Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-06-22
2001-06-05
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S188000
Reexamination Certificate
active
06242235
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to protein stabilization, particularly the stabilization of polymerases in aqueous solutions containing cationic surfactants.
BACKGROUND OF THE INVENTION
Stabilization of enzymes is necessary for the long term storage and utilization in many biochemical and biotechnological processes. Enzymes have been isolated from thermophilic organisms which are stable to denaturation by heat. However, even these highly thermostable enzymes may be inactivated by chemical agents, proteases, or environmental modifications. The utilization of thermostable and other enzymes often requires the concomitant use of denaturing conditions including highly elevated temperatures, aqueous environments with sub-optimal concentrations of cofactors and substrates, and a pH that is suboptimal for maximum enzyme stability.
Many stabilization techniques are known. These techniques include immobilization of the enzyme on solid substrates, chemical modification of the enzyme, genetic engineering of the enzyme and the addition of stabilizing additives. Surfactants are one group of additives that have been shown to stabilize enzymes. Surfactants are surface active compounds that stabilize the interface between the active form of the enzyme, and the liquid environment in which they are contained.
For example, non-ionic detergents have been variously shown to increase the solution stability of various proteins with enzymatic activity (e.g., cAMP-dependent protein kinase, tyrosine hydroxylase, nitric oxide synthase, tryptophan hydroxylase and a sweet potato beta-amylase). Additionally, non-ionic detergents such as TRITON X-100 and Tween 20 have been shown to stabilize the activity of DNA polymerases (See, e.g., Biochem., 14: 789-95 [1975]). European Patent Application 776,970 A1, incorporated herein by reference, discloses the use of non-ionic detergents including polyoxyethylated sorbitan monolaurate (Tween 20) and ethoxylated alky phenol (NP-40) to stabilize the activity of Taq thermostable DNA polymerase.
Low concentrations of the anionic detergent sodium dodecyl sulfate (SDS) have been shown to stabilize enzyme activity. However, due to the possibility of cooperative binding if the optimal concentration of SDS is exceeded in solution, the use of SDS in protein stabilization is limited. It is known, however, that many cationic detergents bind less strongly to proteins than strong anionic detergents such as SDS (See e.g., Nozaki et.al., J. Biol. Chem., 249:4452-59 [1974]). Furthermore, most proteins have fewer cationic binding sites than anionic binding sites.
The utility of enzymes such as DNA polymerases often is limited by the stability of the polymerase in solution. Thus, there is need for additives which improve the stability of enzymes in solution, particularly those additives which improve stability as well as avoid the drawbacks of currently used surfactants.
SUMMARY OF THE INVENTION
The present invention relates to protein stabilization, particularly the stabilization of polymerases in aqueous solutions with cationic surfactants.
In some embodiments, the present invention provides a composition comprising a mixture of a protein having enzymatic activity and a cationic surfactant. The present invention is not limited to any particular enzyme. Indeed, the stabilization of a variety of enzymes is contemplated. In some preferred embodiments, the protein is a polymerase (e.g.,
E. coli
DNA polymerase I, Taq polymerase, Tne polymerase, Tth polymerase, T4 DNA polymerase, RNA polymerase II, SP6 RNA polymerase, T7 RNA polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, etc.). In other embodiments, the enzyme is preferably a kinase, phosphorylase, or phosphatase (e.g., calf intestinal phosphatase).
Likewise, the present invention is not limited to a particular cationic surfactant. Indeed, a variety of cationic surfactants are contemplated. In some embodiments, the cationic surfactant has a Hydrophile-Lipophile Balance (HLB) index number of about 10 to 17. In some preferred embodiments, the cationic surfactant has a HLB index number of about 11 to 16. In other embodiments, the cationic surfactant is a polyethoxylated amine. In some particularly preferred embodiments, the polyethoxylated amine has the following structure:
In some embodiments, z is an integer from about 15 to 20, most preferably 18. In other embodiments, x+y has an average value of about 5 to 15 so that the HLB index number is from about 11 to 16. In some preferred embodiments, x+y has an average value of 5 or 15. In some embodiments, the nitrogen may be substituted with a phosphorous, sulphur or arsenic radical. In still other embodiments, the cationic surfactant is present in the solution or mixture at a concentration of about 0.0005 to 1.0% by volume.
In some embodiments, the mixture or solution includes a buffering reagent. The present invention is not limited to a particular buffering reagent. Indeed, a variety of buffering reagents are contemplated. In some embodiments, the buffering reagent is preferably a MOPS, HEPES, or Tris buffer. In other embodiments, the concentration of the buffer in the solution is from about 10 mM to 70 mM. In some embodiments, the pH is from about 7.0 to 9.2.
In other embodiments, the solution or mixture includes a monovalent salt and/or a divalent salt. The present invention is not limited to any particular salt. Indeed, a variety of salts are contemplated, including, but not limited to, NaCl, KCl, MgCl
2
, and CaCl
2
. In some embodiments, the divalent cation is present at a concentration of about 0.1 to 10 mM. In other embodiments, the monovalent cation is present at a concentration of about 1 to 100 mM.
In still further embodiments, the solution or mixture includes a chelator and/or a reducing agent. The present invention is not limited to particular chelators and reducing agents. Indeed, a variety of chelators and reducing agents are contemplated. Preferred chelating agents include, but are not limited to, EDTA and EGTA. Preferred reducing agents include, but are not limited to, dithiothreitol and &bgr;-mercaptoethanol. In some embodiments, the chelating agent is present at a concentration of about 0.01 to 10 mM. In other embodiments, the reducing agent is present at a concentration of about 0.1 to 20 mM.
In some embodiments, the present invention provides methods for stabilizing proteins with enzymatic activity. In some embodiments, a protein with enzymatic activity (e.g., a polymerase, kinase, phosphatase, or phosphorylase) and a cationic surfactant are provided. In some preferred embodiments, the cationic surfactant has an HLB index number of from about 10 to 17. In particularly preferred embodiments, the cationic surfactant is a polyethoxylated amine, as described above. In other embodiments, the protein with enzymatic activity and cationic surfactant are combined so that the activity of the enzyme is stabilized as compared to the activity of the enzyme in the absence of the cationic surfactant.
Definitions
To facilitate an understanding of the invention, a number of terms are defined below.
As used herein, the term “enzyme” refers to molecules or molecule aggregates that are responsible for catalyzing chemical and biological reactions. Such molecules are typically proteins, but can also comprise short peptides, RNAs, ribozymes, antibodies, and other molecules. A molecule that catalyzes chemical and biological reactions is referred to as “having enzyme activity” or “having catalytic activity.”
As used herein, the terms “stabilization,” “stabilizing,” and “stabilized,” when used in reference to enzyme activity refer to the ability of a material to maintain, enhance, or otherwise inhibit the decline or loss of the activity of an enzyme, often as measured over time (i.e., in the presence of a stabilizer, an enzyme retains its activity for a longer time period than the enzyme in the absence of the stabilizer). “Stabilization of enzyme activity” also refers to the ability of a material to maintain the activity of a
Huang Fen
Shultz John W.
Promega Corp.
Weber Jon P.
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