4 for sucking and discharging medicines. The space on the front side of the main body case 1 is a rectangular work space 6, and above this work space 6, a horizontal arm 7 that horizontally moves in the left-right (X-axis) direction by an actuator (the actuator itself is not shown). Is projected in front of the main body case 1. In the workspace 6, a plate for preparing a reagent and other containers are arranged as shown in FIGS. 1 to 5, for example, according to each dispensing operation.

  The horizontal arm 7 is provided with a groove-shaped guide 9 in the front-rear (Y-axis) direction, and is vertically guided in the vertical (Z-axis) direction, which is guided by the guide 9 and moves horizontally in the Y-axis direction. A column 11 protrudes upward, and a slider 13 with an actuator that moves up and down by an actuator along a vertical groove-shaped guide 12 is attached to a side surface of the column 11. A needle 14 for dispensing a chemical solution is attached to the slider 13 in the vertical direction, and a transfer tube 16 connected to the above-described syringe 3 is connected to the proximal end side of the needle 14.

  A drive device (actuator) for horizontally moving the horizontal arm 7 and the vertical column 11 is built in the main body case 1, and an actuator for driving the two syringes 2 and 3 is built in the dispensing unit 4. Yes. Except for the fact that two syringes are installed, the liquid handler is substantially the same as a conventionally known one.

  As shown in FIG. 13, the two syringes of the liquid handler used in this embodiment are divided into a needle 14 and a syringe 2 for cleaning the transfer tube 16 and a syringe 3 for dispensing chemicals, and valves 17 and 18 respectively. Is provided. The syringe heads of both the syringes 2 and 3 and the valves 17 and 18 are communicated with each other by a flow path 19.

In the syringe structure, as shown in the operation process diagrams A to I in FIG. 13, in the process A, the flow path 19 is formed in a state where the pistons 21 and 22 of the syringes 2 and 3 are respectively stroked to the syringe head side. , washed liquid (water) is filled in the transfer tube 16, in the line of the needle 14.

Then wash liquid syringe 2 for suction while stopping the syringe 3 for dispensing as shown by B process is operated in the direction of suction, sucks the reagent 24 of the washing solution and the reagent container 23 by the needle 14, line the washing liquid of the inner storing in the syringe 2.

  Subsequently, as shown in step C, the dispensing syringe 3 is operated in the suction direction to store the reagent 24 in the syringe 3. The stored reagent 24 is sequentially dispensed into each well (concave portion) of the reagent plate 26 (the reagent plate 26 will be described later) by causing the syringe 3 to act in the pushing direction in the D step, and as in the E step. The dispensing of each plate 26 is completed.

Then after discharging the remaining reagent in the line together with pushing the cleaning liquid by actuating extruding syringe 2 in F step, a washing liquid container 27 by suction actuating the syringe 2 into the syringe 2 and all the lines within the G step by sucking more washing liquid 28, to fill the washing liquid in the line, and the syringe 2. Thereafter, the syringe 2 and the line are cleaned by repeating the pushing and sucking of the syringe 2 at least twice as in the H and I steps, and the initial step A is reset. Such a dispensing method is repeated for each chemical solution constituting the reagent and for each reagent plate 26, and the operation control of the liquid handler itself is performed by a personal computer or other control device based on a predetermined program.

  Conventionally, since one syringe was used and an air gap was adopted as a boundary area in order to avoid mixing of the filling liquid and the reagent in the line, the flow rate could not be obtained because reagent aspiration was performed without breaking the air gap, Even if the flow rate condition is determined, the condition setting cannot be reproducible due to contamination of the inner wall of the transfer tube, and it is necessary to set the flow rate condition each time to adjust the volume that can be dispensed by changing the transfer tube volume. There were disadvantages such as.

On the other hand, in the above method, since two syringes are used, the filling liquid is removed with the other syringe, and the line is filled with the reagent, there is no air gap, so there is no air gap and the flow rate can be increased. The reproducibility of the flow rate condition can be ensured without being hindered. Moreover, since the volume of the transfer tube is fixed and the dispensing volume can be adjusted by changing the volume of the syringe, there is an advantage that the flow rate condition setting is simple.
Other than the above-described liquid handler and syringe, for example, a reagent preparation instrument such as a plate will be described together with the description of the reagent preparation described later.

4). Preparation of Crystallization Reagent (3600 Types) In this embodiment, as shown in the reagent preparation table for initial screening and secondary screening for crystallization reagent, there are 144 types and 3456 types of reagents, respectively. Prior to preparation of the screening reagent, it is necessary to prepare a precipitant and other stock solutions. Therefore, reagent preparation in this embodiment is (1) Stock solution preparation = Precipitant (Precipitant 16 types), Buffer agent (Buffer 6 types), Preparation of additive (Additive 4 types), (2) Premix Buffer (Pre Mix Buffer) = PMB) = PMB Init (Initial = Initial), PMB Opti (Optimize = Secondary, Deployment), PMB Phos (Phosphate = Phosphate), (3) High-throughput kit = HTP Kit ) = HTP Kit Init, HTP kit Opti, HTP kit Opti add (with additive = additive), and HTP kit Phos, each process can be classified into the following steps. Describe.

(1) Preparation of undiluted solution Table 2 shows the formulation for preparing the buffer for preparing the reagent, Table 3 shows the formulation for preparing the precipitant and additive, and Table 4 shows the types of the precipitant, buffer, and additive. Shown with stock number. In the numbers indicated by “XX-XXX” in each table, the first two digits are the stock solution number, 01 = precipitating agent, 02 = buffering agent, 03 = additive, and lower 3 Digits indicate the ID of each solution. The meanings of the other symbols in the table are FW = molecular weight, Conc = concentration, Weight = reagent weight, MilliQ = purified water, and other reagent components are shown in the table or figure as much as possible. And the same in the drawings.

  For preparation of the stock solution, each chemical and water are weighed and mixed with stirring with reference to Tables 2 and 3 (when the buffer is CHS, NaOH is also added). At this time, for easy understanding, the reagents are matched based on the reagent stock solution number and the concentration (Table 4). All reagent stock solutions are filtered (filtered) with Steritop Millipore SCGPS05RE 0.22 μm 500 ml). The pH of the buffer solution is also measured.

(2) PMB (Premix Buffer) Production Method FIG. 1 shows that vials (No. 1 to 39) are placed on the workspace 6 of a liquid handler (see FIG. 12) for producing PMB (PMB Init = for initial screening). The layout view of PMB Init in which the vial box 31 and the 200 ml container (No. 1 to 12) of buffer, titration reagent and purified water (MilliQ) are arranged is shown. At this time, the vial No. 39 is filled with purified water for washing the needle 14, and an injection needle (not shown) is inserted into the peripheral end side position of the hole in vials No. 21 to 26 which are buffer containers. The circulation of the air inside and outside is secured so as to eliminate the pressure and negative pressure phenomenon in the container (vial). Vials (No. 1 to 20, 27 to 36) other than the above contain only the precipitant and the additive.

  After the above set, the PMB Init dedicated control program for the liquid handler is started and the liquid handler is activated to dispense the buffer solution in each vial. Then, the syringe needle is removed and the vial is vibrated to mix the PMB. Then, measure the pH. The prepared PMB is as shown in the composition table shown in Table 5, and the numbers other than pH in the table are the amount (μl) of each chemical solution and the like.

2 and 3 are layout diagrams on the same workspace 6 as FIG. 1, FIG. 2 shows PMB Opti (for secondary screening), and FIG. 3 shows PMB Phos (use of sodium phosphate and potassium phosphate). shows a case, buffer and purified water are each 200 ml, dispensing work method according to the point and liquid handler be filled with purified water to a vial (No.39) is the same as the case of FIG. 1 .

  In contrast to the case of FIG. 1, in the examples shown in FIGS. 2 and 3, the liquid handler is used by using a program dedicated to PMB Opti or PMB Phos, and the vials for inserting the injection needles are No. 1 to 36. Different. And the chemical | medical solution arrange | positioned or dispensed to each vial is as each shown in FIG. 2, FIG. As a result, the composition of PMB produced corresponds to Table 6-1 and Table 6-2 for FIG. 2, and Table 7 for FIG.

(3) Preparation of reagent kit (HTP Kit) Next, a reagent kit (3600 species) was prepared using the stock solution for reagent kit preparation and PMB (premix buffer) prepared as described in (1) and (2) above. How to do will be described. The reagent kit is performed using the above-described liquid handler and a control program by a personal computer corresponding to each kit, and the kit plate 26 shown in FIG. 13 has 12 × as shown in FIG. 4 and FIG. 6 (hole) 72-well type is used.

1) Dispensing paraffin In the wells (recesses) of each plate 26, in order to prevent evaporation of the crystallization reagent and prevent a change in concentration, the reagent floats on the reagent surface and seals contact with the space. Dispense the low specific gravity paraffin.

  FIG. 4 shows the arrangement of two plates 26 and paraffin and purified water for washing needles arranged on the workspace 6 of the liquid handler. The liquid handler is controlled by a program dedicated to paraffin dispensing and performs the dispensing operation. Is called. The plate after paraffin dispensing is stored until the HTP kit is prepared as described below.

2) Preparation of reagent kit for initial screening (HTP kit Init) Fig. 5 shows screw vials with paraffin-dispensed plate 26 (two in 144 wells) and chemical solution for PMB Init for preparation of reagent kit for initial screening. An arrangement diagram on the work space 6 of the box 31 is shown, and a screw vial (No. 1 to 26) is pre-stabbed with an injection needle in the manner described above, and purified water is dispensed into the No. 37 vial. Dispensing is performed in a state where purified water is also disposed in the surplus space of the work space 6. Table 8 shows the reagent arrangement table of PMB Init (vials Nos. 1 to 26 in FIG. 5) used for preparing the HTP kit. The HTP kit prepared as described above has the contents shown in FIG. It consists of 16 types of agents, 6 types of buffering agents, and 4 types of additives.

  Of the reagent preparation tables in Table 1, those indicated by reagent Nos. 1 to 144 are initial screening reagents, and Nos. 1 to 84 are 2-Propanol (isopropanol), MPD (2-methyl-2,4). -Pentanediol), PEG 4,000 (polyethylene glycol 4,000), PEG 20,000 (polyethylene glycol 20,000), Jeffamine M-600 (Jephamine M-600), Na Form (sodium formate), Na Chlor (sodium chloride), Li Sulf (sulfuric acid) Lithium), Amm Sulf (ammonium sulfate), KNa Tart (sodium tartrate), Na Acet (sodium acetate), tri-Na Citr (trisodium citrate), Mg Sulf (magnesium sulfate), Na2 Malon (disodium malonate) 14 kinds of precipitants are used as a standard of combination (standard chemical solution). Acetic acid (acetic acid), Na3 citrate (trisodium citrate), MES (2-morpholinoethanesulfonic acid), HEPES (2- [4- (2hydroxyethyl) -1-piperazinyl] ethane Distributing drug solution that sequentially distributes 6 types of buffer (buffer) consisting of sulfonic acid), Tris (tris (hydroxymethyl) aminomethane), CHES (N-cyclohexyl-2-aminoethanesulfonic acid) ing.

  Titration reagent NaOH is allocated to the buffering agents Acet (acetic acid), MES, and HEPES, and HCl is allocated to the other buffering agents Citr (citric acid), Tris, and CHES. Nos. 1 to 84 are additive-free.

  Next, Nos. 85 to 108 in Table 1 show four types of additives Li Chlor (lithium chloride), Mg Chlor (magnesium chloride), Ca Chlor (calcium chloride), 1, for the precipitant MPD as the reference chemical solution. 4-Dioxane is divided into 4 groups. Each group allocates the above six types of buffer and subdivides the conditions so that a total of 24 types of reagents are prepared.

  Reagent Nos. 109 to 132 in Table 1 are allocated to four types of additive groups in the same manner as above with respect to the precipitant PEG4K as the reference chemical solution, and each group is assigned a total of six types of buffer agents (distribution chemical solutions). It is subdivided into 24 conditions. The Nos. 133 to 138 are assigned the above six types of buffering agents to the 9th (No. 49 to 54) precipitating agent Amm Sulf, and the additive 1,4-Dioxane is assigned to each. It is a thing.

  The reagents in Nos. 85 to 138 in the above table are empirically confirmed that the additive has some positive effect on protein crystallization in addition to the reagent without the additive. The reagent to which the four types of additives shown in the table are added is shown. This increases the number of initial screening reagents by 48 and further subdivides the conditions.

  The following Nos. 139 to 144 increase quantitatively and regularly in a titration reagent ratio of 0.39 to 0.511 with respect to the 15th precipitant Na Phos (sodium phosphate) used in this example. Buffer K2 Phos (dipotassium phosphate) was allocated as a distribution chemical solution, and the additive was not added. Na Phos and K2 Phos used here have both functions of a precipitating agent and a buffering agent, and are not particularly allocated as a buffering agent.

  In the above initial screening reagents, the most desirable values, which are generally confirmed empirically, are selected for the mixing ratio, concentration, pH, etc. of each chemical solution, and the initial screening reagents are used to subdivide these quantitative factors. No particular emphasis is placed on, and subdivision of quantitative elements is realized mainly in secondary screening reagents of No. 145 or lower.

3) Preparation of reagent kit for secondary screening (without additives) (HTP kit Opti) This example is also prepared in the same manner as the above-described initial screening reagent kit, and is a screw vial box with the contents of the PMB Opti reagent arrangement table shown in Table 9 A set of 31 and a pair of plates (72 wells) 26, a point where a precipitating agent (PPT) and purified water (MilliQ) are arranged, and vials Nos. 1 to 36 are used for reagents and pre-inserted with an injection needle The point to leave is different.

  FIG. 8 shows the contents of the components of the reagent kit prepared by this method. In the secondary screening, there are combinations of crystallization reagents for each precipitating agent, and 144 types of precipitating agents are used for each one (4 patterns). , 6 types of buffers (6 patterns), with or without additives. The amount of water varies depending on the presence or absence of additives.

  Reagent Nos. 145 to 2160 in Table 1 correspond to the reagents in this section, and among the initial screening reagents, the first 14 types of precipitating agents (reference drug solutions) are divided into 6 types of buffer agents (distributed drug solutions) every 6 types. Allotted to groups (no additives). And the ratio of the precipitants of No. 1 to 144 was regularly changed in four steps, 0.25, 0.35, 0.45, 0.55 (Note: The precipitation concentration was also changed for each group according to this. 4 groups of small groups are formed and changed quantitatively or regularly), and within each small group, the titration reagent ratio is regularly changed in 6 stages and assigned to the conditions. Is subdivided.

  Of these, only the amount of change in the precipitant ratio and titration reagent ratio is common to the whole, and other numerical values change the change region as appropriate according to each chemical (for example, MPD, PEG20K, Na Form). The change area, etc.) does not change that the change is quantitative and regular. In addition, changes in pH of the buffering agent are appropriately changed according to each buffering agent. The ratio of the titration reagent is increased or decreased depending on whether the titration reagent is acidic or alkaline.

4) Preparation of secondary screening reagent kit (with additives) (HTP kit Opti add) The secondary screening reagent kit with additives is different from the above 3) in terms of the presence or absence of additives. Since other than is common, explanation of common parts is omitted. In addition to the above differences, additives are dispensed into the vial (No. 38) in FIG. 9 and Table 10, and the presence or absence of the additive and the amount of water in FIG. 10 are different.

  Nos. 2161 to 312 in Table 1 allocate the six types of buffering agents (distribution chemicals) to the second and third types of precipitating agents (reference chemicals) MPD and PEG4K, respectively, and four more types Additives are allocated. Each precipitating agent is divided into four additive groups according to the type of additive, and each additive group is further subdivided into six buffer pairs.

  In addition, each buffer group is divided into 6 groups, each divided into 4 steps of the precipitant ratio and precipitating agent concentration that change quantitatively and regularly. The 6 groups that are common to each other are assigned as different types of titration reagent ratios that change quantitatively and regularly in six steps, and the conditions are subdivided. The precipitant ratio and the same concentration in this range repeats substantially quantitative and regular changes for each buffer.

  Nos. 3313 to 3456 in Table 1 are the 9th precipitating agent (reference chemical solution) Amm Sulf, with 6 types of buffering agents assigned 24 times each and 1,4-Dioxane added as an additive to all. It is. Each buffer group allocated to 24 is divided into 4 stages (4 groups) in which the precipitant ratio and the same concentration change in a quantitative and regular manner, and each group has 6 titration reagent ratios. The amount of increase / decrease is changed approximately quantitatively and regularly (with this change, the pH value of the buffering agent also increases / decreases substantially quantitatively).

5) Preparation of reagent kit for secondary screening (using phosphoric acid) (HTP kit Phos) This reagent uses phosphoric acid as a precipitating agent, and all the vials (No. 1 to 36) in FIG. 11 and Table 11 are phosphorous. The acid is dispensed and the concentration and pH are changed sequentially. The phosphoric acid (Phos) in the secondary screening reagent is composed of 144 kinds of combinations of only two kinds of solutions (Na Phos, K2 Phos) with a precipitant and a buffer. Therefore, the reagent composition differs for each well. In this case, Na Phos and K2 Phos may be used for either the precipitant or the buffer.

  In Table 1, Nos. 3457 to 3600, the concentration of Na Phos used as a precipitating agent quantitatively and regularly changed in four stages of 1,1.4, 1.8, and 2.2. The pH of the buffer (titration reagent) changes almost quantitatively and regularly in 6 steps, and this change becomes almost quantitative and regular in a high numerical range every 4 groups, and changes in a total of 6 regions. .

  On the other hand, the blending ratio of Na Phos and K2 Phos with water is the phosphate buffer for each of the above 4 groups, as shown in Table 11, in which 36 types of blending were performed almost quantitatively and regularly at the premix stage. No. 3457-3600 (144 types in total) are allocated at a fixed rate during the buffering action of the liquid. Therefore, although the numerical values related to the quantitative elements are not quantitative changes within the four groups, they remain unchanged in accordance with certain allocation rules. In this example, Na Phos and K2 Phos can be used interchangeably.

  6) In this example, 3600 types of screening reagents configured as described above can be prepared as shown in Table 1. Using these, the initial (144 types) and secondary (secondary) described below can be used. 3456) protein crystallization screening is performed.

  In addition, in the combination of reagents in Table 1, it is desirable that quantitative and regular changes in quantitative elements (including changes in concentration, pH, etc.) for condition subdivision are exact, but protein An error within a range in which the optimum region of the reagent conditions subdivided in the crystallization can be found is acceptable.

5. Protein Crystallization Screening Next, regarding the protein crystallization screening method using the screening reagent kit described above, the protein used for crystallization and the equipment used for crystallization and evaluation (robot), evaluation method and evaluation Various points such as results will be described.

(1) Crystallization experiment, protein used in experiment and equipment used 1) Protein used The following two proteins were used in this experiment.
a. Organism T.Thermophilus (HB8) Number of purified proteins 177 Number of crystallizations 94
b. Organism P.Horikoshii (OT3) Number of purified proteins 256 Number of crystallizations 77

  The above a is a thermophilic bacterium having a growth temperature range of 50 ° C.-82 ° C. (optimum temperature is 70-75 ° C.), and b is a hyperthermophilic archaea having an optimal growth temperature around 95 ° C. .

  In the protein crystallization experiment derived from the cells described above, all of the proteins used are heat resistant. That is, when the heat resistance mechanism of a protein is to be explored, it is used when considering the use of a heat-resistant protein or the like (the heat-resistant protein is resistant to protein denaturation factors other than temperature). In other words, as far as the heat resistance of living organisms and biological materials is concerned, a wide range of research from basic research to applied research can be carried out using a and b as materials.

2) Crystallization method and equipment used for crystal observation (robot)
For the crystallization of this experiment, a protein crystallization method and apparatus (robot = trade name “TERA”) disclosed in Japanese Patent Application Laid-Open No. 2003-107076 according to the proposal of the inventors was used.

  The above-described apparatus can automatically manage all the constituent units including a database for registering the crystallized and crystal observing work schedules and a database of crystal photographs taken. This device is a 1 μl (0.5 μl protein solution + 0.5 μl crystallization reagent) scale oil batch method using a 72-well microplate similar to the “IMPAX” of Douglas, Inc., which is widely used all over the world. In addition to the “dispenser robot” that can perform crystallization, the “crystallization well automatic observation device” that automatically photographs crystals using a microscope, 2,500 crystallization plates and 125 crystallization reagent plates It consists of a “plate storage / conveyance unit” that can be stored, and a “plate setting robot” that links each device.

  All plates are managed by a “barcode printing / reading system” built in for identification, and currently work 24 hours a day, unattended. Crystallization can be processed in 32 plates and 2,304 conditions, and observation in 100 plates and 7,200 conditions per day. In the Crystallization Reagent Repository, as standard crystallization conditions for each protein, there are 3,460 types of secondary screenings that have been refined by developing pH and precipitant concentration for each of the 144 initial screening reagents. Reagents are prepared and stored in advance.

  For this reason, the crystal observation result can be fed back to the improvement of the crystallization condition, and the crystal improvement experiment under a wide range of conditions can be immediately performed from the console of the same personal computer. In addition, if the crystal observation work scheduler is used, the temporal change of the crystallization drop state can be automatically taken as an image and periodically stored. In addition, all information from the fully automated crystallization system is automatically stored in the system “HTPF-DB”, which consists of a program that collectively manages the experimental data of the entire “High Throughput Factory” deployed at the RIKEN Harima Institute. Uploaded requests for crystal observation evaluation and crystallization work can be distributed over the network using a web browser.

(2) Evaluation of crystallization by score The quality of protein crystallization is evaluated by external observation of the crystallization results. In this experiment, however, the evaluation is based on image analysis using a camera in the robot. The evaluation is performed based on the evaluation criteria for each score, for example, as shown in FIG.

  In this example, as shown in the figure, the crystallization state is changed to dry (-1), transparent (0), precipitation (1), jelly-like lump (4), 0.05 mm or more of fine crystals / needle-like fines. Scores are divided into crystals, dirty plate crystals, dirty cluster crystals (5), plate crystals, needle crystals, separable cluster crystals (6), and single crystals of 0.03 mm or more (8). And check the score.

Next, the flow of the crystallization and the result evaluation will be described with reference to the flowchart of FIG.
1) In the case of a new sample, determine whether the volume is 300 μl or more. If 300 μl or more, crystallize in 144 types of initial screening, and if less than 300 μl, crystallize in 72 types of initial screening.
2) Next, a score check is performed using the evaluation criteria shown in FIG.
3) If the total score after 1 week is less than 50 and the maximum score is 4 or more, secondary screening is performed. If the total score is greater than 50, repeat the initial screening.
4) In the secondary screening, 24 types are developed for the reagent No. that scored 4 or more in the initial screening. The development method is optimized based on four levels of concentration per precipitant and six levels of pH per buffer. (For details, see FIGS. 5 to 11 and Tables 8 to 11.)
5) The score is checked again using the evaluation criteria shown in FIG.
6) If the score is 6 or higher in the secondary screening, check the diffraction or crystallize under the same conditions.
7) If the resolution is less than 4.0 mm in the diffraction check, crystallization is completed. If the resolution is 4.0 or higher, conduct advanced experiments.

6). Confirmation of crystallization rate In protein crystallization, if a small amount of other solutions are mixed, the degree of crystal production and reproducibility in crystallization experiments will be affected. However, by using the initial screening reagent prepared by the above-described method, as shown in the graph shown in FIG. It can be seen that crystals are produced. This makes it possible to determine whether the crystallization reagent produced by the above-described method has succeeded in crystallizing proteins at high frequency. In addition, it is possible to produce experimental results for each type of protein. This means that the buffer pH and precipitation agent concentration of each reagent are systematically arranged, and the effects of each buffer, pH, precipitation agent, etc. can be considered from each viewpoint.

  Furthermore, as shown in FIG. 17, the same experimental results as the initial screening reagent experimental results were obtained in the secondary screening. This is presumably because the initial screening reagent and the secondary screening reagent are systematically included in the same class, and thus the same result is obtained when the result is obtained from the viewpoint of the crystallization experiment. However, in FIG. 18, as a result of subdividing the crystallization reagent, the results that were not obtained by the primary screening for 40% of the samples are optimized by executing the secondary screening. On average, crystallization is optimized at 254%. (Note: This ratio is the number of secondary screening score 5 or higher divided by the initial screening score 5 or higher.) This makes it possible to obtain better quality protein crystals that were impossible with conventional screening reagents. It shows that it is possible to produce.

  The present invention is used in protein crystallization tests for analyzing the crystal structure of proteins necessary in research fields such as life science, drug discovery, and food.

FIG. 2 is a layout diagram of chemicals and containers in a rekit handler workspace for preparing a PMB (premix buffer) used for preparing a reagent kit for initial screening of the present invention. It is the layout of the chemical | medical solution and containers in the rekit handler workspace for PMB (however, phosphoric acid is used for a precipitating agent) similarly used for preparation of the reagent kit for secondary screening. Placement of chemicals and containers in the rekit handler workspace for the same PMB used for preparation of secondary screening reagent kits (however, phosphoric acid is used for the precipitant) FIG. 5 is a layout view of plates and liquids for dispensing paraffin to a reagent kit plate. FIG. 5 is a layout diagram of chemical solutions and containers in a workspace for preparing an initial screening reagent kit. It is explanatory drawing which shows the reagent structure of the reagent kit for initial screening. FIG. 5 is a layout diagram of plates, chemical solutions and containers in a workspace for preparing a secondary screening reagent kit without adding an additive. It is explanatory drawing which shows the reagent structure of the reagent kit for secondary screening which does not add an additive. FIG. 5 is a layout diagram of plates, chemical solutions and containers in a workspace for preparing a secondary screening reagent kit without adding an additive. It is explanatory drawing which shows the reagent structure of the reagent kit for secondary screening which does not add an additive. FIG. 5 is a layout diagram of plates, chemical solutions, and containers in a workspace for preparing a secondary screening reagent kit using phosphoric acid as a precipitant. It is a general | schematic perspective view of the liquid handler for preparation of the reagent kit for screening. It is a schematic diagram which shows the structure and action | operation process of a syringe used for a liquid handler. It is the microscope picture of the crystal state which shows an example of the evaluation criteria classified by score for protein crystallization state evaluation. It is a flow figure of protein crystallization experiment and crystallization state evaluation. It can be a graph showing the crystallization rate by score evaluation of the initial screening result of protein crystallization using the screening reagent kit of the present invention. It is a graph which shows the crystallization rate by score evaluation of the secondary screening result of protein crystallization using the screening reagent of this invention. It is a graph which shows the change of the crystallization rate of an initial screening and a secondary screening.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Main body case 2,3 Syringe 4 Dispensing unit 6 Work space 7 Horizontal arm 9 Guide 11 Support column 12 Groove-shaped guide 14 Needle 13 Slider 16 Transfer tube 17, 18 Valve

Claims (11)

  Either one of the precipitating agent and the buffer solution combined with each other is used as a reference chemical solution for combination, and the other chemical solution that is distributed and combined with the reference chemical solution is used as a distributing chemical solution. Reagent kit for initial screening, which is grouped according to the number of types of dispensing drug solution and quantitatively combined sequentially, and dispensed into a large number of wells arranged in a kit container, and the above initial screening For at least one of the reference chemical liquid and the distribution chemical liquid whose combination is specified in the reagent kit for the drug, one or more quantitative elements of the mixing ratio or amount, concentration, pH, mixing ratio or amount of the raw drug for the chemical liquid Substantially quantitatively or regularly sequentially change the reagent conditions and increase the number of samples by multiple times. The combination of chemical and distribution chemical, kit containers made by dispensing each component into a number of wells disposed in the secondary screening reagent kit and protein screening reagent kit composed.   From the combination of the reference drug solution and the distribution drug solution in the initial screening reagent kit, one or two or more additives are further distributed corresponding to each of the combinations where it is desirable to add additives for protein crystallization, 2. The protein screening reagent kit according to claim 1, wherein each additive is grouped and added by dispensing to form an initial screening reagent kit.   In the combination of individual precipitants and buffers in the secondary screening reagent kit, either or both of the mixing ratio and concentration of the precipitants are changed substantially quantitatively or regularly in multiple steps, and the buffer agent By changing either one or both of the pH and the titration reagent ratio substantially quantitatively or regularly in multiple steps, the combinations of the reference drug solution and the distribution drug solution are grouped together, and the reagent conditions are subdivided and the number of reagents is reduced. The reagent kit for screening a protein according to claim 1 or 2 which has been increased.   For combinations where it is desirable to add additives for protein crystallization from among the combinations of the reference drug solution and the distribution drug solution in the secondary screening reagent kit, one or more additives are distributed and added separately for each combination. 4. The reagent screening kit for protein according to claim 1, 2 or 3, wherein each reagent is grouped and added by dispensing.   Part or all of the precipitant is 2-Propanol (isopropanol), MPD (2-methyl-2,4-pentanediol), PEG 4,000 (polyethylene glycol 4,000), PEG 20,000 (polyethylene glycol 20,000), Jeffamine M-600 ( Jeffamine M-600), Na Form (sodium formate), Na Chlor (sodium chloride), Li Sulf (lithium sulfate), Amm Sulf (ammonium sulfate), KNa Tart (sodium tartrate), Na Acet (sodium acetate), tri-Na Claim 1, selected from Citr (trisodium citrate), Mg Sulf (magnesium sulfate), Na2 Malon (disodium malonate) and Na Phos (sodium phosphate), K2 Phos (dipotassium phosphate) Reagent kit for screening 2, 3 or 4 proteins.   Some or all of the buffering agents are Acetic acid (acetic acid), Na3 citrate (trisodium citrate), MES (2-morpholinoethanesulfonic acid), HEPES (2- [4- (2hydroxyethyl) -1-piperazinyl] 6. Screening of the protein of claim 1, 2, 3, 4 or 5 selected from ethanesulfonic acid), Tris (tris (hydroxymethyl) aminomethane), CHES (N-cyclohexyl-2-aminoethanesulfonic acid) Reagent kit.   A part or all of the additive is selected from Li Chlor, Mg Chlor, Ca Chlor, and 1,4-Dioxane. , 5 or 6 protein screening reagent kit.   The protein screening reagent kit according to claim 1, 2, 3, 4, 5, 6 or 7, wherein the number of reagents in the initial screening reagent kit is 144 or more.   The reagent kit for screening proteins according to claim 1, 2, 3, 4, 5, 6 or 7, wherein the number of reagents in the secondary screening reagent kit is 3456 or more.   A protein crystallization screening is performed using the initial screening reagent according to claim 1, 2, 5, 6, 7 or 8, and the reagent condition of the reagent having a high crystallization rate is subdivided. A protein crystallization screening method wherein the protein is screened by selecting from among 1,3,4,5,6,7 or 9 secondary screening reagents. The crystallization state of the protein obtained as a result of the crystallization test is image-recognized, and the image-recognized image is compared with the evaluation reference images of many crystallization states that have been evaluated for each score and recognized in advance. 11. The protein crystallization screening method according to claim 10, wherein the crystallization state of each crystallized protein is determined separately, and the crystallization rate of each screening reagent for the protein is determined based on the score.