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RNA polymerase mutant with improved functions

一种RNA聚合酶、聚合酶的技术，应用在功能改善的RNA聚合酶突变体领域，能够解决设计困难等问题，达到比活性提高、缩短转录反应时间的效果

Active Publication Date: 2011-09-07
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNA tends to form complex higher-order structures at low temperatures, which makes it difficult to design primers that can be detected with high sensitivity in the TRC method

Method used

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  • RNA polymerase mutant with improved functions
  • RNA polymerase mutant with improved functions
  • RNA polymerase mutant with improved functions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1T7

[0069] The cloning of embodiment 1T7 RNA polymerase gene

[0070] Cloning of the T7 RNA polymerase gene was carried out in the following manner.

[0071] (1) Using the T7 phage genomic DNA genomic library (manufactured by SIGMA) as a template plasmid, the T7 RNA polymerase gene was amplified by PCR using the following reagent composition and reaction conditions, divided into the first half and the second half. Furthermore, the following primers were used for the synthetic DNA primers in the reagent composition: primer FF (SEQ ID NO: 1) and primer FR (SEQ ID NO: 2) were used for the amplification of the first half, and primers for the amplification of the second half were Primer RF (SEQ ID NO: 3) and primer RR (SEQ ID NO: 4).

[0072] (Reagent composition) (Total reaction volume: 100μL)

[0073] 200pM synthetic DNA primers each

[0074] 100ng template plasmid

[0075] 0.2mM dNTPs

[0076] 0.025 units / μL TaqDNA polymerase

[0077] (TaKaRa Ex Taq (trade name), manufactured ...

Embodiment 2

[0086] The making of embodiment 2 expression vector

[0087] With the pTrc99A-T7RNApol ( figure 1 ) as a template, the DNA fragment containing the T7 RNA polymerase gene was amplified by PCR method, and combined with the pCDF2 plasmid ( figure 2 )connect.

[0088] (1) with pTrc99A-T7RNApol ( figure 1 ) as a template plasmid, the PCR reaction was carried out using the following reagent composition and reaction conditions.

[0089] (Reagent composition) (Total reaction volume: 100μL)

[0090] 200pM Primer pTrcF (SEQ ID NO: 7)

[0091] 200pM Primer pTrcR (SEQ ID NO: 8)

[0092] 100ng template plasmid

[0093] 0.2mM dNTPs

[0094] 0.025 units / μL TaqDNA polymerase

[0095] (TaKaRa Ex Taq (trade name), manufactured by TAKARA Bio)

[0096] Enzyme with buffer

[0097] (Reaction conditions)

[0098] Using a thermal cycler (manufactured by Perkin-Elmer), after heating at 94° C. for 2 minutes, a temperature cycle of 94° C. for 1 minute; 58° C. for 30 seconds; and 72° C. for 2...

Embodiment 3

[0117] Example 3 Production of Mutant Library (Part 1)

[0118] According to the following process, the pCDF2-T7RNAPHis plasmid ( Figure 4 ) The T7 RNA polymerase gene introduced a mutation.

[0119] (1) with pCDF2-T7RNAPHis ( Figure 4 ) as a template plasmid, the error-prone PCR reaction was carried out using the following reagent composition and reaction conditions.

[0120] (Reagent composition) (Total reaction volume: 100μL)

[0121] 0.1~0.3mM (as needed) MnCl 2

[0122] 200pM primer pTrcFs (SEQ ID NO: 15)

[0123] 200pM primer pTrcRs (SEQ ID NO: 16)

[0124] 100ng template plasmid

[0125] 0.2mM dATP

[0126] 0.2mM dGTP

[0127] lmM dCTP

[0128] lmM dTTP

[0129] 2mM MgCl 2

[0130] 0.01 unit / µL Taq DNA polymerase (GoTaq (trade name), manufactured by Promega)

[0131] Enzymes come with Mg-free buffer

[0132] (Reaction conditions)

[0133] Using a thermal cycler (manufactured by Perkin-Elmer), after heating at 94° C. for 2 minutes, the temperature cycle...

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Abstract

T7 RNA polymerase mutant characterized in that amino acid residues corresponding to at least one selected from a group comprising at least glutamine at position-786, lysine at position-179 and valine at position-685 in the amino acid sequence configuring a wild-type T7 RNA polymerase that is represented in Sequence No. 6 have been substituted with another amino acid and thermal stability and / or specific activity have been improved compared to a wild-type T7-like bacteriophage RNA polymerase.

Description

technical field [0001] The present invention relates to: an RNA polymerase having improved functions by introducing a mutation into a part of the amino acid sequence of a wild-type RNA polymerase, in particular an RNA polymerase having improved thermostability and / or specific activity, encoding the RNA polymerase The gene, the production method of the RNA polymerase, and the method of producing RNA using the RNA polymerase. Background technique [0002] The present invention relates to mutant RNA polymerase obtained from bacteriophage, especially T7 RNA polymerase, which has improved stability and / or specific activity under high temperature conditions compared with wild type. As phages capable of infecting Escherichia coli, there are T3, T7, W31, H, Y, A1, croC21, C22 and C23, among them, the RNA polymerase encoded by T7 phage is T7 RNA polymerase. [0003] As a characteristic of T7 RNA polymerase, first, it has high selectivity for promoter sequences. T7 RNA polymerase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12N9/12
CPCC12N9/1247C12Q2521/119
Inventor 大江正刚佐藤宽井出辉彦
Owner TOSOH CORP
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