Isolation of preferential proteins from cells; obtain cells, incubate with reducing agents and detergent, recover proteins
Inventors
Assignees
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Publication Number
US-7229789-B1
Publication Date
2007-06-12
Expiration Date
Abstract
A process is disclosed for releasing proteins from cells and/or inactivating viruses. In the process, a host cell containing a protein of interest is contacted with a solution of an effective amount of a detergent.
Core Innovation
The patent describes a method of releasing a protein of interest from host cells by suspending the host cells in a buffer and adding an amphipathic charged-amine or amphipathic charged-amine-oxide detergent. The detergent is used in a concentrated form, at least 3 times the desired final concentration after dilution in the buffer. The disclosed approach is directed to releasing intracellular proteins and can include virus inactivation as stated in the provided description.
The document further constrains the buffer and process conditions for the releasing step, including an ionic-strength constraint for the buffer with an explicit range stated in dependent claim language. The disclosure also includes adding glycerol to suspended host cells in a concentrated form, with the intent that the glycerol reaches a desired final concentration after dilution. Additional dependent claim language specifies incubation time and incubation temperature ranges for the cell and detergent mixture.
For detergent selection, the patent includes a list of specified amphipathic charged-amine or charged-amine-oxide detergents for use in the method and provides a narrowing dependent option excluding dimethyltridecylamine. The described disclosure also reports that non-selective extraction can release RNA, DNA, lipids, and cell-derived lipids together with protein, while providing improved separability relative to mechanical lysis as stated in the provided description.
Claims Coverage
The provided independent claims cover two alternative method definitions for releasing a protein of interest from host cells, each anchored by the use of amphipathic charged-amine or amphipathic charged-amine-oxide detergents supplied in a concentrated form at least 3 times the desired final concentration after dilution. A further inventive narrowing is implemented in the dependent claim by selecting from an explicit detergent group and excluding dimethyltridecylamine.
Concentrated amphipathic charged detergent for host-cell protein release
Suspending the host cells in a buffer and adding an amphipathic charged amine or an amphipathic charged amine oxide detergent to the suspended cells, wherein the detergent is in a concentrated form of at least 3 times the desired final concentration of the detergent after dilution in the buffer.
Selected amphipathic charged detergent group in concentrated form
Suspending the host cells in a buffer and adding a detergent to the suspended cells, wherein the detergent is selected from the group consisting of tributylphosphate, dimethyldecylamine, dimethyltridecylamine, dimethylundecylamine, dimethyldidecylamine, dimethyltetradecylamine, dimethylhexadecylamine, dimethyldecylamineoxide, dimethylundecylamineoxide, dimethyldidecylamineoxide, dimethyltetradecylamineoxide and dimethyltridecylamineoxide, and wherein the detergent is in a concentrated form of at least 3 times the desired final concentration of the detergent after dilution in the buffer.
Excluding dimethyltridecylamine from detergent selection
Using the method of releasing a protein of interest from host cells with a detergent that is not dimethyltridecylamine.
Across the independent claim set, the core claim coverage centers on releasing a protein of interest from host cells using an amphipathic charged-amine or amphipathic charged-amine-oxide detergent supplied as a concentrated dosing relative to the desired final concentration after dilution. A further scope refinement is achieved by limiting detergents to an explicit group and excluding dimethyltridecylamine in a dependent claim.
Stated Advantages
Improved separability versus mechanical lysis, as stated in the provided description.
Ability to release protein alongside non-selective extraction of RNA, DNA, and lipids, while still yielding clarified extract containing measurable heterologous protein, as stated in the provided description.
Documented Applications
Releasing intracellular proteins from host cells, including producing clarified extracts containing measurable heterologous protein, as described for Pichia pastoris in the provided description.
Virus inactivation by contacting host cells with the disclosed amphipathic charged-amine or charged-amine-oxide detergents, as stated in the provided description.
Non-selective extraction where RNA, DNA, and lipids (including cell-derived lipids) are extracted alongside protein, as described in the provided description.
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