Reverse transcriptase having improved thermostability
Inventors
Park, Han Oh • Yang, Sung Jun • Joo, Sung Mo • Hwang, Byoung Oh
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Assignees
Member
BioneerBioneer is a biotechnology company specializing in molecular biology, offering solutions in nucleic acid synthesis, molecular diagnostics, protein production, and laboratory automation. The company's expertise covers oligonucleotide chemistry, gene synthesis, sample preparation, molecular diagnostics, and analytical services such as mass spectrometry and sequencing, serving clinical, research, and industrial sectors globally.
Bioneer is a biotechnology company specializing in molecular biology, offering solutions in nucleic acid synthesis, molecular diagnostics, protein production, and laboratory automation. The company's expertise covers oligonucleotide chemistry, gene synthesis, sample preparation, molecular diagnostics, and analytical services such as mass spectrometry and sequencing, serving clinical, research, and industrial sectors globally.
Publication Number
US-9534210-B2
Publication Date
2017-01-03
Expiration Date
Abstract
The present invention relates to a reverse transcriptase having improved thermostability, more precisely a mutant reverse transcriptase with improved thermostability by substitution of one or more amino acids selected from the group consisting of the 63rd glutamine (Q63), the 264th lysine (K264), the 295th lysine (K295), the 306th threonine (T306), the 346th glutamic acid (E346), the 408th proline (P408), the 438th histidine (H438), and the 454th asparagin (N454) of the amino acid sequence of M-MLV originated reverse transcriptase represented by SEQ. ID. NO: 1 with other amino acids. The mutant reverse transcriptase of the present invention demonstrates excellent thermostability, compared with the wild type reverse transcriptase. Therefore, it is advantageous to obtain the target cDNA with stable reverse transcription activity even in the presence of RNA that can form the stable secondary structure at a high temperature.
Core Innovation
The invention relates to a reverse transcriptase derived from an M-MLV originated reverse transcriptase of SEQ ID NO: 1 that has improved thermostability. The improved thermostability is obtained by substitution of the 306th threonine with leucine (T306L), and in some embodiments by additional amino acid substitutions at selected positions.
Mutant reverse transcriptases are disclosed in which one or more residues including Q63, K264, K295, T306, E346, P408, H438, and N454 are substituted. Examples of substituted residues include Q63L, K264L, K295Q, T306L, E346M, P408E, H438Y, and N454F, and preferred mutant types are associated with mutant sequences designated as SEQ ID NO: 2–9.
The reverse transcriptase mutants maintain reverse transcription activity at elevated temperatures and produce stable cDNA even when RNA forms stable secondary structure. The disclosed data emphasize that the T306L mutant, and also K295Q and P408E, show especially improved thermostability for high-temperature reverse transcription.
Claims Coverage
The document includes two independent claims covering thermostable M-MLV originated reverse transcriptase variants, defined by specific amino-acid substitutions, and their use in genes, expression vectors, transformants, and reverse transcription kits. The claim set centers on at least one primary inventive feature (T306L) with optional additional substitutions.
T306L thermostable M-MLV originated reverse transcriptase
A reverse transcriptase having improved thermostability by substitution of the 306th threonine with leucine (T306L) of the amino acid sequence of M-MLV originated reverse transcriptase of SEQ ID NO: 1.
T306L plus selected additional substitutions for improved thermostability
A reverse transcriptase having improved thermostability by substitution of the 306th threonine with leucine (T306L) of the amino acid sequence of M-MLV originated reverse transcriptase of SEQ ID NO: 1, wherein the reverse transcriptase further comprises one or more amino acid substitutions selected from the group consisting of substitution of the 63rd glutamine with leucine (Q63L), substitution of the 264th lysine with leucine (K264L), substitution of the 295th lysine with glutamine (K295Q), substitution of the 346th glutamic acid with methionine (E346M), substitution of the 408th proline with glutamic acid (P408E), substitution of the 438th histidine with tyrosine (H438Y), and substitution of the 454th asparagin with phenylalanine (N454F).
Overall, the claims define thermostable reverse transcriptase variants based on an M-MLV originated SEQ ID NO: 1 sequence with a central T306L substitution, optionally combined with additional specified substitutions at Q63, K264, K295, E346, P408, H438, and N454 to further enhance thermostability.
Stated Advantages
Improved thermostability of an M-MLV originated reverse transcriptase.
Higher reverse transcription activity at 60°C to 70°C than the wild type M-MLV reverse transcriptase.
Stable cDNA production under conditions described as high-temperature reverse transcription, including when RNA forms stable secondary structure.
Documented Applications
Reverse transcription kits that include the thermostable reverse transcriptase.
Genes encoding the thermostable reverse transcriptase, expression vectors containing the gene, and transformants transformed with the expression vector.
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