US2007105154A1PendingUtilityA1
Cell lysis composition, methods of use, apparatus, and kit
Est. expiryNov 1, 2022(expired)· nominal 20-yr term from priority
C11D 3/38636C11D 1/88C11D 1/662B01D 15/3804C12N 1/06C11D 1/44B01D 15/361C11D 1/72C07K 1/36B01J 20/28009C11D 3/3719C11D 3/381
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Claims
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.
Claims
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30 . A method for recovering proteins or peptides from host cells comprising the steps of:
providing a source of cells having a desired protein or peptide; providing a composition comprising 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; and contacting the cells with the composition in an amount effective to effect lysis of the cell and subsequent release of the protein or peptide.
31 . The method according to claim 30 , further comprising the step of separating the released protein or peptide.
32 . The method according to claim 31 , further comprising the step of contacting the released protein or peptide with a substrate that binds the released protein or peptide.
33 . The method according to claim 32 , wherein the substrate comprises a magnetic or non-magnetic resin.
34 . The method according to claim 30 , wherein the composition further comprises lysozyme.
35 . The method according to claim 30 , wherein the cells comprise prokaryotic or eucaryotic cells.
36 . The method according to claim 35 wherein the cells comprise bacterial, yeast, insect or plant cells.
37 . The method according to claim 30 wherein the cells are in the form of a cell culture or a pellet.
38 . The method according to claim 30 wherein the surfactant is selected from the group consisting of non-ionic surfactants, cationic surfactants, and mixtures thereof.
39 . The method according to claim 38 wherein the surfactant is present in an amount ranging from about 0.001 to about 10% (w/v) of the composition.
40 . The method according to claim 38 wherein the non-ionic surfactants comprise ethoxylated alkylphenols.
41 . The method according to claim 40 wherein the ethoxylated akylphenols comprise ethoxylated nonylphenols or octylphenoxypolyethoxyethanol.
42 . The method according to claim 38 wherein the cationic surfactant comprise ethylene oxide condensates of alphatic amines or ethoxylated tallow amines.
43 . The method according to claim 30 wherein the surfactant comprises an ethoxylated amine.
44 . The method according to claim 30 , wherein the surfactant comprises one or more compounds selected from the group consisting of Tomah E-18-5, Tomah E-18-15, Rhodameen VP 532/SPB, Trymeen 6607, Triton X-100.
45 . The method according to claim 30 wherein the cell membrane altering compound is present in an amount effective to substantially lyse or cause pore formation in cell membranes or walls.
46 . The method according to claim 30 , wherein the cell membrane altering compound comprises polymyxin-beta-nonapeptide (PMBN), alkylglycoside or alkylthioglycoside, betaine detergent, quarternary ammonium salt, amines, lysine polymers, magainin, melittin, phospholipase A 2 or phospholipase A 2 activating peptide (PLAP).
47 . The method according to claim 30 wherein the cell membrane compound inhibits phospholipid sensitive Ca +2 dependent protein kinase and attacks cell membranes.
48 . The method according to claim 30 wherein the cell membrane altering compound is an antibiotic.
49 . The method according to claim 48 wherein the cell membrane altering compound comprises a polymyxin B sulfate or vancomycin.
50 . The method according to claim 48 wherein the cell membrane altering compound comprises a mixture of polymyxin B1 and polymyxin B2.
51 . The method according to claim 46 wherein the cell membrane altering compound comprises an alkylglycoside or an alkylthioglycoside.
52 . The method according to claim 51 , wherein the cell membrane altering compound comprises octyl thioglucoside.
53 . The method according to claim 52 , wherein the octyl thioglucoside is present at a final concentration of at least 0.4%, and less than 1% (w/v).
54 . The method according to claim 53 , wherein the octyl thioglucoside is present at a final concentration of between 0.4% and 0.6% (w/v).
55 . The method according to claim 30 , the composition further comprising a buffer salt.
56 . The method according to claim 55 , wherein the buffer salt is present in an amount sufficient to maintain a pH range from about 6.5 to about 9.0.
57 . The method according to claim 30 , wherein the composition further comprising a defoaming agent.
58 . The method according to claim 57 , wherein the composition further comprises an agent to reduce non-specific binding of non-affinity labeled proteins.
59 . The method according to claim 30 , wherein the composition further comprises a lysozyme.
60 . The method according to claim 30 wherein the composition comprises Tomah E-18-15, Triton X100, and octyl beta thioglucopyranoside.
61 . The method according to claim 30 wherein the composition comprises 2% Tomah E-18-15, 2% Triton X100, and 6% octyl beta thioglucopyranoside in 500 mM HEPES (pH 7.5).
62 . A method for recovering proteins or peptides from host cells comprising the steps of:
providing a source of cells having a desired protein or peptide; providing a composition comprising 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; providing a substrate for binding the protein or peptide; contacting the cells with the composition in an amount effective to effect lysis of the cell and release of the protein or peptide; contacting the released protein or peptide with the substrate under conditions effective for binding the release protein with the substrate; washing the protein or peptide bound to the substrate; and recovering the protein or peptide bound to the substrate.
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80 . A high throughput method for recovering proteins or peptides from host cells comprising the steps of
providing one or more sources of cells having a desired protein or peptide; providing a composition comprising 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; and contacting each source of cells with the composition in an amount effective to effect lysis of the cells and subsequent release of the protein or peptide.
81 . The method according to claim 80 , further comprising the step of separating the released protein or peptide from each source cell.
82 . The method according to claim 81 , wherein said step is performed by contacting the released protein or peptide with a substrate that binds to some or all of the release protein or peptide.
83 . The method according claim 82 , wherein the substrate comprises a magnetic or non-magnetic resin.
84 . The method of claim 81 , further comprising measuring the activity or binding of the released protein or peptide.
85 . A high throughput method for recovering proteins or peptides from host cells comprising the steps of
providing one or more source of cells having a desired protein or peptide; providing a composition comprising 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; providing one or more substrates for binding the protein or peptide; contacting each source of cells separately with the composition in an amount effective to effect lysis of the cell and subsequent release of the protein or peptide; contacting the released protein or peptide from each source of cells with the substrate under conditions effective for binding some or all of the released protein with the substrate; washing the protein bound to the substrate; and recovering the protein bound to the substrate.
86 . The method according to claim 85 , wherein the substrate comprises a magnetic or non-magnetic resin.
87 . The method according to claim 85 , further comprising the step of measuring the activity or binding of the released protein or peptide.
88 . A high throughput method for screening a library of proteins or peptides from sources of host cells, each source of host cell having a vector that encodes a protein or peptide member of the library, the method comprising the steps of:
providing a library of proteins or peptides from sources of host cells, each source of host cells having a vector that encodes a protein or peptide of the library; providing a composition comprising 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; providing one or more substrates for binding the protein or peptide; contacting each source of cells with the composition in an amount effective to effect lysis of the cell and subsequently release of the protein or peptide; contacting the released protein or peptide from each source of cells with the substrate under conditions effective for binding some or all of the released protein or peptide with the substrate; washing the protein or peptide bound to the substrate, and recovering the protein or peptide bound to the substrate.
89 . The method according to claim 88 , wherein the protein or peptides are mutants of a particular protein or peptide of interest.
90 . The method according to claim 89 , further comprising the step of measuring the activity or binding properties of the protein or peptide.
91 . The method according to any one of claims 80 , 85 or 88 wherein the composition comprises Tomah E-18-15, Triton X100, and octyl beta thioglucopyranoside.
92 . The method according to claim 80 , 85 , or 88 wherein the composition comprises 2% Tomah E18-15, 2% Triton X100, and 6% octyl beta thioglucopyranoside in 500 mM HEPES (pH 7.5).
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