Substantially pure reverse transcriptases and methods of prooduction 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 enzym...

AI Technical Summary

Benefits of technology

[0008] In addition to these various chemical treatments a number of physical methods of disruption have been used. These physical methods include osmotic shock, e.g., suspension of the cells in a hypotonic solution in the presence or absence of emulsifiers (Roberts, J. D., and Lieberman, M. W., Biochemistry 18:4499-4505 (1979); Felix, H., Anal. Biochem. 120:211-234 (1982)), drying (Mowshowitz, D. B., Anal. Biochem. 70:94-99 (1976)), bead agitation such as ball milling (Felix, H., Anal. Biochem. 120:211-234 (1982); Cull, M., and McHenry, C. S., Meth. Enzymol. 182:182:147-153 (1990)), temperature shock, e.g., freeze-thaw cycling (Lazzarini, R. A., and Johnson L. D., Nature New Biol. 243:17-20 (1975); Felix, H., Anal, Biochem. 120:211-234 (1982)), sonication (Amos, H., et al., J. Bacteriol. 94:232-240 (1967); Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York, John Wiley & Sons (1993), pp. 4.4.1-4.47) and pressure disruption, e.g., use of a french pressure cell (Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York, John Wiley & Sons (1993), pp. 16.8.6-16.8.8). Other approaches combine these chemical and physical methods of disruption, such as lysozyme treatment followed by sonication or pressure treatment, to maximize cell disruption and protein release (Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York, John Wiley & Sons (1993), pp. 4.4.1-4.47).

[0012] Instead of attempting to remove nucleic acids from preparations of reverse transcriptase enzymes, a more reasonable and successful approach would be to prevent contamination of the enzymes by nucleic acids from the outset in the purification process. Such an approach would be two-pronged: 1) preventing release of nucleic acids from the bacterial cells during permeabilization of the cells to release the enzymes; and 2) preventing contamination of the enzymes during the purification process itself. Furthermore, an optimal method would obviate the need for centrifugation in the process, thus allowing large-scale, and even continuous, production of nucleic acid-free reverse transcriptase enzymes. The present invention provides such methods, and reverse transcriptase enzymes produced by these methods.

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.