Recombinant DNA encoding a reverse transcriptase derived...

Details Recombinant DNA encoding a reverse transcriptase derived... Recombinant DNA encoding a reverse transcriptase derived...

1999-09-15

2003-07-15

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S320100, C435S252330, C435S252300, C435S069100, C435S325000, C536S023100, C536S023200

Reexamination Certificate

active

06593120

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to recombinant proteins, particularly to viral reverse transcriptase enzymes produced by recombinant DNA technology, and specifically relates to reverse transcriptase derived from Moloney Murine Leukemia virus (MMLV) that is expressed from recombinant DNA in a bacterial host cell and that includes multiple histidine residues.
BACKGROUND OF THE INVENTION
Retroviruses are a group of viruses whose genetic material consists of single-stranded RNA. Following adsorption and entry of retroviral RNA into the host cell, the viral RNA is used as a template for synthesis of a complementary DNA (cDNA) strand. The cDNA is then made double-stranded through the action of an enzyme having DNA polymerase activity; this double-stranded DNA integrates into the host genome. The RNA-directed DNA polymerase activity responsible for the synthesis of cDNA from the viral RNA template is commonly called reverse transcriptase (“RT”).
A number of retroviruses have been implicated as the causative agents of various cancers, and other diseases. A retrovirus, human immunodeficiency virus-1 (HIV-1), is the causal agent of acquired immunodeficiency syndrome (AIDS). Also, reverse transcriptase enzymes have become important reagents in molecular biology because of their ability to make cDNA from almost any RNA template. Reverse transcriptase is commonly used to make nucleic acids for hybridization probes and to convert single-stranded RNA into a double-stranded DNA for subsequent cloning and expression.
Reverse transcriptases have been used as a component of transcription-based amplification systems. These systems amplify RNA and DNA target sequences up to 1-trillion fold and have been previously described in detail (see Burg et al., PCT Patent Application WO 89/01050 (1988) and U.S. Pat. No. 5,437,990; Gingeras et al., PCT Patent Application WO 88/10315 (1988); Gingeras et al., European Patent Application EPO 0373960 (1989); Davey & Malek, European Patent Application EPO 0329822 (1988); Malek & Davey, PCT Patent Application WO 91/02814 (1989); Davey et al., U.S. Pat. Nos. 5,409,818 and 5,554,517; Davey et al., U.S. Pat. No. 5,466,586; Malek et al., U.S. Pat. No. 5,130,238; Kacian et al., European Patent Application EPO 0408295 A2 (1990) and U.S. Pat. Nos. 5, 399,491, 5,480,784, 5,824,518, 5,888,779 and 5,554,516), the experimental details of which are hereby incorporated by reference herein.
Some transcription-based amplification methods are especially convenient because the amplification reactions are isothermal. These systems are particularly suited for diagnostic tests in clinical laboratories. For example, detection of pathogens causing infectious diseases and gene sequences associated with cancers or genetic diseases are important uses of such tests. Reverse transcriptases are also employed as an initial step in some protocols in which the polymerase chain reaction (PCR) amplifies an RNA target (see Malek et al., U.S. Pat. No. 5,130,238 (1992); and Mocharla et al., 1990,
Gene
99:271-275). In RT-PCR procedures, the reverse transcriptase is used to make an initial cDNA copy of the RNA target, which is then amplified by successive rounds of DNA replication using PCR.
Retroviral reverse transcriptases have three enzymatic activities: RNA-directed DNA polymerase activity, DNA-directed DNA polymerase activity, and RNAse H activity (Verma I., 1977,
Biochim. Biophys. Acta
473: 1-38). The latter activity specifically degrades RNA contained in an RNA:DNA duplex. RNA strand degradation in RNA:DNA intermediates by RNAse H is an important component of some transcription-based amplification systems. RNAse H-mediated degradation of RNA is distinguishable from unwanted degradation due to contaminating nucleases, which interferes with amplification.
A disadvantage of the transcription-based amplification systems is their sensitivity to even trace amounts of nucleases. Because a number of important diseases may yield samples containing very few target nucleic acid molecules, detection of small amounts of target is often crucial for an accurate and timely diagnosis. Indeed, target amplification methods are most valuable when the number of target molecules is low. With low levels of input target nucleic acids, unwanted degradation of RNA targets, or of RNA or DNA reaction intermediates, can lead to amplification failures and consequent inaccurate diagnosis. Ribonuclease contamination is also a problem in RT-PCR reactions, because RNA target loss can result in amplification failure.
Ribonucleases are relatively ubiquitous and occur in high concentrations in a variety of biological materials, including in retrovirus preparations and cells commonly used to express recombinant proteins. Ribonucleases (“RNases”) frequently contaminate RT preparations from a variety of sources and can interfere with cDNA synthesis, probe preparation and other uses besides target amplification. Often, an RNase inhibitor is added to a reaction mixture to minimize the deleterious effects of this contamination (e.g., see Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual
(2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), pp. 8.11-8.13).
Commonly-used substances that inhibit or inactivate RNases, including detergents, chaotropes, organics, metals, proteases and metals are inappropriate for use in target amplification systems because they also inhibit the enzymes used for amplification. RNase-inhibiting proteins, e.g. human placental RNase inhibitor (Blackburn et al., 1977,
J. Biol. Chem.
252: 5904) or rat liver RNase inhibitor (Gribnau et al., 1969,
Arch. Biochem. Biophys.
130: 48-52), may be unstable, are expensive, and can contribute additional interfering substances, such as nucleic acids and RNases that are not inhibited by the inhibitor.
In addition to nucleases, traces of other enzymes, nucleic acids, and certain buffer salts may interfere with amplification reactions. While these substances are merely undesirable for many uses of RT, because of the nature of the amplification reaction, it is critical that RT preparation contain as little contaminating substances as possible.
Reverse transcriptases have been isolated and purified from various sources. When RT is isolated directly from virus particles, cells or tissues, high costs may preclude their widespread use in diagnostic tests (e.g., see Kacian et al., 1971,
Biochim. Biophys. Acta
46: 365-83; Yang et al., 1972,
Biochem. Biophys. Res. Comm.
47: 505-11; Gerard, et al., 1975,
J. Virol.
15: 785-97; Liu et al., 1977,
Arch. Virol.
55 187-200; Kato et al., 1984,
J. Virol. Methods
9: 325-39; Luke et al., 1990,
Biochem.
29: 1764-69 and Le Grice et al., 1991,
J. Virol.
65: 7004-07). Also, these methods have not assured removal of inhibitors or contaminants that interfere with target amplification reactions. Another important consideration for a variety of reverse transcriptase uses is the RT-associated RNase H activity. The amount of RNase H activity and coordination of RNase H activity with the RNA- and DNA-dependent RT activities are important features that affect an enzyme's utility for various purposes, including transcription-based amplification systems. Too much or too little activity, inappropriate activity (e.g., non-specific RNase activity), or poorly-coordinated RNase H and DNA synthesis activities can all lead to reduced performance. Proper balance of the synthetic and degradative activities must be maintained; this is not only a function of the particular RT used, but also depends on the ability of a purification protocol to remove inappropriate RNase and/or DNase activities.
Reverse transcriptase genes have been cloned and expressed in bacterial hosts. Attempts to clone and express in
E. coli
a gene encoding reverse transcriptase from avian myeloblastosis virus (AMV-RT) did not lead to production of significant amounts of purified enzyme. This is probably because AMV-RT consists of two polypeptide chains (&agr; and &bgr;) which must form a dimer and undergo specific post-translat

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