CN104330426A - Device and method for screening crystallization condition of high-throughput protein under strong magnetic field - Google Patents

Abstract

The invention provides a high-throughput protein crystallization condition screening device and method under a strong magnetic field. Several small pools loaded with liquid reagents are evenly arranged on the base plate of the mother liquid. A corresponding through hole, the capillary is assembled in each through hole, and the protein sample plate equipped with the capillary can be placed in the temperature control system; the groove base and the groove top cover are both discs with grooves on the surface, the groove base and The grooved side of the groove top cover is opposite, the grooved position corresponds to the position of the through hole on the protein sample plate, and the groove is filled with vacuum grease. The invention provides a new crystallization environment, realizes the screening of high-throughput protein crystallization conditions in a strong magnetic field environment, does not need to take out or freeze the crystals, and can significantly reduce the amount of protein samples crystallized in the magnet.

Description

Device and method for screening high-throughput protein crystallization conditions under strong magnetic field

technical field

The invention relates to a high-throughput protein crystallization condition screening method and device.

Background technique

The 21st century is an era of life sciences, and the study of protein structure and function is of undoubted importance. As the most important technology, XRD technology has resolved 89% of the structure in PDB, and growing high-quality crystals has always been the bottleneck of this technology. Although the structures in the PDB are increasing geometrically every year, the success rate of obtaining high-quality crystals has not improved, and this bottleneck problem has not been fundamentally resolved.

In recent years, superconducting magnet technology has developed rapidly, and the field strength conditions with different gradients of superconducting magnets provide a new way for life science research. Protein crystallization under magnetic field provides a new method and idea for solving the bottleneck problem of analyzing protein molecular structure by X-ray single crystal diffraction. When protein crystallization is carried out in a superconducting magnet, the two aspects of magnetic field strength and magnetic field gradient work together to have an important impact on protein crystallization and crystal quality. The magnetic field strength reduces the growth rate and dissolution rate of protein crystals, changes the crystal morphology, and improves the crystal quality; the magnetizing force generated by the magnetic field gradient can change the actual gravitational state of the protein solution, by affecting the solute transport and shape in protein crystallization. Nucleation and crystal growth process to improve crystal quality.

However, although magnetic fields provide a special environmental resource with unique advantages for protein crystallization, the utilization of this resource has been limited. The main reason for the limitation comes from the high-intensity magnetic field itself: the size of the room temperature cavity that a high-intensity magnetic field can provide is limited, so that the existing high-throughput protein crystallization device cannot be used in a magnetic field, resulting in the inability to achieve high-throughput protein crystallization under magnetic field conditions. Screening of crystallization conditions. In order to make full use of the special resource of high-intensity magnetic field in the direction of protein crystallization, it is necessary to invent a high-throughput protein crystallization condition screening method and its device under high-strength magnetic field to fill the research gap of high-throughput protein crystallization condition screening in magnets , and provide a new solution for solving the bottleneck problem of X-ray protein single crystal diffraction technology.

Contents of the invention

In order to overcome the deficiencies of the prior art, the present invention provides a high-throughput protein crystallization condition screening method and its device in a strong magnetic field environment, making full use of the small space occupied by the capillary to form a capillary that can be placed in a small space in a magnetic field Each capillary in the array is pre-packed with the target protein through the siphon action of the capillary and dried for use, and then the crystallization condition screening reagent is sucked into the capillary pre-packed with the target protein through the siphon action again and placed in a temperature-controlled In a controlled magnetic field, high-throughput screening of crystallization conditions can be achieved. The present invention can realize high-throughput protein crystallization condition screening under high-intensity magnetic field conditions while ensuring that the magnetic field environment conditions of all samples are consistent, and can effectively reduce the amount of protein samples used in screening, so that the method has the advantages of With remarkable practicability, it can become a crystallization method based on a strong magnetic field with promotional value. The invention not only provides a solution for high-throughput protein crystallization condition screening under magnetic field, but also provides a new protein crystallization condition screening technology for protein crystallization, which can be used in other environmental conditions besides magnetic field.

The technical solution adopted by the present invention to solve the technical problem is: a high-throughput protein crystallization condition screening device for a strong magnetic field environment, including a mother liquor base, a protein sample plate, a groove base, a groove top cover, a capillary, and a temperature Control System. The materials used in the mother liquid base, the protein sample plate, the groove base, the groove top cover, the capillary and the temperature control system are all antimagnetic materials; the mother liquid base plate is evenly arranged with several small pools loaded with liquid reagents, The protein sample tray has through holes corresponding to the positions of the small cells on the mother liquor chassis, the capillaries are assembled in each through hole, and the protein sample tray equipped with capillaries can be placed in the temperature control system; the described The groove base and the groove top cover are both discs with grooves on the surface. The grooved sides of the groove base and the groove top cover are opposite to each other. The groove position corresponds to the position of the through hole on the protein sample plate, and the groove is filled with vacuum. fat.

The capillary fits in the through holes of two parallel protein sample discs.

The present invention also provides a method for screening high-throughput protein crystallization conditions in a strong magnetic field environment, comprising the following steps:

1) Prepare a protein solution, and then use the capillary action of the capillary to draw the protein solution into the capillary;

2) Dry the protein solution in the capillary at the set drying temperature. During the drying process, the temperature control range is 0-60°C, the temperature control accuracy is ±0.1°C, and the drying time is 2-7 days;

3) Installing the completely dried capillary in the protein sample tray to form a capillary array for screening protein crystallization conditions; correspondingly inserting the array into the mother liquor base pre-installed with the screening reagent, and absorbing the screening reagent solution from the mother liquor base by capillary phenomenon;

4) Insert the two ends of the capillary into the vacuum grease on the base of the groove and the top cover of the groove respectively to realize the sealing of the capillary and place it in the magnet cavity at 0-60°C for 2-7 days for crystallization;

5) After the crystallization is completed, observe whether protein crystals grow in the capillary, and use an X-ray diffractometer to conduct in-situ diffraction to identify protein crystals and collect diffraction data.

Said protein refers to water-soluble protein.

In the step 4) the capillary is placed under electric field, hypergravity conditions or conventional environmental conditions to screen the protein crystallization conditions.

The beneficial effects of the present invention are: 1. The present invention belongs to a novel screening method in terms of screening technology, and the innovation is reflected in the process of first drying the protein sample and then mixing it with the screening reagent. This process is different from all existing protein crystallization experimental techniques, and provides a new crystallization environment, so it is hopeful to discover new crystallization conditions that could not be found in the past, and can be extended to other various environmental situations (including conventional environmental conditions) 2. Utilization The small size of the space occupied by the capillary forms a capillary array that can be placed in the narrow space of the magnetic field, and for the first time realizes the screening of high-throughput protein crystallization conditions in a strong magnetic field environment; The crystal is taken out or frozen, which reduces the damage of the crystal; 4. It can significantly reduce the amount of protein samples crystallized in the magnet.

Description of drawings

Figure 1 is a schematic diagram of the overall structure of a high-throughput crystallization device;

Fig. 2 is a schematic diagram of partial parts of the high-throughput crystallization device, wherein Fig. 2-a is a schematic diagram of the top cover of the groove, Fig. 2-b is a schematic diagram of protein sample tray 1, Fig. 2-c is a schematic diagram of protein sample tray 2, Fig. 2 -d is a schematic diagram of the mother liquor base, and Figure 2-e is a schematic diagram of the groove base;

In the figure 1-nut; 2-groove top cover; 3-protein sample plate one; 4-protein sample plate two; 5-square fixed shaft; 6-mother liquor base; 7-groove base.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, and the present invention includes but not limited to the following embodiments.

The invention relates to a method and device for screening high-throughput protein crystallization conditions in a strong magnetic field environment. Realize high-throughput crystallization condition screening. The present invention combines high-throughput protein crystallization with superconducting magnets for the first time, and can preliminarily screen crystallization conditions of proteins in superconducting magnets. The invention has the advantages of low technical cost, easy operation, the same magnetic field experiment environment, and in-situ diffraction of the experiment results.

In order to achieve a strict comparison between the magnet and the magnet in vitro control, the same water bath was used to control two temperature controllers with the same shape and size, which were used for the crystallization experiment of the experimental group and the control group, respectively. Errors from crystallization ambient temperature are minimized. The temperature control range of the temperature control device is 277-303±0.1K.

A high-throughput protein crystallization condition screening device used in a strong magnetic field environment, including a mother liquid base, a protein sample plate, a groove base, a groove top cover, a capillary, and a temperature control system. The top cover of the groove, the protein sample tray and the base of the mother liquid are arranged in order from top to bottom (the position of the base of the groove is the same as that of the base of the mother liquid, and the two realize different experiments). Compared with existing screening devices, the crystallization condition screening device can be used in special physical environments, such as strong magnetic fields, electric fields, hypergravity environments, space and conventional environments. Compared with the existing automatic instruments, the crystallization condition screening device is more convenient and economical.

The materials used in the experimental device are diamagnetic materials.

There are 96 small pools on the mother liquid base plate, which are evenly arranged along the circumference, and can be loaded with 1 to 96 kinds of liquid reagents of 10 to 500 ul.

The protein sample disc has 96 small holes, which are evenly arranged along the circumference and correspond to the positions of the small pools on the mother liquor chassis one by one. The diameter of the small hole matches the outer diameter of the capillary, and 1 to 96 capillaries can be loaded.

The groove base and the groove top cover are disks with circumferential shallow grooves, and the groove positions correspond to the capillary tube positions. The tank is filled with vacuum grease, and after the capillary absorbs the screening reagent, insert the two ends of the capillary into the vacuum grease in the base and top cover grooves respectively to realize the sealing of the capillary.

The temperature control system is composed of a temperature control water jacket, a water bath, a single chip microcomputer, and temperature control software. The temperature control range is 0～60℃, and the temperature control accuracy is ±0.1℃.

A method for screening high-throughput protein crystallization conditions in a strong magnetic field environment, comprising the steps of:

1. Prepare the protein solution, and then use the capillary action of the capillary to draw the protein solution into the capillary. Compared with the present invention, the conventional method is to drop the protein solution into a commercial crystallization plate after it is prepared. Therefore, the present invention has the advantages of small size of the experimental device and the configuration of the solution without the need for automatic equipment.

2. Set an appropriate drying temperature according to the characteristics of the protein, and dry the protein solution in the capillary at the set drying temperature. The temperature control range of the temperature control box is 0-60°C, and the temperature control accuracy is ±0.1°C. Depending on the concentration of the protein solution and the type of protein, the drying time is 2 to 7 days. Compared with the present invention, the conventional crystallization method does not have the step of drying the protein solution. The drying process of the present invention can ensure the stability of the protein before mixing with the crystallization reagent, and after preparing the dried samples in large batches at one time, in subsequent experiments used directly in the process. Conventional methods require re-preparation of the solution and re-dropping of the solution for each experiment, so the present invention can make the follow-up experiment process more convenient.

3. Take out the completely dried capillary and install it on the protein sample plate to form a capillary array for protein crystallization condition screening. The array is correspondingly inserted into the stock solution base pre-loaded with the screening reagent, and then the screening reagent solution is respectively drawn from the stock solution base by capillary phenomenon again. The conventional step corresponding to this step is to mix the protein solution and the crystallization screening reagent manually or by machine. Compared with this conventional step, the present invention is simpler and more convenient than the conventional manual operation process, and does not require expensive automatic equipment.

4. After the suction is completed, seal the capillary with vacuum grease and place it under temperature control conditions. The temperature control range is the magnet chamber at 0-60°C, and the crystallization time is 2-7 days. Compared with the traditional crystallization plate, the high-throughput protein crystallization condition screening device used in this step is smaller in size, and because it is a small-angle rotationally symmetrical structure, it can ensure that the magnetic field level of each sample is consistent. However, the traditional crystallization plate is not only large in size (it cannot be placed in a smaller magnetic field chamber), but also has a relatively low degree of symmetry, which cannot make every crystallization droplet have the same magnetic field conditions.

5. After the crystallization is complete, observe whether protein crystals grow in the capillary. The identification of protein crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer, without removing the crystals or doing other treatments. When X-ray diffraction is performed on conventional protein crystals, most of the crystals need to be frozen and stored, and the in-situ diffraction of conventional crystallization plates will be interfered by the crystallization plates, and the background scattering intensity is relatively high.

Said protein refers to water-soluble protein.

The method for screening protein crystallization conditions can also be applied to screening protein crystallization conditions under other environmental conditions (including electric field, hypergravity conditions, conventional environmental conditions, etc.).

Example 1: The high-throughput crystallization device realizes the high-throughput crystallization condition screening of lysozyme in the magnet and compares the crystal quality with the conventional environment

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample tray 1, protein Sample tray two (one and two share the capillary), groove top cover, and nut. 2 sets of device assembly;

The second step: prepare the lysozyme solution, and use the capillary action of the capillary to draw the lysozyme solution into the capillary;

Step 3: Put the capillary tube containing the lysozyme solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 2 days;

Step 4: Take out the completely dried capillary and install it on the protein sample disk to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease and place one group of crystallization devices in a special physical environment under temperature control conditions, and the other group in a temperature control box in a conventional environment. The temperature is 20°C. Crystallization time 2 days;

Step 6: After the crystallization is complete, observe whether lysozyme crystals grow in the capillary. The identification of lysozyme crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer without removing or doing crystals. other processing.

The results are as follows: using the high-throughput protein crystallization condition screening method using a strong magnetic field, the lysozyme crystals grown under different crystallization conditions are different. The high-throughput protein crystallization condition screening method and its device used in a strong magnetic field environment can achieve the purpose of screening protein crystallization conditions in a magnet. In the comparison of the quality of lysozyme crystals in a strong magnetic field and in a conventional environment, in situ diffraction was performed on the crystals grown in the two groups of capillaries, and the diffraction patterns of the crystals were obtained. The diffraction data showed that the quality of the lysozyme crystals in the magnet was higher than that in the conventional environment. Conditioned crystal quality.

Example 2: High-throughput crystallization device realizes catalase high-throughput crystallization condition screening in electric field and compares with conventional crystal quality

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample tray 1, protein Two sets of sample plate (first and second common bearing capillary), groove top cover, nut, device assembly 2 groups;

The second step: prepare the catalase solution, and use the capillary action of the capillary to draw the catalase solution into the capillary;

Step 3: Put the capillary tube containing the catalase solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 3 days;

Step 4: Take out the completely dried capillary and install it on the protein sample plate to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease, place one group of crystallization devices in an electric field under temperature control conditions, and place the other group in a temperature control box in a conventional environment. The temperature control box is controlled at 20°C. Crystallization time 7 days;

Step 6: After the crystallization is completed, observe whether catalase crystals grow in the capillary. The identification of catalase crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer without the crystal Take it out or do other processing.

The results are as follows: using the high-throughput protein crystallization condition screening method using the electric field, the catalase protein crystals grown under different crystallization conditions are different. The high-throughput protein crystallization condition screening method and its device used in the electric field environment can achieve the purpose of screening protein crystallization conditions in the electric field. The crystals grown in the two groups of capillaries were diffracted in situ, and the crystal diffraction patterns were obtained. The diffraction data showed that the quality of catalase crystals in the electric field was higher than that under conventional conditions.

Example 3: The high-throughput crystallization device realizes the high-throughput crystallization condition screening of Somali sweet protein in a high-gravity environment in the magnet and compares it with conventional crystal quality

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample tray 1, protein Sample tray two (one and two share the capillary), groove top cover, and nut. 2 sets of device assembly;

The second step: prepare the Somali sweet protein solution, and use the capillary action of the capillary to draw the Somali sweet protein solution into the capillary;

Step 3: Put the capillary tube containing the Somali sweet protein solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 3 days;

Step 4: Take out the completely dried capillary and install it on the protein sample disk to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease, place one group of crystallization devices in an electric field under temperature control conditions, and place the other group in a temperature control box in a conventional environment. The temperature control box is controlled at 20°C. Crystallization time 3 days;

Step 6: After the crystallization is complete, observe whether Somali sweet protein crystals grow in the capillary. The identification of Somali sweet protein crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer without the crystal Take it out or do other processing.

The results are as follows: Using the high-throughput protein crystallization condition screening method in a strong magnetic field and high gravity environment, the Somali sweet protein crystals grown under different crystallization conditions are different. The high-throughput protein crystallization condition screening method and its device used in a high-gravity environment can achieve the purpose of screening protein crystal conditions in a high-gravity environment. The crystals grown in the two groups of capillaries were subjected to in-situ diffraction, and the diffraction patterns of the crystals were obtained. The diffraction data showed that the quality of catalase crystals in the high-gravity environment was higher than that under conventional conditions.

Example 4: The high-throughput crystallization device realizes the high-throughput crystallization condition screening of concanavalin protein in a conventional environment

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample plate 1, protein Sample tray two (one and two share capillary tubes), groove top cover, nut, and a set of device assembly;

The second step: prepare the protein solution of concanavalin bean, and absorb the protein solution of concanavalin bean into the capillary by the capillary action of the capillary;

Step 3: Put the capillary tube containing the concanavalin protein solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 4 days;

Step 4: Take out the completely dried capillary and install it on the protein sample disk to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease and place it in a temperature control box under temperature control conditions. The temperature of the temperature control box is 20°C. Crystallization time 4 days;

Step 6: After the crystallization is completed, observe whether concanavalin protein crystals grow in the capillary. The identification of concanavalin protein crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer without the crystal Take it out or do other processing.

The results are as follows: using a high-throughput protein crystallization condition screening method using a strong magnetic field, different crystallization conditions can produce different conditions of concanavalin protein crystals. The high-throughput protein crystallization condition screening method and its device used in a conventional environment can achieve the purpose of screening protein crystallization conditions in a conventional environment. The in-situ diffraction was performed on the crystal grown in the capillary, and the diffraction pattern of the crystal was obtained, indicating that the experimental device can realize the in-situ diffraction of the crystal.

Example 5: High-throughput crystallization device realizes screening of proteinase K high-throughput crystallization conditions in a magnet

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample plate 1, protein Sample tray two (one and two share the capillary), groove top cover, and nut. 1 set of device assembly;

The second step: preparing proteinase K solution, using the capillary action of the capillary to draw the proteinase K solution into the capillary;

Step 3: Put the capillary tube containing the proteinase K solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 4 days;

Step 4: Take out the completely dried capillary and install it on the protein sample plate to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease and place it in the magnet chamber under temperature control conditions. The temperature control box is controlled at 20°C. Crystallization time 3 days;

Step 6: After the crystallization is completed, observe whether proteinase K crystals grow in the capillary. The identification of proteinase K crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer without removing or doing crystallization. other processing.

The results are as follows: using a high-throughput protein crystallization condition screening method using a strong magnetic field, different crystallization conditions produce different proteinase K crystals. The high-throughput protein crystallization condition screening method and its device used in a strong magnetic field environment can achieve the purpose of screening protein crystallization conditions in a magnet. The in-situ diffraction was performed on the crystal grown in the capillary, and the diffraction pattern of the crystal was obtained, indicating that the experimental device can realize the in-situ diffraction of the crystal.

Example 6: High-throughput crystallization device realizes screening of glucose isomerase high-throughput crystallization conditions in an electric field

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample plate 1, protein Sample tray two (one and two share the capillary), groove top cover, and nut. 1 set of device assembly;

The second step: prepare the glucose isomerase solution, and use the capillary action of the capillary to draw the glucose isomerase solution into the capillary;

Step 3: Put the capillary tube containing the glucose isomerase solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 3 days;

Step 4: Take out the completely dried capillary and install it on the protein sample disk to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease and place it in an electric field environment under temperature control. The temperature of the temperature control box is controlled at 20°C. Crystallization time 3 days;

Step 6: After the crystallization is completed, observe whether glucose isomerase crystals grow in the capillary. The identification of glucose isomerase crystals and the collection of diffraction data can be directly carried out in situ diffraction by X-ray diffractometer without the crystal Take it out or do other processing.

The results are as follows: using the high-throughput protein crystallization condition screening method using the electric field, the glucose isomerase crystals grown under different crystallization conditions are different. The high-throughput protein crystallization condition screening method and its device for screening protein crystallization conditions in an electric field environment can achieve the purpose of screening protein crystallization conditions in an electric field. The in-situ diffraction was performed on the crystal grown in the capillary, and the diffraction pattern of the crystal was obtained, indicating that the experimental device can realize the in-situ diffraction of the crystal.

Example 7: The high-throughput crystallization device realizes the high-throughput crystallization condition screening of trichosanthin protein in a conventional environment

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample plate 1, protein Sample tray two (one and two share capillary tubes), groove top cover, nut, and a set of device assembly;

The second step: prepare the trichosanthin protein solution, and use the capillary action of the capillary to absorb the trichosanthin protein solution into the capillary;

Step 3: put the capillary tube containing the trichosanthin protein solution into a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 20°C, and the drying time is 4 days;

Step 4: Take out the completely dried capillary and install it on the protein sample disc to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease and place it in a temperature-controlled incense box under temperature-controlled conditions. The temperature of the temperature-controlled box is controlled at 20°C. Crystallization time 4 days;

Step 6: After the crystallization is complete, observe whether trichosanthin protein crystals grow in the capillary. The identification of trichosanthin protein crystals and the collection of diffraction data can directly use the X-ray diffractometer for in-situ diffraction without removing the crystals or doing them. other processing.

The results are as follows: Using the screening method of high-throughput protein crystallization conditions in the conventional environment, the crystals of trichosanthin protein crystals grown under different crystallization conditions are different. The high-throughput protein crystallization condition screening method and its device used in a conventional environment can achieve the purpose of screening protein crystallization conditions in a magnet. The in-situ diffraction was performed on the crystal grown in the capillary, and the diffraction pattern of the crystal was obtained, indicating that the experimental device can realize the in-situ diffraction of the crystal.

Example 8: The high-throughput crystallization device realizes the high-throughput crystallization condition screening of heat shock protein 90 ^N in the magnet and compares the crystal quality with the conventional environment

Step 1: Assembly of high-throughput protein crystallization condition screening device. From bottom to top, the protein crystallization condition screening device is the mother liquid base (the groove base is used for sealing in special environments), square fixed shaft, protein sample plate 1, protein Sample tray two (one and two share the capillary), groove top cover, and nut. 2 sets of device assembly;

The second step: prepare the heat shock protein 90 ^N solution, and use the capillary action of the capillary to absorb the heat shock protein 90 ^N solution into the capillary;

Step 3: Put the capillary tube containing the heat shock protein 90 ^N solution in a drying vessel placed in a temperature-controlled box to dry. The drying temperature is 4°C, and the drying time is 2 days;

Step 4: Take out the completely dried capillary and install it on the protein sample plate to form a capillary array for screening protein crystallization conditions. Correspondingly insert the array into the mother liquid base pre-installed with screening reagents, and then use capillary phenomenon to draw the screening reagent solution from the mother liquid base respectively. The screening reagent used is the commercial screening reagent Index;

Step 5: After the suction is completed, seal the capillary with vacuum grease and place one group of crystallization devices in a special physical environment under temperature control conditions, and the other group in a temperature control box in a conventional environment. The temperature is 4°C. Crystallization time 2 days;

Step 6: After the crystallization is complete, observe whether heat shock protein 90 ^N crystals grow in the capillary. The identification of heat shock protein 90 ^N crystals and the collection of diffraction data can be directly carried out in situ by X-ray diffractometer without Fish out the crystals or do other processing.

The results are as follows: using the high-throughput protein crystallization condition screening method using a strong magnetic field, the heat shock protein 90 ^N crystals grown under different crystallization conditions are different. The high-throughput protein crystallization condition screening method and its device used in a strong magnetic field environment can achieve the purpose of screening protein crystallization conditions in a magnet. In the mass comparison of heat shock protein 90 ^N crystals in strong magnetic field and conventional environment, the crystals grown in the two groups of capillaries were subjected to in situ diffraction, and the diffraction patterns of the crystals were obtained. The diffraction data showed that the heat shock protein 90 N in the magnet ^N crystal quality is higher than that of conventional conditions.

Claims (5)

1. A high-throughput protein crystallization condition screening device under a strong magnetic field, comprising a mother liquid base, a protein sample tray, a groove base, a groove top cover, a capillary and a temperature control system, characterized in that: said mother liquid base, protein The materials used in the sample plate, the groove base, the groove top cover, the capillary and the temperature control system are all anti-magnetic materials; the mother liquid base plate is evenly arranged with several small pools loaded with liquid reagents, and the protein sample plate is There are through holes corresponding to the positions of the small pools on the mother liquor chassis one by one, the capillaries are assembled in each through hole, and the protein sample plate equipped with capillaries can be placed in the temperature control system; the groove base and the groove The top cover is a disc with grooves on the surface, the grooved base and the grooved top cover are opposite to each other, the grooved position corresponds to the position of the through hole on the protein sample plate, and the groove is filled with vacuum grease. 2 . The device for screening high-throughput protein crystallization conditions under a strong magnetic field according to claim 1 , wherein the capillary is assembled in the through holes of two parallel protein sample discs. 3. A method for screening high-throughput protein crystallization conditions under the strong magnetic field of the device according to claim 1, characterized in that it comprises the steps of: 1) Prepare a protein solution, and then use the capillary action of the capillary to draw the protein solution into the capillary; 2) Dry the protein solution in the capillary at the set drying temperature. During the drying process, the temperature control range is 0-60°C, the temperature control accuracy is ±0.1°C, and the drying time is 2-7 days; 3) Installing the completely dried capillary in the protein sample tray to form a capillary array for screening protein crystallization conditions; correspondingly inserting the array into the mother liquor base pre-installed with the screening reagent, and absorbing the screening reagent solution from the mother liquor base by capillary phenomenon; 4) Insert the two ends of the capillary into the vacuum grease on the base of the groove and the top cover of the groove respectively to realize the sealing of the capillary and place it in the magnet cavity at 0-60°C for 2-7 days for crystallization; 5) After the crystallization is completed, observe whether protein crystals grow in the capillary, and use an X-ray diffractometer to conduct in-situ diffraction to identify protein crystals and collect diffraction data. 4. The method for screening high-throughput protein crystallization conditions under a strong magnetic field according to claim 3, characterized in that: said protein refers to a water-soluble protein. 5. the high-throughput protein crystallization condition screening method under the strong magnetic field according to claim 3, is characterized in that: described step 4) place capillary tube under electric field, hypergravity condition or routine environment condition and carry out protein crystallization condition filter.