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Enzyme stabilization by cationic surfactants

A technology of surfactants and cations, applied in the direction of enzyme stability, enzymes, transferases, etc., can solve problems such as polymerase stability limitations

Inactive Publication Date: 2001-10-03
PROMEGA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Utilization of enzymes such as DNA polymerases is often limited by the stability of the polymerase in solution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Ability of Taq polymerase to amplify DNA segments with and without detergent

[0071] This example describes a PCR-based assay for determining the stability of detergents (ie, surfactants) to polymerases. The conditions specified therein are: the polymerase cannot produce a detectable amplification product in the absence of detergent, but can produce a detectable amplification product in the presence of a stable detergent such as Tween 20.

[0072] Prepare the following reaction mixture:

[0073] 100 μl of 2 mM dNTP mix

[0074] 2ng / μl pGEM luc 10μl

[0075] Primer A (1μg / μl) 10μl

[0076] Primer B (1μg / μl) 10μl

[0077] 100 μl of 10 X Taq buffer

[0078] 25mM MgCl 2 100μl

[0079] Nanopure water 670μl

[0080] Total 1000μl

[0081] pGEM luc (Part#E1541) and 25mM MgCl were purchased from Promega, Madison WI 2 (Part#E1902). The 10X Taq buffer formulation was: 500 mM KCl, 100 mM Tris-Cl (pH 9.0 at 25°C). 10X Taq buffer was prepared by dissolving KCl and Trizm...

Embodiment 2

[0092] Surfactant Screening

[0093] In this example, the surfactant was screened for its ability to stabilize the enzyme. Dissolve the following compounds in nanopure water to a final concentration of 10% (w / v or v / v, depending on whether the substance is solid or liquid): tetradecyltrimethylammonium bromide (Sigma T4762), sulfo Dioctyl succinate (Sigma D-4422), cholic acid (Sigma C-1254 lot 56H0339), taurocholic acid (Sigma T-4009, lot 15H5001), 3-[(3-cholamidopropyl) di Methylamino]-1-propanesulfonic acid inner salt (Sigma C-3023, Lot 86H5022), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonate ( Sigma C-3649, lot 35H5065), cetylpyridinium chloride (Sigma C-9002, lot 77H1047), Tween 20 (SigmaP-1379) and Triton X-100.

[0094] Prepare 10X buffers with each of these surfactant solutions. The 10X buffer composition for each surfactant is: 500mM KCl, 100mM Tris-HCl pH9.0 (at 25°C), 1% surfactant (by adding the above detergent solution at 1 :1...

Embodiment 3

[0109] Screening of other surfactants

[0110] In this example, other surfactants were screened for their ability to stabilize proteins. Prepare a nanopure aqueous solution (10% w / v or v / v) of: N-dodecyl-n,n'-dimethyl-3-ammonium-1-propanesulfonate (Sigma D-4516 , lot 95H5045), Mega 10 (Sigma D-6277, lot 37H5041), N-octadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (Sigma O-8004, lot 44H5006 ), SB 3-10, N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (Sigma T-7763, lot 96H5001), dimethyl di-decabromide Octyl ammonium, Triton X-200 (Sigma X200, lot 75H0989), Triton W-30 (Sigma Chem. Co. W-30, lot 18F0766), Triton X-301 (Sigma X301, lot 13H7706), Triton 770 (Sigma 770, lot 18F0768).

[0111] Add 99 μl aliquots of the master reaction mixture described in Example 1 to separate 0.2 ml tubes, then add 1 μl of 10% surfactant solution to each tube and purify in the absence of detergent 2 μl of Taq polymerase (10 U / μl). Control reactions consisted of tubes ...

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PUM

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Abstract

The present invention provides methods and compositions for protein stabilization, particularly the stabilization of polymerases in aqueous solutions with cationic surfactants. The present invention further provides cationic surfactants, including polyethoxylated amines, that stabilize thermostable and thermolabile enzymes in solution. These surfactants stabilize the activity of various enzymes, including thermostable DNA polymerases, thermolabile DNA polymerases and reverse transcriptases.

Description

field of invention [0001] The present invention relates to the stabilization of proteins, in particular the stabilization of polymerases in aqueous solutions containing cationic surfactants. Background of the invention [0002] In many biochemical and bioengineering processes, enzymes need to be stabilized for long-term storage and utilization. Enzymes have been isolated from thermophilic organisms that are stable to heat denaturation. However, even these highly thermostable enzymes can be inactivated by chemical reagents, proteases, or environmental changes. When working with thermostable enzymes, as well as other enzymes, it is often necessary to simultaneously use several denaturing conditions, including greatly elevated temperatures, aqueous environments containing suboptimal concentrations of cofactors and substrates, and suboptimal concentrations for maximum enzyme stability. optimal level of pH. [0003] Many stabilization techniques are known. These techniques in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12NC12N9/96
CPCC12N9/1252C12N9/1241C12N9/1276C12N9/12C12N9/96
Inventor J·W·舒尔茨F·黄
Owner PROMEGA CORP
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