Cell lysis composition, methods of use, apparatus, and kit

a cell lysis and composition technology, applied in the field of cell lysis composition, methods for extracting and purifying proteins, apparatuses and kits, can solve the problems of additional costly and time-consuming purification steps for the removal of contaminants

Inactive Publication Date: 2007-05-10
PROMEGA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0069] contacting each source of cells with the composition in an amount effec...

Problems solved by technology

Lysis by physical methods produces membrane fragments and small DNA molecules caused by shearing of the chromosomal DNA, either of which can interfere with subsequent...

Method used

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  • Cell lysis composition, methods of use, apparatus, and kit
  • Cell lysis composition, methods of use, apparatus, and kit
  • Cell lysis composition, methods of use, apparatus, and kit

Examples

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example 1

Preparation of Cell Lysis Reagent

[0145] In this Example, several representative cell lysis reagents are described. For cell pellets, a cell lysis reagent at 1× concentration aqueous formulation is preferably used. For cell media, a cell lysis reagent at 10× concentration is preferable.

(a) Cell Lysis Reagent at 1× Concentration:

[0146] This 1× aqueous formulation is useful for extracting proteins or peptides from cell pellets (frozen or non-frozen). The amount of the formulation added to the pellets is generally based on the optical density of the cells. For example, 200 ul of 1× formulation is used for the lysis of cells with an OD600 of 1.8 / 1 ml. The formulation contains the following components:

100 mM HEPES, pH 7.5,

1% Triton X-100 (Sigma, St. Louis, Mo., Cat#T-9284)

1% Mazu DF204 (defoaming agent, PPG Industries, Gurnee, Ill., Cat#213306-2)

0.4% Tomah (purified Tomah E-18-15, Bioaffinity systems, Roscoe, Ill., Cat#016483)

10 mM imidazole (Sigma, St. Louis, Mo.; Cat#I-239...

example 2

Release of Renilla Luciferase from E. coli

[0148] Cytoplasmic protein, as measured by the enzyme Renilla Luciferase, is released from E. coli cells when the cells are treated with a solution containing detergent and Polymyxin B. Surprisingly, this release of enzyme is not accompanied by general cell lysis as measured by observation of the optical density of the culture during the treatment.

[0149]E. coli bacteria expressing His-tagged Renilla Luciferase were grown in Luria Broth [L Broth]+10 ng / ml tetracycline [Tet] [50 ml of media in a 250 ml flask] at 37° C. overnight in a shaking incubator rotating at 200 RPM. The E. coli strain was prepared by transforming E. coli with a vector expressing histidine-tagged Renilla luciferase. The vector was constructed by conventional methods. See Maniatis et al., “Molecular Cloning: A Laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Following overnight culture the bacterial cells were diluted 1:100...

example 3

Screening of Detergents for the Ability to Release Enzymes into Media from E. coli Without Inactivation of the Enzyme

[0158] In this example, a number of detergents were tested for their ability to release cytoplasmic protein from E. coli cells alone and in combination with Polymyxin B.

[0159] The following stock detergent solutions were prepared:

Two grams of deoxycholic acid, sodium salt [Sigma D 6750, 102H0811] was dissolved in deionized water to produce a 4% (v / v) DOC solution.

Two grams of Lauryl Sulfate, sodium salt [Sigma L 4390, 73H0057] was dissolved in deionized water to produce a 4% (v / v) SDS solution.

Two grams of Tomah E-14-5 [Tomah Chemical Company, lot 71002-1] was dissolved in deionized water to produce a 4% (v / v) Tomah E-14-5 solution.

Two grams of Tomah E-14-2 [Tomah Chemical Company, lot 70224-1] was dissolved in deionized water to produce a 4% (v / v) Tomah E-14-2 solution.

Two grams of Tomah E-18-15 [Tomah Chemical Company, lot 60911-1] was dissolved in deion...

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Abstract

Cell lysis compositions, methods for extracting and isolating proteins and peptides from a host cells using the compositions, kits and apparatus for extracting and isolating protein and peptide molecules from host cells and for detecting for the presence of a protein or peptide. The composition allows for the extraction and isolation of proteins and peptides from host cells without the need for mechanical disruption and with or without isolation of the cells from cell medium. The composition includes at least one surfactant having a hydrophobic-lipophilic balance value in the range from about 11 to about 16; and at least one cell membrane altering compound.

Description

Cross-reference [0001] This application claims the benefit of U.S. provisional application No. 60 / 422,931, filed Nov. 1, 2002, which is incorporated by reference in its entirety.FIELD OF THE INVENTION [0002] The present invention relates to a cell lysis composition, methods for extracting and purifying proteins, apparatus and kit for extracting target proteins from host cells including cell media and cell pellets. In particular, the present invention relates to a composition for extracting proteins from host cells without the need for mechanical disruption. BACKGROUND OF THE INVENTION [0003] Recombinant DNA technology provides a valuable means of synthesizing large amounts of desirable eukaryotic proteins such as mammalian hormones, interferons, and enzymes. While organisms can be readily manipulated in order to produce the desired protein, the host organism does not normally secrete the protein product into the culture medium. Thus lysis of the organisms, e.g., bacteria, followed b...

Claims

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Application Information

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IPC IPC(8): C40B40/10C40B30/06C12P21/06B01D15/36B01D15/38C07H19/00C07H21/00C07K1/36C11D1/44C11D1/66C11D1/72C11D1/88C11D3/37C11D3/38C11D3/386C12NC12N1/06C12N1/08
CPCB01D15/361B01D15/3804B01J20/28009C07K1/36C11D1/44C11D1/662C11D1/72C11D1/88C11D3/3719C11D3/381C11D3/38636C12N1/06
Inventor ENGEL, LAURIESHULTZ, JOHN W.JOHNSON, TONNY M.ZIMMERMAN, KRISTOPHERBOZEK, LAURA L.STEVENS, JUDITH N.
Owner PROMEGA
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