Substantially pure reverse transcriptases and methods of production thereof

Abstract

The present invention provides substantially pure reverse transcriptases, which are preferably substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in synthesizing, amplifying or sequencing nucleic acid molecules, including through the use of the polymerase chain reaction, particularly RT-PCR.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation of U.S. application Ser. No. 10 / 640,662, filed Aug. 14, 2003, which is a divisional of U.S. application Ser. No. 09 / 533,548, filed Mar. 23, 2000, now U.S. Pat. No. 6,630,333, which claims the benefit of U.S. Provisional Patent Application No. 60 / 126,050, filed Mar. 23, 1999, the disclosures of each of which are incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention is in the fields of molecular biology, protein chemistry and protein purification. Specifically, the invention provides compositions comprising reverse transcriptases (RTs) and methods for the production of such reverse transcriptase enzymes. Such methods provide for reverse transcriptases that are substantially free from contamination by nucleic acids and other unwanted materials or proteins. Compositions comprising the reverse transcriptase enzymes of ...

AI Technical Summary

Benefits of technology

"The present invention provides methods for producing reverse transcriptases that are free from contamination by nucleic acids and other unwanted materials. These reverse transcriptases can be used in various applications such as synthesis, amplification, and sequencing of nucleic acids. The invention also provides compositions comprising these reverse transcriptases. The invention is based on the discovery that certain techniques for disrupting bacterial cells can lead to the release of intracellular proteins, which can then be purified and used for various applications. The invention provides methods for disrupting bacterial cells using chemical and physical methods, as well as combinations of both. The invention also includes the use of detergents and proteins or protamines to enhance the effect of the treatment. Overall, the invention provides a way to produce high-quality reverse transcriptases for various applications."

Problems solved by technology

However, these methods possess distinct disadvantages as well.

This need for centrifugation limits the batch size capable of being processed in a single preparation to that of available centrifuge space; thus, large production-scale preparations are impracticable if not impossible.

Furthermore, physical methods, and many chemical techniques, typically result in the release from the cells not only of the desired intracellular proteins, but also of undesired nucleic acids and membrane lipids (the latter particularly resulting when organic solvents are used).

These undesirable cellular components also complicate the subsequent processes for purification of the desired proteins, as they increase the viscosity of the extracts (Sambrook, J., et al., in: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), pp.

One problem associated with these approaches is that the enzyme preparations are typically contaminated with nucleic acids (e.g., RNA and DNA).

Since reverse transcriptase enzymes are routinely used in techniques of amplification and synthesis of nucleic acid molecules (e.g., the Polymerase Chain Reaction (PCR), particularly RT-PCR) the presence of contaminating DNA or RNA in the enzyme preparations is a significant problem since it can give rise to spurious amplification or synthesis results.