CN110551703A

CN110551703A - Preparation and application methods of novel material BEM

Info

Publication number: CN110551703A
Application number: CN201910855146.7A
Authority: CN
Inventors: 韩烨; 赵芳坤; 肖华志; 周志江
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2019-12-10

Abstract

The invention discloses a preparation and application method of a novel material BEM, and relates to a method for preparing a purified immobilized material by using an inclusion body, wherein the method comprises the step of boiling an inclusion body obtained by escherichia coli recombinant expression by using HCl or trichloroacetic acid (TCA) to obtain the BEM (bacterial enhanced matrix, the main component is cell wall) for purifying immobilized recombinant protein with an AmcA label. The method can prepare useless inclusion bodies into a purification and immobilization material for purifying and immobilizing the recombinant protein with the AcmA label. The method can prepare the inclusion body into a purified immobilization material BEM and can replace GEM as the purified immobilization material.

Description

preparation and application methods of novel material BEM

Technical Field

The invention relates to a preparation method and an application method of a novel material BEM, in particular to a method for preparing the novel material BEM by using cell fragments for purifying a recombinant protein immobilized with an AcmA label.

Background

the immobilization of the enzyme has important significance for the application of the enzyme, and the expensive enzyme can be recycled by the immobilization, and simultaneously, the introduction of new protein impurities into a reaction system can be avoided. Immobilization of enzymes is widely used in the fields of industrial catalysis, fermentation, food, and the like. The enzyme for immobilization may be obtained by fermentation using an enzyme-producing strain, isolation and purification from a natural product, or by recombinant expression. Recombinant expression can increase the yield of the enzyme, and purification by affinity chromatography or the like can be performed by adding an appropriate tag. These processes require multiple steps and also require chemicals such as ammonium sulfate, imidazole, etc. Therefore, the methods have the defects of long period, enzyme activity loss and the like.

Escherichia coli is an important engineering bacterium and is widely applied to laboratories and factories. Coli is the most common recombinant expression host, and a large amount of protein is recombinantly expressed in e. Coli recombinant expression belongs to overexpression, a large amount of protein cannot be folded correctly, and inclusion bodies are easily formed. After sonication and centrifugation, the resulting pellet is composed mainly of inclusion bodies and cell debris, including also cell wall components. The precipitate is generally considered to be discarded. The major component of the E.coli cell wall is the peptidoglycan to which the protein anchoring domain at the C-terminus of the peptidoglycan hydrolase AcmA of lactococcus lactis can specifically bind. . By utilizing this property, non-living bacteria enhanced substrates (BEM) can be obtained by acid boiling of the inclusion bodies. BEM can be used to purify immobilized recombinant proteins with AcmA tags.

Disclosure of Invention

The method aims to provide a preparation method and an application method of a novel material BEM, and solves the problems of long period and enzyme activity loss of a method for purifying and immobilizing recombinant protein with AcmA in the prior art.

The technical scheme of the invention is as follows:

a novel material BEM prepared by cell debris is used for purifying recombinant protein immobilized with AcmA label, and the purification method comprises the following steps:

1) Preparing recombinant alpha-amylase with an AcmA label, wherein the supernatant is used as a source for purifying immobilized enzyme, and the precipitate is used as a raw material for preparing BEM;

2) Boiling the inclusion body with 0.1M-0.2M HCl or TCA for 30-60 min;

3) Centrifuging to collect precipitate, and washing the precipitate with 50mM Tri-HCl (pH7.2) to obtain BEM;

4) BEM purification of immobilized recombinant alpha-amylase;

5) SDS-PAGE analysis.

In the step 1), escherichia coli BL21 with plasmid pET28 a-alpha-amylase-AcmA is cultured, IPTG induction is utilized, thalli are collected, ultrasonic crushing and centrifugation are carried out, recombinant alpha-amylase with an AcmA label is obtained, the obtained supernatant is used as a source of purified immobilized enzyme, and the sediment inclusion body is used for preparing BEM.

in step 2), the inclusion bodies are resuspended in 0.1M-0.2M HCl (or TCA) and boiled for 30-60 minutes.

The precipitate obtained by the boiling of the previous step was collected by centrifugation in step 3), and the precipitate was washed well with 50mM Tri-HCl (pH7.2) to obtain the prepared BEM.

In step 4), the prepared BEM was combined with the supernatant prepared in step 1) to purify the recombinant α -amylase for 30-50 minutes, centrifuged at 4000rmp, and the precipitate was washed well with 50mM Tri-HCl (pH 7.2).

The immobilization of BEM preparations and purifications was analyzed in step 5) by SDS-PAGE.

Has the advantages that: the invention takes an inclusion body obtained in escherichia coli recombinant expression as a raw material to prepare a novel BEM material for purifying and immobilizing recombinant protein with an AcmA label, and the BEM material can purify and immobilize the recombinant protein with the AcmA label. The method can prepare the inclusion body into a purified immobilization material BEM and can replace GEM as the purified immobilization material. The method has the advantages of simple and easy operation, low cost and full utilization of the waste inclusion body.

Drawings

FIG. 1 preparation of BEM using inclusion bodies for purification of recombinant alpha-amylase;

m: a protein Marker; 1. 2: whole mycoprotein composition before and after induction; 3. 4: after induction, ultrasonically crushing, centrifugally separating to obtain supernatant and precipitate; 5: preparing BEM by using the inclusion body; 6: preparation of inclusion bodies BEM purified immobilized recombinant alpha-amylase.

Detailed Description

Example 1: preparation of BEM

culturing Escherichia coli BL21 with plasmid pET28 a-alpha-amylase-AcmA, inducing with IPTG, collecting thallus, ultrasonic crushing and centrifuging, analyzing with SDS-PAGE, obtaining recombinant alpha-amylase with AcmA label as shown in figure 1, and using the obtained supernatant as purified immobilized enzyme source to precipitate inclusion body for preparing BEM.

the inclusion bodies were resuspended in 0.1M-0.2M HCl (or TCA) and boiled for 30-60 minutes. The boiled precipitate was collected by centrifugation, and the precipitate was washed thoroughly with 50mM Tri-HCl (pH7.2) to give a prepared BEM, which was analyzed by SDS-PAGE and removed most of the protein as shown in FIG. 1.

in this example, purified immobilized material BEM was prepared using inclusion bodies as raw materials.

Example 2: BEM purification immobilization recombination alpha-amylase

The prepared BEM was combined with the supernatant containing the recombinant α -amylase for 30 to 50 minutes, centrifuged at 4000rmp, and the precipitate was washed well with 50mM Tri-HCl (pH7.2) to purify the recombinant α -amylase. The immobilization of BEM was analyzed by SDS-PAGE, and as a result, as shown in FIG. 1, the objective protein labeled with AcmA was bound by the prepared BEM.

This example uses the prepared BEM to purify immobilized AcmA-tagged recombinant alpha-amylase.

Claims

1. A preparation method and an application method of a novel material BEM are characterized in that the novel material BEM is prepared by using cell fragments and used for purifying a recombinant protein immobilized with an AcmA label, and the method comprises the following steps:

1) Preparing recombinant alpha-amylase with an AcmA label, taking supernatant as a source for purifying immobilized enzyme, and taking a precipitate inclusion body as a raw material for preparing BEM;

2) boiling the inclusion body with 0.1M-0.2M HCl or TCA for 30-60 min;

4) BEM purification of immobilized recombinant alpha-amylase;

5) SDS-PAGE analysis BEM preparation and purification immobilization.

2. The method for preparing and using the novel material BEM according to claim 1, characterized in that the step 1) is to culture Escherichia coli BL21 with plasmid pET28 a-alpha-amylase-AcmA, induce with IPTG, collect thalli, then carry out ultrasonic disruption and centrifugation to obtain recombinant alpha-amylase with AcmA label, the obtained supernatant is used as a source of purified immobilized enzyme, and precipitate inclusion body for preparing BEM.

3. The method for preparing and using the novel material BEM according to claim 1, wherein the step 2) is to resuspend the inclusion bodies in 0.1M-0.2M HCl or TCA and boil for 30-60 min.

4. The method for preparing and using BEM as claimed in claim 1, wherein the step 4) is performed by combining the prepared BEM with the supernatant prepared in the step 1) to purify the recombinant α -amylase for 30-50 minutes, centrifuging at 4000rmp, and washing the precipitate with 50mM Tri-HCl (pH 7.2).