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Expression optimization and purification method of helicobacter pylori serine proteinase

A technology of serine protease and Helicobacter pylori, which is applied in the field of genetic engineering, achieves good application prospects, high-efficiency solution, and improved soluble expression

Pending Publication Date: 2019-07-23
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this patent has improved the expression of recombinant serine protease to a certain extent and it is soluble expression (70-85% of recombinant Helicobacter pylori serine protease is expressed in soluble form), there is still room for further improvement

Method used

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  • Expression optimization and purification method of helicobacter pylori serine proteinase
  • Expression optimization and purification method of helicobacter pylori serine proteinase
  • Expression optimization and purification method of helicobacter pylori serine proteinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Contains the construction of the E.coil BL21 (DE3) engineered bacterium of recombinant vector pET-22b-HtrA-His

[0033] Including the following steps:

[0034] 1. According to the recombinant vector pET-22b-HtrA (patent CN108531468A) obtained in the early stage of the laboratory, Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to perform site-directed mutation of the HtrA gene fragment in the existing plasmid , and then add a 6×His tag (CATCATCACCATCACCAT) to the C-terminus of the gene fragment to obtain a recombinant serine protease gene fragment HtrA-His containing a histidine tag.

[0035] 2. Using pET-22b-HtrA as the expression vector and BL21 as the expression host, insert the recombinant protein gene fragment HtrA-His between the 5'Nde I and 3'Sal I of the multi-cloning site restriction site, and then successfully connect The expression vector is introduced into the host, and the E.coil BL21 engineering bacteria containing the recombinant ve...

Embodiment 2

[0037] Example 2 Expression of E.coil BL21 (DE3) Engineering Bacteria Containing Purpose Recombinant Vector pET-22b-HtrA-His and Optimization of Expression Conditions

[0038] 1. Activation and preservation of bacteria

[0039] The original strains of E.coil BL21 engineering bacteria obtained in Example 1 were taken out for activation, single clones were selected and stored in glycerol tubes to ensure the purity and reliability of the experimental strains, including the following steps:

[0040] 1. Relevant culture medium preparation

[0041] LB liquid medium (1L formula): tryptone 10g, yeast extract 5g, sodium chloride 10g;

[0042] LB solid medium (1L formula): tryptone 10g, yeast extract 5g, sodium chloride 10g, agar powder 15g.

[0043] 2. Preparation of solid LB plates

[0044] (1) Ingredients for solid LB medium, sterilized at 121°C for 30 minutes;

[0045] (2) The ultra-clean workbench is sterilized by ultraviolet light for 30 minutes, and the alcohol lamp is lit in t...

Embodiment 3

[0089] The purification method of embodiment 3 recombinant Helicobacter pylori serine protease

[0090] In this example, by adjusting the elution gradient and elution system, the optimal elution conditions for the target protein in affinity chromatography are explored and determined, which specifically includes the following steps:

[0091] 1. Acquisition of purified sample protein solution

[0092] According to the optimal induction expression conditions of the above-mentioned engineering bacteria, a large amount of wet bacteria was obtained and lysed by repeated freezing and thawing methods; after being fully lysed, centrifuged at 4°C and 10,000 rpm for 10 minutes, discarded the precipitate, and collected the supernatant, which was Desired purification load sample protein solution.

[0093] 2. Packing and leak detection of affinity chromatography column

[0094] Put about 1mL of nickel column packing into the column by wet packing method, and at the same time turn on the n...

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Abstract

The invention discloses an expression optimization and purification method of helicobacter pylori serine proteinase. The expression optimization and purification method comprises the steps of constructing a target protein expression vector, performing induced expression of target protein under optimized condition, performing separation and purification on the target protein and the like. Accordingto the expression optimization and purification method disclosed by the invention, the target protein expression vector containing a His label is constructed and is successfully conveyed into host bacteria; besides, four factors of pre-inducing time, inducing temperature, the final concentration of an inducer and inducing time influencing growth of thalli of engineering bacteria and inducing expression quantity of the target protein are optimized through designing an orthogonal experiment, the optimal inducing expression condition is obtained, and the expression quantity of the target proteinis greatly increased; and an affinity chromatography method and an eluting system of the target protein are also optimized, so that the purity of the target protein reaches 98% and is notably increased than that by a traditional method, and the expression optimization and purification method has good application prospect on large-scale mass production of recombination protein.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for expression optimization and purification of Helicobacter pylori serine protease. Background technique [0002] Helicobacter pylori can infect gastric tissues and cause ulcers, and is the causative agent of gastric ulcers and gastric cancer. Helicobacter pylori infection usually occurs in childhood, the bacteria is widely distributed, and many people are carriers of the bacteria, and the complications caused by it include gastritis, gastric ulcer and duodenal ulcer. In addition, the bacteria can also increase the risk of gastric cancer in carriers. [0003] Currently, gastritis and gastric ulcer can be treated with antibiotics, and these treatments often require the use of 2 to 3 different types of antibiotics, but still cannot cure all patients. There is currently no effective treatment for stomach cancer, and the spread of antibiotic resistance is comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/57C12N15/70
CPCC12N9/52C12N15/70C12Y304/21014
Inventor 赵肃清王甜甜肖益热陈莉莉叶远志
Owner GUANGDONG UNIV OF TECH
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