TWI513969B - Method and apparatus for protein crystallization and crystal growth - Google Patents

Description

Method and device for crystal formation and crystal growth of protein

The present invention relates to a technique and apparatus for crystal formation and crystal growth of a protein, which can obtain a sufficiently large crystal in a short time and can be used for X-ray crystallography.

Crystallization of proteins has been generally performed by X-ray crystallography to understand its biological functions. There are generally two main techniques for crystal formation, namely hang drop and sitting drop. Both are based on the gas diffusion of the solvent. Because the droplets are small, in a closed or semi-open chamber, the conditions of the droplets dynamically change with the environment of the counter solvent. Crystallization is formed according to crystal nucleation and crystal growth, in which crystal nuclei are formed and grown by taking protein molecules from solution (see Acc. Chem. Res. 2009, 42,621-629 ). In general, the crystallization of the protein is carried out under static conditions to avoid any perturbation, resulting in a large number of small crystals or irregular aggregation, which would take a long time, at least from day to month. Although there have recently been research and development of high-capacity technologies to investigate various crystallization conditions ( Ac. Chem. Res. 2003 , 36, 157-163), technologies for increasing the possibility of single crystal proteins and accelerating crystal formation continue to be under development.

Protein nucleation involves two processes, namely, a cluster of protein molecules and the reorganization of protein molecules in a cluster ( Ac. Chem. Res. 2009 , 42, 621-629; J. Am. Chm. Soc. 1998 , 120, 5539-5548; J. Am. Chm. Soc. 1999 , 121, 1627-1635.; J. Cryst Growth. 1999 , 197, 257-262). One way to increase the crystal nucleus is to apply an external stimulus, such as electricity, super-waves, and light fields, to the protein supersaturated solution. The effect of these stimuli is to vary the density of protein molecules in solution to form clusters and/or to rotate the clustered molecules to form a lattice structure. When the nucleus triggering mechanism is utilized at precise positions in an ultra-short interval to avoid excessive interference, a fine single crystal can be efficiently obtained. The following contents are, for example, the use of ultrashort laser pulses for crystal formation under different power conditions. Lysozyme and glucose isomerase trigger the nucleus with a femtosecond laser pulse under pulse energy conditions (1.95 nJ/pulse), but the probability of producing a single crystal is low. Also, the same femtosecond laser is applied to the formation of protein crystals under high energy conditions. This condition produces visible cavitation bubbles due to photo-ablation of the protein solution.

The technique of accelerating crystal growth after nucleation is also highly demanded to obtain large crystals with high efficiency and to study protein structure. The limitation of crystal growth is due to the diffusion of protein molecules into the crystal. For small droplets, the protein molecules are also limited to a small amount.

The present invention provides a technique and a device for crystal formation and crystal growth of a protein, which can obtain a sufficiently large crystal in a short time and can be used for X-ray crystal analysis.

An apparatus for obtaining protein crystals according to an embodiment of the invention includes: flying The second laser optical system produces a protein crystal for the protein droplets; and an ink jet ejector acts on the protein droplets to accelerate the crystal growth of the protein crystal.

An apparatus for obtaining protein crystals according to an embodiment of the invention includes a substrate, a sample chamber, a femtosecond laser optical system, an optical unit, and an inkjet injector. The substrate is light transmissive and has a protein droplet on it. The sample chamber covers the protein droplets on the substrate with a window at the top of the sample chamber. Femtosecond laser optics that emit single or multiple femtosecond laser pulses. An optical unit that focuses the femtosecond laser pulse on the air and liquid interface of the protein droplet. An ink jet ejector produces a plurality of droplets of solution into which the protein crystals are injected.

A method of obtaining protein crystals according to an embodiment of the present invention comprises: irradiating a protein droplet with a femtosecond laser pulse; and injecting a plurality of solution droplets into the protein crystal.

A method for obtaining protein crystals according to an embodiment of the present invention comprises: providing a protein droplet; irradiating the air and liquid interface of the protein droplet with a pulse of a focused femtosecond laser to form a protein crystal nucleus; A droplet of solution is applied to the protein droplet to stimulate the protein droplet and control the protein concentration in the protein droplet to accelerate the growth of the protein crystal.

A method for obtaining protein crystals according to an embodiment of the present invention comprises: preparing a protein droplet on a transparent substrate; irradiating the protein droplet with a focused femtosecond laser pulse; and providing a plurality of solutions by using an inkjet injector Droplets give the protein droplets.

The above described features and advantages of the invention will be apparent from the following description.

1‧‧‧protein droplets

2‧‧‧Substrate

3‧‧‧ sample chamber

4‧‧‧ window

5‧‧‧ solution droplet

6‧‧‧Inkjet injector

7‧‧‧ Temperature Controller

8‧‧‧Porous material

9‧‧‧ Piezoelectric solution droplet generator

10‧‧‧Liquid container

11‧‧‧Pulse generator

12‧‧‧ femtosecond laser

13‧‧‧ Optical unit

14‧‧‧ Objective lens

15‧‧‧ camera

16‧‧‧ computer

17‧‧‧Circular light source

31‧‧‧Ray beam

32‧‧‧protein droplets

41, 42, 43‧ ‧ microscope images

44~49‧‧‧ crystal

51~53‧‧‧crystal size line

S21~S24‧‧‧Steps

1 is a schematic view showing the structure of an apparatus for obtaining protein crystals according to an embodiment of the present invention.

2 is a schematic flow chart of a method for obtaining protein crystals (crystal nuclei) and crystal growth according to an embodiment of the present invention.

3 is a schematic diagram of the mechanism for obtaining protein crystals in accordance with an embodiment of the present invention.

4 is a schematic diagram showing the actual growth process of protein crystals in accordance with an embodiment of the present invention.

Figure 5 is a graphical representation of crystal size versus time during protein crystal growth in accordance with one embodiment of the present invention.

The present invention utilizes a femtosecond laser to produce a protein single crystal core. The protein is then continuously supplied to accelerate the growth of the crystal, which may, for example, utilize an ink jet injector to achieve continuous supply of the protein solution. Thus, the present invention can actually obtain some or a single protein crystal, and can control crystal growth, thereby accelerating the crystal growth rate of the protein.

Some embodiments are provided below to illustrate, but the invention is not limited to the embodiments shown.

1 is a schematic view showing the structure of an apparatus for obtaining protein crystals according to an embodiment of the present invention. Participate in Figure 1, protein droplet 1, which is for example 500nl, sit On a substrate 2. The substrate 2 is a light transmissive material, for example, borosilicate glass, and has a thickness of, for example, 0.17 mm. The sample chamber 3, for example of a plastic material, is placed on the substrate 2 to cover the protein droplets 1 to maintain proper humidity to prevent rapid evaporation of the protein droplets 1. The structure of the sample chamber 3 is combined by a side wall and a top cover. One end of the side wall is disposed on the substrate 2 such that the sample chamber 3 covers the protein droplet 1 on the substrate 2. The top cover of the sample chamber 3 has a window 4, for example about 4 mm in diameter, to enable the addition of a plurality of solution droplets 5 to the protein droplet 1 through the window 4. The plurality of solution droplets 5 are, for example, droplet-shaped jets, for example, which can be produced by the ink jet injector 6. The conditions of the environment around the sample chamber 3 are controlled by the temperature controller 7 and the wet porous material 8. The inkjet injector 6 is, for example, a piezoelectric-driving type injector that produces small droplets to form a solution droplet 5. The droplets are about the extent of picolitter. The ink jet injector 6 includes a piezoelectric solution droplet generator 9, a liquid container 10, and a pulse generator 11 to drive the piezoelectric solution droplet generator 9. The liquid from which the solution droplets 5 are produced is supplied from the liquid container 10. The linearly polarized femtosecond laser 12 provides a single pulse, such as a wavelength of 800 nm, a pulse width of 130 fs, and a measured energy of 2 nJ/pulse. This single pulse is focused by the optical optical unit 13 on the protein droplet 1. The optical unit 13 includes, for example, an objective lens (10X, NA.0.4) 14. The optical unit 13 can be generally regarded as a focusing unit to focus the laser pulses. The position of the laser focus, the protein droplet 1 and the resulting protein crystals are all monitored by the camera 15 of the CCD. The data of the camera 15 is recorded on the computer 16. The computer 16 controls the pulse generator 11, the temperature controller 7, and the femtosecond laser 12. The droplet size of the solution droplet 5 is monitored by the annular light source 17 during the entire process.

The driving frequency of the ink jet injector 6 is generally 10 Hz. The droplet size of the solution droplet 5 is generally, for example, 10 pl. The solution droplet 5 is added through the window 4 at the top of the sample chamber 3 In. This sample chamber 3 is designed to effectively prevent rapid evaporation of the protein droplets 1. A porous material 8 having salt saturated water is attached to the sample chamber 3 to control the humidity. In addition, the ink jet injector 6 is continuously added to the liquid through the window 4 at the top of the sample chamber 3, so that the size of the protein droplet 1 can be controlled to remain approximately the same because the droplet evaporation and liquid addition are balanced. In the general case, the solution droplet 5 contains the same components and ratio as the protein droplet 1, that is, the same protein liquid. However, the proportion of its components may be adjusted as appropriate depending on the needs. In another embodiment, the solution droplet 5 contains the same material as the protein droplet 1, but the concentrations may be the same or different. Thus, for example, such a droplet smaller than 500 nl can be maintained for a long period of time.

When the substrate 2 of glass is moved by the motor platform, the device can be easily modified to reach a plurality of protein droplets 1. Thus, if the top cover of the sample chamber 3 is relatively fixed, the window 4 can be modified into a slit shape or a plurality of windows 4. However, if the top cover of the sample chamber 3 is movable, a window 4 is sufficient for the corresponding movement.

The above apparatus is suitable for the formation of protein crystals, and produces a substantially single crystal by a single pulse laser. Further, the apparatus also causes the protein droplets 1 to be mixed all the time, and the ink jet ejector 6 allows the protein concentration in the droplets to be maintained at a constant value.

In another embodiment, FIG. 2 is a schematic flow chart of a method for obtaining protein crystals (crystal nuclei) and crystal growth according to an embodiment of the present invention. In step S21, it prepares a protein droplet 1 on a substrate 2. This substrate 2 is, for example, a light-transmissive glass sheet. At step S22, the protein droplets 1 on the substrate 2 are covered by the sample chamber 3.

At step S23, a single pulse provided by the femtosecond laser 12 focuses on the protein solution Drop the surface of 1. That is to say, the laser is focused on the interface between the air and the liquid to produce protein crystals. To ensure that the laser can focus on the interface between the air and the liquid, a laser can be applied at the edge of the droplet, as shown in Figure 3, where the laser beam 31 can be easily focused on the air-liquid-substrate boundary 32. This focusing condition enables a high probability of crystal formation. During this time, the appearance of the crystal is still undetermined because it exceeds the resolution of the objective lens that monitors the crystal nucleus of the protein. Also, the position of the nucleus is unpredictable, and the appearance of the nucleus is generally far from the laser focus point.

In step S24, it is the growth of protein crystals. A plurality of solution droplets 5 are added to the protein droplet 1 during the required period. Not only water, salt and/or protein saturated water is also used for protein droplets 1 to control the condition of protein droplets 1 and accelerate the growth of protein crystals.

The verification of protein crystal formation is illustrated by the use of lysozyme. 3 is a schematic diagram of the mechanism for obtaining protein crystals in accordance with an embodiment of the present invention. Referring to Figure 3, the lysozyme protein droplet 1 is dropped onto the glass substrate 2. The composition of protein droplet 1 is, for example, 500 nl, 50 mg/ml is dissolved in 0.5 M NaCl, 50 mM sodium acetate, pH 4.5. The substrate 2 is covered by the sample chamber 3. The multiple pulses of the femtosecond laser have an energy of 2-200 nJ/pulse and are focused on the boundary 32 between the aforementioned air-liquid-substrate. A plurality of solution droplets 5 were continuously added at a lapse of 10 Hz with a 25 mg/ml lysozyme aqueous solution (water as a solvent).

4 is a schematic diagram showing the actual growth process of protein crystals in accordance with an embodiment of the present invention. Referring to Fig. 4, crystals obtained from protein droplets 1 of lysozyme under three conditions can be seen from a series of microscope images 41, 42, 43 under the conditions of laser irradiation and addition of protein solution droplets. Under the conditions of laser exposure only Under the conditions of no addition of protein solution droplets, and no laser irradiation and addition of protein solution droplets. The microscope images on the left, middle, and right sides are the conditions for applying laser irradiation for 5, 30, and 60 minutes, respectively. In the microscope images 41, 42, when a single laser pulse is focused on the liquid-air boundary surface of the droplet, after 5 minutes, the quadrilateral crystals 44, 45, 46, 47, 48, 49 are irradiated for 5 minutes. , began to appear in the droplets of lysozyme. When a single laser pulse is focused on a liquid state (data not shown), no visible crystals are formed. The crystal growth rate is faster than the addition of the lysozyme solution droplets under the conditions of adding the lysozyme solution droplets, as in the crystal 44, 45, 46 of the microscope image 41 relative to the crystal 47 in the microscope image 42, 48, 49.

Under one pulse, although the probability of generating a plurality of crystals is 1/6 and the probability of no crystal generation is 1/6, the accuracy of producing a crystal is 4/6. In contrast, under the same conditions but without laser, no crystals are formed, as shown by microscope image 43.

As a result of the microscope image 41, 25 mg/ml of lysozyme was dissolved in water, and inkjet was continuously injected into the droplets, and the volume was about 10 pl/droplet at a frequency of 10 Hz, so that the initial volume of the sitting drop was maintained.

In 60 minutes, inkjet was added to 360 nl of lysozyme solution to the complete droplet. The size of the droplets remains almost the same, which means that the injected solvent is substantially evaporated, as in the microscope image 43.

Figure 5 is a graphical representation of crystal size versus time during protein crystal growth in accordance with one embodiment of the present invention. As shown in Fig. 5, as shown by the crystal size line 51 of the dot, the single tetragonal lysozyme crystal grows rapidly at about 45 nm/s, and becomes 0.185 mm after 60 minutes (between the {110} faces). . On the other hand, in the absence of dissolution Under static conditions of the bacteriocin solution, the crystal growth rate was 7.4 nm/s and was 0.027 mm after 60 minutes of incubation, as indicated by the crystal size line 52 of the triangular point. Continuous injection of protein by inkjet accelerates the growth of lysozyme crystals, which is about 6 times. Further, if there is no laser irradiation and no lysozyme is continuously injected, the crystal growth rate is 0 nm/s as indicated by the crystal size line 53 of the box point.

The lysozyme molecules were continuously added by ink jet to maintain the protein concentration. The complete droplet (500 nl) contains 0.025 mg of lysozyme. A 10 pl drop was continuously injected at a frequency of 10 Hz over 60 minutes, and 360 nl of the lysozyme solution (25 mg/ml) was injected into a complete 500 nl solution, which means that 0.009 mg of lysozyme was added. On the other hand, the approximate crystal size is 0.185 x 0.185 x 0.250 mm, so that the lysozyme consumption is estimated to be 0.009 mg in crystal growth. This is the same amount as the injected lysozyme. Since the evaporation of the solvent of the droplets is balanced with the injection, even if the protein solute is consumed as the crystal grows, the protein concentration is almost constant, for example, 50 mg/ml. Therefore, even if 1/3 of the initial lysozyme is consumed in crystal growth, the crystal size increase is positive as the culture time as shown by the crystal size line 51. The change in the curve may be due to the evaporation of the solvent and the equilibrium of the injection being produced in a semi-closed sample chamber or by an inkjet mixing effect. In fact, the crystal is slowly rotated and moved during the loading process, which is driven by the ink jet. Conversely, this mixing effect is not obtained under static conditions, as shown by crystal size line 52. The amount of crystal growth lysozyme during the 60 minute culture period under static conditions was estimated to be 0.00003 mg. Thus, more than 99% of the lysozyme remains in a water-soluble state, and the concentration is almost constant (50 mg/ml) in 60 minutes. This value is the same as in the case of the injected protein. From this result, it can be seen that continuous injection of protein produces mild stimulation of the protein solution of the droplet, which is indeed a driving force for rapid crystal growth. power.

The present invention proposes a novel protein crystallization technique based on laser-induced nucleation and ink-assisted crystal growth. This crystallization technology also contributes to the study of protein structure because it can achieve high productivity. Both laser-induced nucleation and ink-assisted crystal growth can be applied to a large number of sitting drops.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and any one of ordinary skill in the art can make some changes and refinements without departing from the spirit and scope of the present invention. The scope of the invention is defined by the scope of the appended claims.

1‧‧‧protein droplets

2‧‧‧Substrate

3‧‧‧ sample chamber

4‧‧‧ window

5‧‧‧ solution droplet

6‧‧‧Inkjet injector

7‧‧‧ Temperature Controller

8‧‧‧Porous material

9‧‧‧ Piezoelectric solution droplet generator

10‧‧‧Liquid container

11‧‧‧Pulse generator

12‧‧‧ femtosecond laser

13‧‧‧ Optical unit

14‧‧‧ Objective lens

15‧‧‧ camera

16‧‧‧ computer

17‧‧‧Circular light source

Claims (17)

A device for obtaining protein crystals, comprising: a light transmissive substrate for supporting protein droplets; a sample chamber disposed on the light transmissive substrate and covering the protein droplets to maintain humidity, wherein the sample chamber has a top portion a window; a femtosecond laser system that produces a protein crystal for protein droplets; a focusing unit for focusing a femtosecond laser pulse on the air and liquid interface of the protein droplet; and an inkjet injector through which the window Acting on the protein droplets to accelerate the crystal growth of the protein crystal. The apparatus for obtaining protein crystals according to claim 1, wherein the ink jet type injector injects a plurality of solution droplets into the protein crystal. The apparatus for obtaining protein crystals according to claim 2, wherein the plurality of solution droplets injected stimulate the protein droplets. The apparatus for obtaining protein crystals according to claim 1, wherein the plurality of solution droplets control a protein concentration of the protein droplets. A device for obtaining protein crystals, comprising: a substrate, the substrate is light transmissive and has a protein droplet thereon; a sample chamber covering the protein droplet on the substrate, wherein the sample chamber has a window at the top; a second laser system that emits a single or multiple femtosecond laser pulses; a focusing unit that focuses the femtosecond laser pulse on the air and liquid interface of the protein droplet; and an inkjet injector that produces a plurality of solutions Droplets are injected into the crystal of the protein. The apparatus for obtaining protein crystals according to claim 5, wherein the plurality of solution droplets balance evaporation of the protein droplets. The apparatus for obtaining protein crystals according to claim 5, wherein the plurality of solution droplets comprise the same substance as the protein droplets, but the concentrations are the same or different. A method for obtaining protein crystals, comprising: providing protein droplets on a substrate; using a focused femtosecond laser pulse to illuminate an air and liquid interface of the protein droplets; and injecting a plurality of solutions into the protein droplets drop. A method of obtaining protein crystals according to claim 8 wherein the step of injecting the plurality of solution droplets into the protein crystal comprises using an ink jet injector. The method for obtaining protein crystals according to claim 8, wherein the plurality of solution droplets balance evaporation of the protein droplets. A method of obtaining protein crystals, comprising: providing a protein droplet; illuminating an air-liquid interface of the protein droplet with a focused femtosecond laser pulse to form a protein crystal nucleus; and providing a plurality of solution droplets to the protein The droplets stimulate the protein droplets and control the protein concentration of the protein droplets to accelerate the growth of the protein crystals. A method of obtaining protein crystals according to claim 11, wherein the femtosecond laser pulse is a single pulse. The method for obtaining protein crystals as described in claim 11 of the patent application, Wherein the plurality of solution droplets comprise the same components as the protein droplets, but the ratios of the components are the same or different. The method of obtaining protein crystals according to claim 11, wherein the plurality of solution droplets balance evaporation of the protein droplets. A method for obtaining protein crystals, comprising: preparing a protein droplet on a light transmissive substrate; irradiating the protein droplet with a focused femtosecond laser pulse; using an ink jet injector to inject a plurality of solution droplets Give the protein droplets. The method for obtaining protein crystals according to claim 15, wherein the plurality of solution droplets comprise the same components as the protein droplets, but the ratios of the components are the same or different. The method for obtaining protein crystals according to claim 15, wherein the plurality of solution droplets balance evaporation of the protein droplets.