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Low-temperature high-yield single subunit RNA (ribonucleic acid) polymerase, purification method and application

An RNA polymerase and purification method technology, applied in the field of in vitro RNA synthesis research and production, can solve problems such as easy degradation, achieve the effects of saving cost, ensuring RNA yield, and reducing degradation risk

Active Publication Date: 2020-11-24
RNASYN BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the problem that RNA is easily degraded at high temperature, and develops a technique for RNA transcription and synthesis using low-temperature single-subunit RNA polymerase VSW3 RNA polymerase at low temperature, providing low-temperature high-yield single-subunit RNA polymerase, its purification Method and application in RNA synthesis

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  • Low-temperature high-yield single subunit RNA (ribonucleic acid) polymerase, purification method and application
  • Low-temperature high-yield single subunit RNA (ribonucleic acid) polymerase, purification method and application
  • Low-temperature high-yield single subunit RNA (ribonucleic acid) polymerase, purification method and application

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Embodiment 1

[0034] Example 1: VSW3 RNA polymerase gene synthesis and protein expression purification

[0035] The expression and purification of low-temperature single-subunit RNA polymerase VSW3 RNA polymerase is characterized in that VSW3 RNA polymerase comes from low-temperature bacteriophage, and normal expression and purification methods are easy to form inclusion bodies, and low-temperature expression methods must be strictly used. The production process of expression and purification of VSW3 RNA polymerase includes the following steps:

[0036] (1) Synthesize the VSW3 RNA polymerase gene, and construct the VSW3 RNA polymerase expression vector plasmid;

[0037] Gene synthesis RNA polymerase, the base sequence of the VSW3 RNA polymerase is shown in SEQ ID NO.2 of the sequence listing;

[0038] The amino acid sequence of the VSW3 RNA polymerase is shown in SEQ ID NO.3 in the sequence table, and it is cloned into the prokaryotic expression vector pCold by seamless cloning, and the re...

Embodiment 2

[0049] Example 2: In vitro RNA synthesis by VSW3 RNA polymerase

[0050] In the present invention, we first obtained the transcription promoter specifically recognized by VSW3 RNA polymerase through software prediction and experimental technology, as shown in SEQ ID NO. The ability to synthesize RNA efficiently, which is conducive to the stability of the RNA synthesis process. For future industrial application, we identified the RNA synthesis efficiency of VSW3 RNA polymerase at each enzyme concentration at each temperature (see figure 2 , image 3 , Figure 4 ) and compare it with T7RP (see Figure 5 ),Depend on figure 2 It can be seen that VSW3 RP can synthesize RNA at 4 °C to 30 °C, and the RNA production increases with the increase of temperature, reaching the peak at 25 °C, indicating that the most suitable RNA transcription synthesis temperature of VSW3RP is 25 °C, which is determined by image 3 It can be seen that when the heavy concentration of the enzyme is 0....

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Abstract

The invention discloses a low-temperature high-yield single subunit RNA (ribonucleic acid) polymerase, namely VSW3RNA polymerase, which is applied to efficient synthesizing of RNA at low temperature.The single subunit RNA polymerase is RNA polymerase expressed by a low-temperature phage VSW-3, and can be used for transcribing and synthesizing RNA by taking DNA (deoxyribonucleic acid) as a template within the temperature range of 4-30 DEG C; when the temperature is 25 DEG C, the efficiency of synthesizing RNA by the VSW3RNA polymerase is the highest, and the yield of the VSW3RNA polymerase even exceeds that of T7RP which is widely applied to RNA in-vitro transcription synthesis at present. A base sequence for coding the enzyme is shown in a sequence table SEQ ID NO. 1, an amino acid sequence of the enzyme is shown in a sequence table SEQ ID NO. 2, and a specific promoter sequence recognized by the enzyme is shown in a sequence table SEQ ID NO. 5. The VSW3RNA polymerase is found to be afirst single subunit RNA polymerase which has RNA synthesis capability comparable to or even superior to that of T7RP, and the most suitable temperature for transcribing and synthesizing RNA is 25 DEG C and is 12 DEG C lower than the transcription temperature of 37 DEG C of T7RP, thus an important enzyme tool is provided for efficiently synthesizing RNA at low temperature, and the increasing requirements of scientific research and medical markets on RNA products can be met.

Description

technical field [0001] The invention belongs to the field of in vitro RNA synthesis research and production, and in particular relates to a low-temperature high-yield single-subunit RNA polymerase and a purification method and application. Background technique [0002] Due to the continuous expansion of RNA research and application development, high requirements are put forward for the quality and quantity of RNA synthesis in vitro. High-quality RNA synthesis in vitro is currently the biggest bottleneck in RNA research and application. Due to the characteristics of RNA polymerase transcription and synthesis of RNA, the enzymatic synthesis of RNA is far from being as efficient as DNA synthesis, while chemical synthesis of RNA is limited to small RNAs (several to dozens of nucleotides in length) and some complex nucleotides When modifying RNA, the cost of chemical synthesis rises rapidly as the length of RNA increases (Hogrefe R I, 2013; E. Paredes, 2017); while protein-coding...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/70C12P19/34
CPCC12N9/22C12N15/70C12P19/34C07K2319/21C07K2319/20Y02A50/30
Inventor 朱斌夏恒
Owner RNASYN BIOTECH CO LTD
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