Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1997-05-07
1999-08-10
Weber, Jon P.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
435188, 435193, 435 912, C12N 912, C12N 910, C12N 996, C12P 1934
Patent
active
059358343
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel reverse transcriptase composition having improved storage stability. More particularly, the present invention is concerned with a reverse transcriptase composition having improved storage stability, comprising a reverse transcriptase, an effective stabilizing amount of at least one organic stabilizing reagent selected from the group consisting of trehalose and a nucleic acid containing a transcriptional initiation site recognizable by the reverse transcriptase, and an effective stabilizing amount of a metal salt capable of producing bivalent positive ions in an aqueous solution of the metal salt. The present invention is also concerned with a method for improving storage stability of a reverse transcriptase, which comprises adding to a reverse transcriptase an effective stabilizing amount of at least one organic stabilizing reagent selected from the group consisting of trehalose and a nucleic acid containing a transcriptional initiation site recognizable by the reverse transcriptase, and an effective stabilizing amount of a metal salt capable of producing bivalent positive ions in an aqueous solution of the metal salt.
Conventionally, for maintaining the stability of a reverse transcriptase, it has been necessary to store the reverse transcriptase at a temperature as low as -20 to -80.degree. C. According to the present invention, however, a reverse transcriptase can be stably stored in the form of the above-mentioned composition for a prolonged period of time at a temperature up to at least 4.degree. C. without suffering denaturation of the reverse transcriptase. Further, by virtue of such a relatively high temperature usable for stable storage, the viscosity of the reverse transcriptase composition of the present invention can be advantageously maintained at a low level, so that it becomes possible to accurately dispense the composition by a quantity corresponding to a desired enzyme activity, thereby achieving high reproducibility in experiments using the reverse transcriptase.
The reverse transcriptase composition of the present invention, which has improved storage stability, can be advantageously used in the fields of genetic engineering and virology, with respect to various techniques in which the reverse transcriptases are employed. Particularly, in a method generally employed for the determination of a virus, in which a reverse transcriptase activity is used as an index, the transcriptase composition of the present invention can be advantageously used as a standard substance for determining the amount of virus.
2. Prior Art
Reverse transcriptase is an enzyme having the activity of synthesizing from an RNA template a DNA having a nucleotide sequence complementary to the RNA. This enzyme was discovered in the recent development of molecular biology. Examples of reverse transcriptases include those which are derived from avian myeloblast virus (AMV), Ras associated virus type 2 (RAV-2), mouse molony murine leukemia virus (M-MuLV) and human immunodefficiency virus type 1 (HIV-1). These reverse transcriptases are commercially readily available.
Further, on a laboratory scale, a reverse transcriptase can be easily obtained by directly separating the enzyme from a virus. For example, a reverse transcriptase can be easily prepared by treating particles of retroviruses, such as human immunodefficiency virus Type 2 (HIV-2), human T-cell leukemia virus type 1 (HTLV-1), simian AIDS virus (SIV), feline AIDS virus (FIV) and Rous related avian sarcoma virus, with a surfactant, such as Triton X-100 (manufactured and sold by Sigma Chemical Company, USA). The above-mentioned reverse transcriptases have been widely used in the synthesis of a cDNA from a messenger RNA.
Reverse transcriptase is also used in the PCR (polymerase chain reaction) method which is recently a most widely used nucleic acid amplification technique. Specifically, the PCR method is a technique to amplify at least one type of nucleic acid using a therm
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G.L. Rowley, et al., "Stabilization and Activation of Recombinant Human Immunodeficiency Virus-1 Reverse Transcriptase-P66", pp. 673-679, Biochemical and Biophysical Research Communication, Vol. 167, No. 2, Mar. 16, 1990.
Asahi Kasei Kogyo Kabushiki Kaisha
Weber Jon P.
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