Constant current diffusion protein crystallization method and open constant current diffusion protein crystallization array chip and its fabrication method and application

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: 2017-12-26
武汉介观生物科技有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] 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 diffusion protein crystallization method and open constant current diffusion protein crystallization array chip and its fabrication method and application
  • Constant current diffusion protein crystallization method and open constant current diffusion protein crystallization array chip and its fabrication method and application
  • Constant current diffusion protein crystallization method and open constant current diffusion protein crystallization array chip and its fabrication method and application

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Experimental program
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Embodiment 1

[0052] 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.

[0053] The open constant current diffusion protein crystallization array chip contains 4x4 protein crystallization units, and the basic material uses porous polydimethylsiloxane (PDMS), and its preparation adopts the following steps:

[0054] 1) Draw with L-Edit and make such as Figure 4 and Figure 5 The optical template: mainly includes two parts: 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.

[0055] 2) Fabrication of a patterned mold: the silicon wafer was ultrasonically cleaned with isopropanol and acetone for 5 minutes respectively, and boiled with a mixture of concentrated sulfuric acid and hy...

Embodiment 2

[0061] 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:

[0062] 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.

[0063] 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).

[0064] 3) Continue to pass through the protein aqueous solution at an injection speed of 1...

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Abstract

The invention relates to a protein crystallization method based on constant current diffusion, an open constant current diffusion protein crystallization array chip and a manufacturing method and application thereof. The open constant current diffusion protein crystallization array chip includes a substrate and a substrate fixed on the substrate; several hole-shaped protein crystallization units communicating with the external environment are distributed on the substrate to form an array of crystallization units; Several microfluidic main channels are distributed; each crystallization single-pass radial microfluidic diffusion channel communicates with the corresponding microfluidic main channel. The protein solution was injected into the microfluidic main channel in a constant flow, while the precipitant solution in the open crystallization unit was static. The advantages of the present invention are: 1. A high protein concentration gradient is maintained between the microfluidic main channel and the crystallization unit, so that protein molecules are continuously diffused into the crystallization unit, so that the protein crystallization speed is fast and the crystal cluster density is high; 2. The protein crystals formed in the crystallization unit are not subject to shear force, and can be extracted and analyzed conveniently; 3. The protein consumption is small, the operation is flexible, and it is suitable for high-pass screening of crystallization conditions; 4. The chip is simple to manufacture and the structure Compact, low cost, and mass-producible.

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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Patent Type & Authority Patents(China)
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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