Constant current diffused protein crystallization method, open type constant current diffused protein crystallization array chip and manufacture method and application of chip

A protein crystallization and array chip technology, which is applied in the biological field, can solve problems such as difficulty in promoting protein molecular crystallization, limited total amount of protein molecules, small protein crystal volume, etc., to achieve convenient extraction or analysis, high crystal cluster density, and crystal size big effect

Active Publication Date: 2013-10-23
武汉介观生物科技有限责任公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] A common problem in the above experiments is that when the volume of the liquid is constant, the total amount of protein molecules contained is very limited, and it is difficult to promote the crystallization of protein molecules
If the concentration of protein is increased, the crystallization efficiency can be increased, but the obtained protein crystals are small in size and low in cluster density, so it is difficult to be directly used for diffraction analysis

Method used

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  • Constant current diffused protein crystallization method, open type constant current diffused protein crystallization array chip and manufacture method and application of chip
  • Constant current diffused protein crystallization method, open type constant current diffused protein crystallization array chip and manufacture method and application of chip
  • Constant current diffused protein crystallization method, open type constant current diffused protein crystallization array chip and manufacture method and application of chip

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Figure 1-6 Shown is the first preferred embodiment of the constant current diffusion protein crystallization method and the open constant current diffusion protein crystallization array chip of the present invention. The open constant current diffusion protein crystallization array chip contains 4×4 protein crystallization units, and the basic material uses porous polydimethylsiloxane (PDMS), and its preparation adopts the following steps:

[0047] 1) Draw with L-Edit and make such as Figure 4 and Figure 5 The optical template: mainly includes two parts:

[0048] Figure 5 Main channel design drawing, Figure 6 Design diagrams for diffusion channels. Wherein, the width of the main channel is 100 microns, and the width of the diffusion channel is 5 microns.

[0049] 2) Fabrication of the mold for the design pattern: the silicon wafer was ultrasonically cleaned with isopropanol and acetone for 5 minutes respectively, and boiled with a mixture of concen...

Embodiment 2

[0054] Example 2: Figure 1-6 As shown, the constant current diffusion protein crystallization method of the present invention and the open constant current diffusion protein crystallization array chip are used for the screening test of lysozyme protein crystallization conditions, and the following steps are adopted:

[0055] 1) Configure 20 mg / ml and 120 mg / ml lysozyme protein solutions, and use a circulating syringe pump to simultaneously feed these two protein aqueous solutions into the liquid inlet, and the injection speed is set to 8 μl / min.

[0056] 2) After the diffusion tube of the chip is filled with protein solution, add NaCl solution and CH to 16 crystallization units according to different concentration conditions. 3 COONa / CH 3 COOH mixed solution (the typical concentration range of NaCl is 0.5 mol / L-4 mol / L, CH 3 COONa / CH 3 The ratio range of the COOH mixed solution is 1:1-10:1).

[0057] 3) Continue to pass through the protein aqueous solution at an injection...

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Abstract

The invention relates to a constant current diffused protein crystallization method, an open type constant current diffused protein crystallization array chip and a manufacture method and application of the chip. The open type constant current diffused protein crystallization array chip comprises a base and a substrate fixed on the base, wherein a plurality of hole-shaped protein crystallization units communicated with the external environment are distributed on the substrate so as to form a crystallization unit array, a plurality of microflow main channels are also distributed on the substrate, and each crystallization unit is communicated with a corresponding microflow main channel through radial microflow diffused channels. Protein solution is injected into the microflow main channels in a constant current manner, and precipitator solution in the open type crystallization units is static. The open type constant current diffused protein crystallization array chip has the advantages that 1. a high protein concentration gradient is maintained between the microflow main channel and the crystallization units, so that the protein molecules are continuously diffused into the crystallization units, the protein is crystallized rapidly, and the crystal cluster has high density; 2. the protein crystals formed in the crystallization units are free from the influence of the shear force and can be conveniently extracted and analyzed; 3. the consumption of the protein is small, the operation is flexible, and the method is suitable for high-pass screening under crystallization condition; and 4. the chip is simple to manufacture, has a compact structure, is low in cost and can be produced in a large scale.

Description

technical field [0001] The invention relates to a method for efficiently obtaining protein crystallization and screening of crystallization conditions. Protein crystallization is the most direct and effective method for comprehensively understanding the advanced structure and function of proteins. It is of great significance for the study of biological processes and new drug design, and belongs to the field of biotechnology . Background technique [0002] The study of protein structure and function is of great significance to the understanding of life processes at the molecular level and the design of new drugs. The crystal analysis of protein is the most direct and effective method to fully understand the three-dimensional structure of protein, so it has attracted widespread attention. [0003] In order to efficiently obtain protein crystals that can be used for crystal analysis, it is necessary to use a small amount of protein solution for reasonable condition screening (...

Claims

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Application Information

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IPC IPC(8): C07K1/14
Inventor 陈勇丁一李俊君石剑
Owner 武汉介观生物科技有限责任公司
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