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Kit for screening protein crystals by taking salt as precipitator

A protein crystallization and precipitant technology, used in biological testing, microbial determination/inspection, material testing, etc., can solve the problems of low concentration ratio range, unfavorable protein solubility and stability, and few types of reagents. Good shape, improve the efficiency of crystallization screening, large size effect

Inactive Publication Date: 2013-12-11
ZHONGSHAN HYGENE BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, the limitation of SaltRx-HT is that the combination of precipitants used is freely selected, and its concentration ratio range is relatively low, which has room for improvement in the success rate of protein crystallization screening; Less, not conducive to improving the solubility and stability of proteins

Method used

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  • Kit for screening protein crystals by taking salt as precipitator
  • Kit for screening protein crystals by taking salt as precipitator
  • Kit for screening protein crystals by taking salt as precipitator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Condition screening of lysozyme (HEWL) using the prepared Secondary Salt Screen conditions. The specific formula is shown in Table 3.

[0048]Step 1: Dissolve the six times recrystallized lysozyme (HEWL, lysozyme, lys, No. 100940) purchased from Japan Seikagaku Company in a sodium acetate buffer solution with a pH of 4.6 and a concentration of 0.1M, and prepare a concentration of 20 mg / mL lysozyme solution, filtered through a 0.22 μm sterile filter to remove impurities. It is lysozyme protein solution.

[0049] Step 2: In this example, the prepared Secondary Salt Screen96 was used to screen the crystallization conditions of lysozyme.

[0050] Step 3: Crystallization experiment Using a sitting drop plate, add 90 μL of pool solution to each deep well, use an automatic pipetting system to mix 1 μL of screening crystallization agent and 1 μL of lysozyme protein solution into a crystallization drop, and drop it in a small well. Sit in the drop well, and seal th...

Embodiment 2

[0054] Example 2: Conditional screening of proteinase K (proteinase K) using the prepared Secondary Salt Screen conditions. The specific formula is shown in Table 3.

[0055] Step 1: Dissolve proteinase K (pK, No.P6556) purchased from Sigma-Aldrich in sodium citrate buffer solution with a pH of 6.5 and a concentration of 0.5M to prepare proteinase K with a concentration of 15 mg / mL The solution was filtered through a 0.22 μm sterile filter to remove impurities. That is proteinase K protein solution.

[0056] Step 2: In this example, the prepared Secondary Salt Screen 96 was used to screen proteinase K for crystallization conditions.

[0057] Step 3: Crystallization experiment Using a sitting drop plate, add 90 μL of pool solution to each deep well, use an automatic pipetting system to mix 2 μL of screening crystallization agent and 2 μL of proteinase K protein solution into a crystallization drop, and drop it in a small well. Sit in the drop well, and seal the crystallizati...

Embodiment 3

[0061] Example 3: Concanavalin was screened using the prepared Secondary Salt Screen conditions. The specific formula is shown in Table 3.

[0062] Step 1: Dissolve concanavalin (con, No.L7647) purchased from Sigma-Aldrich in N-2-hydroxyethylpiperazine-N'-2 with a pH of 7.5 and a concentration of 0.1M - In sodium ethanesulfonate buffer, prepare a concanavalin solution with a concentration of 10 mg / mL, and filter with a 0.22 μm sterile filter to remove impurities. It is concanavalin solution.

[0063] Step 2: In this example, the prepared Secondary Salt Screen 96 was used to screen the crystallization conditions of concanavalin.

[0064] Step 3: Crystallization experiment Using a sitting drop plate, add 90 μL of pool solution to each deep well, and use an automatic pipetting system to mix 1.5 μL of screening crystallization agent and 1.5 μL of concanavalin solution into crystallization droplets , drop in a small drip hole, and seal the crystallization plate.

[0065] Step 4...

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Abstract

The invention provides a kit for screening protein crystals by taking salt as a precipitator. The kit comprises a buffer solution, a precipitator and an additive, wherein the salt with relatively high crystallization efficiency is selected as the precipitator in the crystal kit; and meanwhile, a few of additive molecules are added to improve the solubility and stability of the protein, so as to obtain relatively high screening efficiency. By adopting the kit, the success rate of screening of protein crystallization conditions can be effectively improved.

Description

technical field [0001] The invention relates to a 96-condition salt kit for protein crystallization condition screening. Background technique [0002] At present, for the screening of protein crystallization conditions, the salt screening kit of primary choice is the SaltRx-HT (condition 96) salt screening kit from Hampton Research, and the specific components are shown in Table 1. It consists of two parts: salt and pH matrix buffers. Salt was the only major crystallization agent (precipitant) utilized, and different choices of crystallization salt concentration, pH, and salt were based on data mining of the Biomacromolecular Crystallization Database (BMCD) and other crystallization databases, crystallization reports, and Hampton Research work in crystallization laboratories builds on this body of work: up to 35% of protein crystallization kits involve salt as the primary crystallization reagent. Salts mainly include sodium salts, ammonium salts, magnesium salts, potassium...

Claims

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Application Information

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IPC IPC(8): C12Q1/34C12Q1/37C12Q1/30C12Q1/42C12Q1/40G01N33/68
Inventor 尹大川刘悦周伯儒郭云珠张辰艳陈瑞卿陶浩然黄林军
Owner ZHONGSHAN HYGENE BIOPHARM
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