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Heat-resistant reverse transcriptase mutant

A technology of reverse transcriptase and mutant, applied in the field of thermostable reverse transcriptase mutants, which can solve problems such as poor thermotolerance and inactivation

Active Publication Date: 2019-09-27
TAKARA HOLDINGS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, reverse transcriptases produced by Moloney murine leukemia virus or avian myeloblastic leukemia virus have poor heat resistance, and they are inactivated under such temperature conditions that inhibit RNA secondary structure formation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Example 1: Preparation of Reverse Transcriptase Mutants-1

[0157] (1) Preparation of MMLV reverse transcriptase mutants O1-O3 and P12, P13 (T55G, T55A, T55S, T55D, T55K)

[0158] According to Experimental Method 1-(1), an artificial gene encoding a mutant protein in which threonine at position 55 in the wild-type amino acid sequence of MMLV reverse transcriptase was replaced with glycine was prepared. Using the artificial gene thus obtained, protein expression and purification were performed according to Experimental Method 1-(1). The reverse transcriptase mutant with a threonine to glycine substitution mutation at position 55 was designated "01" as used herein. The reverse transcriptase mutant in which threonine at position 55 was replaced with alanine and the reverse transcriptase mutant in which threonine at position 55 was replaced with serine were named "O2" and "O3", respectively. The reverse transcriptase mutant in which the threonine at position 55 was replaced...

Embodiment 2

[0201] Example 2: Heat resistance evaluation test of reverse transcriptase mutants-1

[0202] The heat resistance of the reverse transcriptase mutants and wild-type reverse transcriptase prepared in Example 1-(1) was tested according to the experimental method 1-(2). The results are shown in Table 1.

[0203] [Table 1]

[0204]

[0205] As shown in Table 1, after heat treatment at 44°C and 50°C for 15 minutes, mutants O1, P12 and C3 in particular had 1.3-4.7 times higher residual activity compared to reverse transcriptase with wild-type amino acid sequence. Mutants O3 and P13 had 1.1-1.3 times higher residual activity compared to wild-type reverse transcriptase after heat treatment at 44°C for 15 minutes.

Embodiment 3

[0206] Example 3: Heat resistance evaluation test of reverse transcriptase mutants-2

[0207] Amino acid substitutions of the present invention were examined in combination with known mutations that have been reported to be involved in thermotolerance or novel mutations that were first discovered by the present invention to be involved in thermotolerance. Specifically, Example 1-(3) and (5), Example 1-(6) and (8), Example 1-(9) and (11), were tested according to Experimental Method 1-(2). Prepared in embodiment 1-(12) and (14), embodiment 1-(15) and (17), embodiment 1-(18) and (20) and embodiment 1-(21) and (22) Thermotolerance of reverse transcriptase mutants. The results are shown in Table 2.

[0208] [Table 2]

[0209]

[0210] As shown in Table 2, it has been reported that any combination of the mutations D1, LT, K2, K3 and K4 involved in thermotolerance with the amino acid substitution C3 of the present invention increases the residual activity after heat treatment ...

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Abstract

Provided are: a reverse transcriptase mutant including an amino acid mutation at a position corresponding to position 55 of the amino acid sequence of wild-type reverse transcriptase derived from the Moloney murine leukemia virus, wherein the reverse transcriptase mutant is characterized in that the amino acid mutation is a substitution from threonine to another amino acid, and the other amino acid is selected from the group consisting of amino acids having a nonpolar aliphatic side chain and amino acids having a polar acidic functional group side chain; a nucleic acid that encodes the mutant; a method for producing the mutant and the nucleic acid that encodes the mutant; a method for synthesizing cDNA in which the mutant is used; and a composition and kit including the mutant.

Description

technical field [0001] The present invention relates to thermostable reverse transcriptase mutants. Furthermore, the present invention relates to methods for increasing the heat resistance of existing reverse transcriptases and methods for generating thermostable reverse transcriptase mutants. Background technique [0002] Reverse transcriptase (RTase) usually has RNA-dependent DNA polymerase activity, which is the activity of synthesizing cDNA from template RNA, and ribonuclease H (RNase H) activity, which is the activity of degrading the RNA strand of RNA / DNA hybrids ). [0003] Since reverse transcriptases have RNA-dependent DNA polymerase activity, they can be used for sequencing mRNA (which directly reflects the amino acid sequence of proteins expressed in living organisms), construction of cDNA libraries, RT-PCR, and the like. For these purposes, reverse transcriptases produced by Moloney murine leukemia virus or avian myeloblastic leukemia virus are often used. [...

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
CPCC12N15/09C12N9/1276C12N15/52C12Q1/686C12Y207/07049C12Q2521/107
Inventor 石川一彦上森隆司高津成彰
Owner TAKARA HOLDINGS
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