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Expression and purification method of recombinant Tn5 transposase

An expression and purification, transposase technology, applied in the field of genetic engineering, can solve the problems of Tn5 enzyme degradation and miscellaneous proteins, low amount of Tn5 transposase enzyme, protein conformation error, etc., to ensure functional activity, convenient expression process, The effect of increasing production

Active Publication Date: 2018-11-30
XIAMEN LIFEINT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In the existing methods for the expression and purification of Tn5 transposase, the purified Tn5 enzyme often has the phenomenon of degradation and foreign proteins, and at the same time, the amount of Tn5 transposase obtained is low, the protein conformation is wrong, and the enzyme activity is low.
There is a significant gap between the actual production and application of the transposase after amino acid optimization and the theory

Method used

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  • Expression and purification method of recombinant Tn5 transposase
  • Expression and purification method of recombinant Tn5 transposase
  • Expression and purification method of recombinant Tn5 transposase

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Construction of pET22b-Strep-SUMO-Tn5 expression vector

[0038] The Tn5 transposase gene was amplified by PCR technology, using SEQ ID: NO: 1 and SEQ ID: NO: 2 as primers, in a 50 μl reaction system (with the artificially synthesized SEQ ID NO: 7 being the Tn5 transposase gene As a reaction template), KODpolymerase (manufactured by Japan Toyobo Co., Ltd., product number KOD-101 is a DNA polymerase) was used. The amplification conditions were 94°C for 30s; 94°C for 15s; 58°C for 15s; 74°C for 50s; 74°C for 3min; a total of 32 cycles. PCR product 1 was obtained.

[0039] The PCR product 1 in the above steps was recovered, electrophoresed on a 1.5% (W / V) agarose gel, and the target band was excised for gel recovery.

[0040]The target fragment with Strep tag II tag (as shown in SEQ ID: NO: 5) was artificially synthesized.

[0041] PCR technology was used to amplify the SUMO tag gene, using SEQ ID: NO: 3 and SEQ ID: NO: 4 as primers, in a 50 μl reaction syste...

Embodiment 2

[0048] Example 2: Expression and purification of pET22b-Strep-SUMO-Tn5 vector

[0049] Strain preparation of the pET22b-Strep-SUMO-Tn5 expression vector: take 100 ng of the pET22b-Strep-SUMO-Tn5 expression vector obtained in Example 1. Add to 100 μl DE3 competent cells, ice-bath for 30 minutes, heat shock at 42°C for 45 seconds, insert back on ice for 2 minutes, and add 150 μl anti-antibiotic-free LB medium. 37°C, 220rpm, after pre-shaking for 45min, the plate coated with LB ampicillin was inverted overnight.

[0050] Induced expression: pick the above monoclonal colony and add to 100ml self-inducing medium (1% Tryptone (w / v); 0.5% Yeast extract (w / v); 0.5% Glycerol (v / v); 0.5% Lactose (w / v) / v); 0.05% Glucose (w / v); 0.05% MgSO 4 ·7H 2 O(w / v); 6.25mmol / LKH 2 PO 4 ;31mmol / LK 2 HPO 4 ·3H 2 O; 25mmol / L (NH 4 ) 2 SO 4 ), shake the bacteria overnight at 30°C and 250rpm.

[0051] purification:

[0052] ① Lysis of bacteria: Take 100ml of the above-mentioned overnight bac...

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Abstract

The invention discloses an expression and purification method of recombinant Tn5 transposase. The expression and purification method comprises the following steps: preparing a recombinant vector comprising Tn5 transposase, wherein the recombinant vector is also provided with a strep tag II tag and a SUMO tag; incuding the expression; and purifying the fusion protein by utilizing the strep tag II and SUMO tag, and obtaining the high-purity Tn5 enzyme protein. By adopting the expression and purification method, the degradation of protein bands can be avoided, and the Tn5 transposase proteins canbe accurately assembled and folded by virtue of the characteristics of the SUMO tag.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing and purifying recombinant Tn5 transposase. Background technique [0002] Transposons are DNA fragments that can move and replicate autonomously in the genome. With the deepening of people's understanding of the results and functions of transposons at the molecular level, many transposons have been transformed into tools for genetic analysis and applied to genes. Functional analysis, gene transformation and gene therapy. Transposase is an important factor to realize the function of transposon. Due to the low activity and low expression of natural transposase, the transposition ability of transposon is not high. [0003] Tn5 transposase is an important tool in the transposase series for high-throughput library construction for next-generation sequencing. The expression and activity of the wild-type Tn5 transposase are low, which hinders the development a...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N9/12
CPCC07K2319/20C07K2319/22C12N9/1241C12N15/62C12N15/70
Inventor 崔伟斌叶国栋陈茂立许剑雄韩大雄郭奇伟肖剑萍蔡逸民杨燕燕李顺杰董康梅朱莎莎张丽芳宋丹
Owner XIAMEN LIFEINT TECH CO LTD
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