Recombinant vector and expression and purification method of heat-labile UNG fusion protein

A recombinant vector, expression and purification technology, applied in the field of expression and purification of heat-labile UNG fusion protein, can solve the problems of wrong conformation of expressed protein, low expression amount, degradation of UNG enzyme, and miscellaneous protein.

Pending Publication Date: 2021-02-09
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing UNG protein expression and purification methods, there are often adverse results such as conformation errors of expressed proteins, low expression levels, degr

Method used

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  • Recombinant vector and expression and purification method of heat-labile UNG fusion protein
  • Recombinant vector and expression and purification method of heat-labile UNG fusion protein
  • Recombinant vector and expression and purification method of heat-labile UNG fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction of pCold-SUMO-rCod UNG vector

[0057] The rCOD UNG gene sequence was amplified by PCR, using SEQ ID NO: 1 and SEQ ID NO: 2 as primers, using SEQ ID NO: 3 (synthesized by Shanghai Sangon Bioengineering Co., Ltd.) as a template, and using PFX as a DNA polymerase to amplify The amplification system was 50ul, and the amplification conditions were 98°C, 2min; 98°C, 15s; 64°C, 15s; 72°C, 15s; 72°C, 2min, a total of 35 cycles, and PCR product 1 was obtained.

[0058] Electrophoresis was performed on 1.2% (w / v) agarose gel, and the target band was excised for gel recovery to obtain product 1.

[0059] Digest 3ug of pCold-SUMO plasmid with 20U each of PstI-HF and KpnI-HF, double-digested vector pCold-SUMO (SEQ ID NO:4), react in water bath at 37°C for 4 hours, cut out the target strip after 1.2% agarose gel electrophoresis The band was recovered by gel to obtain product 2, and the digested product was transformed into TOP10 competent cells (purchased...

Embodiment 2

[0065] Example 2: Small expression of 6XHis-SUMO-rCod-UNG

[0066] The strain preparation of the pCold-6XHis-SUMO-rCod-UNG expression vector: take 25ng of the pCold-6XHis-SUMO-rCod-UNG expression vector obtained in Example 1, and add it to 100 μL of 5 kinds of plasmids containing different molecular chaperones (see image 3 ) BL21 (DE3) competent cells (purchased from Shanghai Nearshore Technology Co., Ltd.), ice-bathed for 30 minutes, then heat-shocked at 42°C for 90 seconds, inserted back on ice for 2 minutes, and coated with ampicillin (30 μg / ml) and chlorine Mycin (10 μg / ml) double-resistant LB medium, 37 ° C constant temperature incubator inverted overnight.

Embodiment 3

[0075] Example 3: Purification of 6XHis-SUMO-rCod-UNG

[0076] ① Large-scale induction of protein expression: Take the pCold-6XHis-SUMO-rCod-UNG expression strain obtained in Example 2, cultivate it overnight to obtain seed liquid, and transfer it to 1L ampicillin (30 μg / ml) and chlorine-containing solution at a ratio of 1:100. In liquid LB of mycin (10μg / ml), culture on a shaker at 37°C at 200rpm until OD600 reaches 0.5-0.7, take it out and cool it on ice for 15min, then add IPTG with a final concentration of 0.25mM, place at 15°C at 200rpm Shaker cultured for 16h to induce protein expression.

[0077] ② Lyse the cells: collect the cells by centrifugation for the first time, place the collected cells at -80°C, and freeze them for more than 30 minutes. Take out the frozen cells and resuspend the cells with pre-cooled 50ml His-tag Binding buffer (induced expression bacteria Liquid volume: Histag Binding buffer volume = 20:1). The cells were sonicated in an ice-water bath at 5...

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Abstract

The invention discloses a recombinant vector and an expression and purification method of heat-labile UNG fusion protein. The method comprises the following steps: cloning a gene sequence of Cod UNG to a pCold-sumo plasmid to construct a recombinant plasmid pCold-sumo-Cod UNG, transforming the recombinant plasmid into an Escherichia coli BL21 (DE3) competent cells, simultaneously transforming a plasmid pGTf2 which expresses chaperone into the competent cells, and carrying out low-temperature-induced expression to obtain the soluble sumo-Cod UNG fusion protein. According to the invention, the problem of insoluble Cod UNG expression products is successfully overcome.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing and purifying a thermolabile UNG fusion protein. Background technique [0002] Today, many clinical and scientific studies rely on the detection and quantification of trace analytes in limited sample volumes, and the analysis of DNA and RNA often uses real-time quantitative polymerase chain reaction, digital polymerase chain reaction, and next-generation sequencing. Analysis of tiny amounts of DNA and RNA molecules in limited sample volumes (e.g., liquid biopsies and single cells) often requires pre-amplification to generate sufficient quantities of target molecules to enable single-cell analysis of multiple targets in a reproducible, specific, and sensitive manner. Heavy quantification. As this may require several orders of magnitude doubling of the target sequence, subsequent sample handling becomes a potential contamination hazard, where high concent...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N9/24C12N15/56
CPCC12N15/70C12N9/2497C07K2319/21C07K2319/95C12Y302/02027C12N9/2402C12N2800/101
Inventor 张永有王丹宋娜杰
Owner XIAMEN UNIV
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