Rapid purification method for recombinant pyrolytic enzyme

A high-temperature cracking and purification method technology, which is applied in the field of rapid purification of recombinant high-temperature cracking enzymes, can solve the problems of total volume impact and convenience limitation, and achieve high purification and concentration efficiency, simple operation, and high thermal denaturation removal rate.

Inactive Publication Date: 2018-03-13
KUNMING UNIV OF SCI & TECH
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In order to release the expressed target protein from Escherichia coli, commonly used methods include sonication, high-pressure homogenization, freeze-thawing, etc. Since many expressed target proteins are heat-la...

Method used

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  • Rapid purification method for recombinant pyrolytic enzyme
  • Rapid purification method for recombinant pyrolytic enzyme
  • Rapid purification method for recombinant pyrolytic enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: The rapid purification method of this recombinant high-temperature lyase is as follows:

[0016] 1. Will carry pET28a (containing high temperature lyase TSPpgh Gene, TSPpgh molecular weight is 19KDa) recombinant BL21 strain was inoculated into 100 mL LB culture medium (containing kanamycin final concentration 50 μg / mL) at a mass ratio of 1%, and cultured on a shaker at 37°C until its OD600 value was about 0.6-0.8;

[0017] 2. Add isopropyl-β-D-thiogalactopyranoside IPTG (final concentration: 1mM), place in 28°C, 80rpm shaker for 6 hours;

[0018] 3. Centrifuge 100mL of the fermented liquid after induction at 4500g for 20min, take the cell pellet, suspend the cell in 9mL of 0.05mol / L phosphate buffer with a pH of 7.2, and take 1mL of the cell suspension in a 3mL centrifuge tube , take 6 tubes for thermal cracking, heat treatment in 55-80°C water bath respectively, set up a tube every 5°C, sample thermal cracking time is 20min, ultrasonic crushing tre...

Embodiment 2

[0028] Example 2: Detection of the activity of lyases obtained at different heating temperatures

[0029] 1. The effect of lyase on the growth of thermobacterial strain TC16

[0030] (1) Culture conditions of the host strain TC16

[0031] The high-temperature bacteria test strain TC16 was transferred to the DSM88 medium (recipe in Table 3) with an inoculation amount of 1% by mass, and cultured in a constant temperature shaker at a temperature of 60°C and a speed of 150 rpm until TC16 grew to an OD600 of 0.6 , take out the strain as the spare strain for the experiment;

[0032] Table 3 DSM88 liquid medium formula table

[0033] ;

[0034] (2) Cleavage detection of test strain TC16 by pyrolyase

[0035] The lysis effect of the lyase expressed by Escherichia coli collected by centrifugation in step 4 of Example 1 on the test strain TC16 is used as an evaluation of the difference in the remaining enzyme activity of the lyase in the high-temperature lysis of the bacteria, and...

Embodiment 3

[0043] Embodiment 3: The rapid purification method of this recombinant high-temperature lyase is as follows:

[0044] 1. Use BIOFLO415 fermenter to ferment recombinant Escherichia coli strain BL21 (containing high temperature lyase TSPpgh Gene), the volume of fermentation broth is 10L, the fermentation conditions are 150rpm / min, 37℃, 5L / min; the expression is induced by isopropyl-β-D-thiogalactoside IPTG (final concentration: 1mM) After the end, the temperature in the fermenter is directly raised to 65°C for 20 minutes, which is used to pyrolyze Escherichia coli, and to inactivate, denature and precipitate a large amount of miscellaneous proteins; Ultrafiltration of the broken liquid through a 0.45 μm filter membrane, and the filtrate is collected; 1 mL of the filtrate is filtered and concentrated through a 3KDa ultrafiltration membrane bag, and the concentrated liquid is the preliminary purified high-temperature lyase enzyme liquid;

[0045] 2. Use the Coomassie Brillia...

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Abstract

The invention discloses a rapid purification method for recombinant pyrolytic enzyme. According to the method, escherichia coli is used as an expression host of recombinant pyrolytic enzyme, the recombinant pyrolytic enzyme is subjected to inducible expression in host cells of escherichia coli, thalli are collected centrifugally, and escherichia coli is broken by heating to release expressed pyrolytic enzyme; meanwhile, host protein of escherichia coli is denatured, devitalized and precipitated at high temperature, which is favorable for purifying pyrolytic enzyme resisting to thermal denaturation; further, high-purity pyrolytic enzyme is obtained rapidly through the process of bacteria debris catching and protein concentration of pyrolytic enzyme through ultra-filtration. The rapid purification method is easy to operate; the escherichia coli protein thermal denaturation removal rate is high; the purification concentration efficiency is high; the method is adaptable to industrial production and marketing promotion and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a rapid purification method of recombinant high-temperature lyase. Background technique [0002] Protein purification is an important step in the extraction of biological enzymes. The usual purification methods include affinity chromatography, molecular sieve, organic solvent fractionation, isoelectric point precipitation, etc. The steps involved in the above methods vary according to the characteristics of the protein. In many cases, it is necessary to combine multiple methods to obtain the target protein, and the more purification steps, the recovery efficiency of the protein will be greatly affected. It can be seen that improving the extraction efficiency of protein is always the core issue in protein extraction. In industrial applications, The protein extraction process can even have a major impact on the quality and cost of the product. [0003] Escherichia coli is ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12R1/19
CPCC12N9/88
Inventor 林连兵刘洋荣露娟邓先余王峰郭军
Owner KUNMING UNIV OF SCI & TECH
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