WO1994005308A1 - Purification de composes amphiphiles - Google Patents

Purification de composes amphiphiles Download PDF

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Publication number
WO1994005308A1
WO1994005308A1 PCT/US1993/007694 US9307694W WO9405308A1 WO 1994005308 A1 WO1994005308 A1 WO 1994005308A1 US 9307694 W US9307694 W US 9307694W WO 9405308 A1 WO9405308 A1 WO 9405308A1
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Prior art keywords
peptide
amino acid
lys
seq
ala
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Jon I. Williams
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Magainin Pharmaceuticals Inc
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Magainin Pharmaceuticals Inc
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Priority to AU50147/93A priority Critical patent/AU5014793A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians

Definitions

  • This invention relates to the purification of
  • amphiphilic compounds More particularly, this invention relates to the purificiation of amphiphilic compounds, in particular amphiphilic peptides, from a starting material, such as, for example, biological tissues, cell lysates, solutions containing chemically synthesized peptides, or materials containing peptides made by recombinant
  • Amphiphilic peptides in general, have distinct hydrophobic and hydrophilic regions due to the presence of hydrophobic and hydrophilic amino acids in the peptide chain, and many are capable of forming an alpha-helical structure. Such amphiphilic peptides may also possess important biological or therapeutic properties; i.e., such peptides may inhibit, prevent, or destroy the growth or proliferation of target cells or viruses. Such peptides consequently may be used as antibiotics, anti-microbial agents, anti-fungal agents, anti-parasitic agents,
  • the peptides may be administered to a human or non-human animal or to plants, in order to inhibit growth of a target cell or virus or virally-infected cell in a human or non-human animal, or in a plant.
  • biologically active amphiphilic peptides may be used in vitro as preservatives or sterilants to protect materials from microbial or viral contamination.
  • amphiphilic peptides are membrane-active; i.e., the peptides are able to pass into and/or through cell membranes and disrupt cellular functions.
  • Biologically active amphiphilic peptides in many instances are also ion-channel forming peptides, whereby the peptides enable ions having pharmacological properties, such as, for example, fluoride ions, peroxide ions, bicarbonate ions, silver ions, zinc ions, mercury ions, arsenic ions, copper ions, antimony ions, gold ions, thallium ions, nickel ions, selenium ions, bismuth ions, and cadmium ions, to pass through cell membranes.
  • the peptide may potentiate the activity of the ion having pharmacological properties, and the ion having pharmacological properties may concomitantly potentiate the peptide.
  • Examples of biologically active amphiphilic peptides include magainin peptides, CPF peptides, XPF peptides, PGLa peptides, melittin peptides, cecropins, sarcotoxins, and certain synthetic amphiphilic peptides.
  • Many amphiphilic peptides are obtained naturally from various organisms.
  • magainin peptides may be obtained from the skin of the African clawed frog Xenopus laevis
  • melittins may be obtained from the hemolymph of the honeybee Apis mellifera.
  • a biological material such as animal tissue or cell lysate, which contains the peptides.
  • peptides may be made by recombinant gene expression
  • chaotropic agents such as urea, guanidine hydrochloride, guanidinium isothiocyanate, and the like, followed by one or more chromatographic steps involving an immobile solid phase material.
  • a procedure requires expensive materials and often requires a considerable amount of time.
  • Individual purification steps which do not require expensive reagents and which provide high levels of peptide product recovery are very desirable for both experimental and commercial scale purification processes for
  • Zasloff, PNAS, Vol. 84, pgs. 5449-5453 (1987), discloses the isolation of magainin peptides from the skin of the frog Xenopus laevis using batch isolation on CM52 (Whatman) ion exchange media in 0.2M ammonium acetate, pH5.1-5.2, followed by isolation from a Bio-Gel P-30 (Bio-Rad) size exclusion chromatography column, and purification by HPLC on a Vydac C4 reverse phase column.
  • chloroform methanol mixtures
  • recovery of the proteolipid fraction is accomplished by evaporation of the organic mixture, resuspension in chloroform: methanol (2:1),
  • amphiphilic peptide or protein amphiphilic peptide or protein.
  • amphiphilic peptide or protein comprises contacting a material containing at least one amphiphilic peptide or protein with a mixture of an aprotic organic solvent and an alcohol in an amount effective to form a single miscible solution.
  • the miscible solution is
  • the aqueous solution is added in an amount effective to form a first phase, which includes an aqueous solution containing the amphiphilic peptide or protein, and a second phase which includes the organic solvent.
  • the at least one amphiphilic peptide or protein then is recovered from the first phase.
  • the aprotic organic solvent is chloroform or diethyl ether.
  • the aprotic organic solvent is chloroform.
  • the alcohol may be, in one embodiment, an alkanol, and in particular may be selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, isopropanol, isobutyl alcohol and butanol, including 1-butanol and 2-butanol, and pentanol (including N-pentanol).
  • the mixture contains the aprotic organic solvent and alcohol in a volume ratio of from about 1:5 to about 5:1, preferably from about 1:2 to about 2:1.
  • the mixture of aprotic organic solvent and alcohol is added to the material, which may be an aqueous solution containing the at least one amphiphilic peptide or protein or a dry material containing the amphiphilic peptide or protein, in an amount sufficient to form a single miscible solution.
  • the single miscible solution which is formed by the addition of the aprotic organic solvent and alcohol to the remainder material, is capable of being separated into a first aqueous phase and a second organic phase upon the addition of a separate aqueous solution.
  • the aqueous solution which is added to the miscible solution in an amount effective to form a first aqueous phase containing the at least one amphiphilic peptide or protein, and a second organic phase, may, in one embodiment, be water or an aqueous salt solution.
  • the aqueous material is an aqueous salt solution, and more preferably a lithium chloride solution, a potassium chloride solution, or a sodium chloride solution.
  • the salt may be at a concentration of from about .04M to about 1M, preferably about 0.1M. Applicant has found, surprisingly, that when an aqueous salt solution is added to the single miscible solution, the at least one amphiphilic peptide or protein which is soluble in the single miscible solution is
  • the second organic phase which contains an organic solvent, such as, for example, chloroform, with some portion of an alcohol and some portion of water, contains hydrophobic materials and insoluble precipitates but
  • amphiphilic molecules can dissolve in chloroform: alcohol mixtures, the aqueous solubility of such amphiphilic
  • the at least one amphiphilic peptide or protein may be recovered from the final aqueous phase by means known to those skilled in the art.
  • recovery means can include, but are not limited to, application of one or more methods selected from the group consisting of liquid chromotography, high performance liquid chromatography, reextraction, diafiltration, ultrafiltration, crystallization, or other chemical processing to remove unwanted molecular species such as salt ions.
  • a material containing at least one amphiphilic peptide or protein is contacted with a solution including an aprotic organic solvent.
  • the solution is preferably at an acidic pH.
  • the contacting of the material with the solution at an acidic pH provides an extract solution containing the at least one amphiphilic peptide or protein. At least a portion of the aprotic organic solvent then is removed from the extract solution to provide a remainder material
  • amphiphilic peptide or protein containing at least the at least one amphiphilic peptide or protein.
  • the remainder material then is contacted with an aprotic organic solvent and an alcohol such as hereinabove described, and processed as hereinabove described in order to obtain a purified amphiphilic peptide or protein.
  • the solution at an acidic pH is the solution at an acidic pH
  • the solution at an acidic pH further includes an acid, which in ionic strength and chemical acid character provides a solution which preferably has a pH no greater than 4.0.
  • the acid is selected from the group consisting of trifluoroacetic acid and trichloroacetic acid. Most preferably, the acid is trifluoroacetic acid.
  • the trifluoroacetic acid is present in an amount of about 0.1% (vol./vol.).
  • amphiphilic peptide or protein with a solution including an aprotic organic solvent at an acidic pH enables the at least one amphiphilic peptide or protein to be dissolved in the solvent, thereby providing an extract solution containing the at least one amphiphilic peptide or protein.
  • This extract solution then is separated from any insoluble materials if such are present. The insoluble materials may be re-extracted one or more additional times to increase the yield of the at least one amphiphilic peptide or protein.
  • at least the aprotic organic solvent eg.,
  • acetonitrile which contains hydrophobic materials, is removed from the solution.
  • the aprotic organic solvent may be removed by a variety of means, such as, for example, by evaporation, distillation, lyophilization, or the like.
  • the extract solution may be evaporated to dryness, thereby leaving a dry, solid remainder material containing the at least one amphiphilic peptide or protein.
  • a sample mixture containing at least one amphiphilic peptide, or protein can be prepared as a material which is exposed to a chloroform:methanol mixture so as to form a single miscible solution containing the at least one amphiphilic peptide, or protein, and then mixed with a portion of an aqueous salt solution.
  • Amphiphilic peptides, or proteins would preferentially partition into the primarily aqueous phase.
  • the primarily aqueous phase can subsequently be reduced in volume by vacuum evaporation, distillation, lyophilization or the like to reduce or eliminate any organic solvent components to the primarily aqueous phase and then re-exposed to a second
  • the initial organic solvent mixture used during the initial organic extraction step of a multiple extraction procedure can be carried out with chloroform:methanol in a volume ratio of 1:2 and in subsequent organic extraction steps in a volume ratio of 2:1.
  • the initial organic extraction step can use chloroform:n-butanol in a volume ratio of 4:1
  • a second organic extraction step could use chloroform: methanol in a volume ratio of 1:2
  • subsequent organic extraction steps could use chloroform:methanol in a volume ratio of 2:1.
  • the method of the present invention is particularly applicable to the purification of amphiphilic peptides, or proteins, many of which are biologically active.
  • the amphiphilic peptide is a basic (positively charged) polypeptide having at least sixteen amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
  • the hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one amino acid other than a hydrophobic amino acid (preferably at least two amino acids) and generally by no greater than four amino acids, and the amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.
  • the hydrophilic amino acids are generally also in groups of two adjacent amino acids in which at least one of the two amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic amino acids being spaced from each other by at least one amino acid other than a
  • hydrophilic amino acid preferably at least two amino acids and generally no greater than four amino acids, and the amino acids between pairs of hydrophilic amino acids may or may not be hydrophobic.
  • the polypeptide comprises a chain of at least four groups of amino acids, with each group consisting of four amino acids. Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid.
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser, Thr, and homoserine (Hse).
  • the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2, 4-diaminobutyric acid
  • Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different.
  • the polypeptide chain may comprise 5 or 6 groups of this
  • each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
  • the polypeptide chain of the at least one amphiphilic peptide or protein preferably has at least 12 amino acids, and no greater than 50 amino acids. It is to be understood, however, that the polypeptide does not have to consist entirely of the groups described above.
  • the polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
  • the groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
  • the biologically active polypeptide may comprise a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
  • each of the at least four groups of amino acids which are in the peptide chain is of the
  • B are hydrophobic amino acids, one of C or D is a basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid.
  • the resulting polypeptide chain therefore, may have one of the following sequences:
  • X. is D; C-D- or B-C-D-, Y 1 is -A or -A-B or -A-B-C X 2 is A-, D-A- or C-D-A- Y 2 is -B, -B-C or B-C-D
  • X 3 i s B-, A-B-, D-A-B- Y 3 is -C, -C-D, -C-D-A
  • X 4 is C-, B-C-, A-B-C- Y 4 is -D, -D-A, -D-A-B
  • n is at least 4.
  • amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other.
  • the peptide may have amino acids extending from either end of the chain.
  • the chains may have a
  • Other amino acid sequences may also be attached to the "Ala” and/or the "Lys" end.
  • the chain may have, for example, a C-D sequence before the first A-B-C-D group.
  • other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains.
  • amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
  • the peptide may be a magainin peptide.
  • a magainin peptide is either a magainin such as magainin I, II or III or an analogue or derivative thereof.
  • the magainin peptides preferably include the following basic peptide structure X 12
  • R 11 is a hydrophobic amino acid
  • R 12 is a basic hydrophilic amino acid
  • R 13 is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid
  • R 14 and R 14a are hydrophobic or basic hydrophilic amino acids
  • R 15 is glutamic acid or aspartic acid, or a hydrophobic or a basic hydrophilic amino acid
  • n is 0 or 1.
  • R 13 is a hydrophobic or neutral hydrophilic amino acid
  • R 14a is a hydrophobic amino acid
  • R 15 is glutamic acid or aspartic acid.
  • a magainin peptide may include the following structure:
  • R 11 , R 12 , R 14 and R 14a are as previously defined.
  • a magainin peptide may also have the following
  • R 16 where R 16 is a basic hydrophilic amino acid or asparagine or glutamine.
  • R 16 -R 17 where R 17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
  • R 17 is a neutral hydrophilic amino acid.
  • a magainin peptide may also have the following
  • X 12 , Y 12 and Z 12 are as previously defined and a is 0 or 1 and b is 0 or 1.
  • the magainin peptides may also include the following basic peptide structure X 13 :
  • R 1 1 -R 14 -R 12 -R 11 -R 1 1 -R 12 - wherein R 11 , R 12 , R 13 , R 14 , and
  • R 14a are amino acids as hereinabove described.
  • the magainin peptide may also include the following structure X 13 -Z 13 ; wherein X.. -. is the hereinabove described basic peptide structure and Z,-, is
  • R 11 , R 14 , R 14a, R 15 , R 16 , and R 17 are as hereinabove described, and n is 0 or 1, and each n may be the same or different.
  • the magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids.
  • a magainin peptide preferably has from 21 to 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include
  • magainin peptides having the following primary sequences as given in the accompanying sequence listing as well as appropriate analogues and derivatives thereof:
  • magaininin peptides refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs.
  • the peptide may be a PGLa peptide or an XPF peptide.
  • a PGLa peptide is either PGLa or an analogue or
  • the PGLa peptides preferably include the following basic peptide structure X 14 :
  • the PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end, or at both the amino and carboxyl end.
  • a PGLa peptide may have the
  • R 11 and R 14 are as previously defined.
  • a PGLa peptide may also have the following structure:
  • R 11 is as previously defined.
  • a PGLa peptide may also have the following structure:
  • X 14 , Y 14 , and Z 14 are as previously defined, a is 0 or 1 and b is 0 or 1.
  • An XPF peptide is either XPF or an analogue or
  • the XPF peptides preferably include the following basic peptide structure X 16 :
  • the XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids.
  • structure of XPF may include additional amino acids at the amino end or at the carboxyl end, or at both the amino and carboxyl ends.
  • an XPF peptide may include the following structure:
  • R 11 and R 14 are as previously defined.
  • An XPF peptide may include the following structure:
  • An XPF peptide may also have the following structure:
  • the peptide may be a CPF peptide or appropriate analogue or derviative thereof.
  • CPF peptides as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
  • the CPF peptide may be one which includes the following basic peptide structure X 20 :
  • R 21 is a hydrophobic amino acid
  • R 2 2 is a hydrophobic amino acid or a basic hydrophilic amino acid
  • R 23 is a basic hydrophilic amino acid
  • R 24 is a hydrophobic or neutral hydrophilic amino acid
  • R 25 is a basic or neutral hydrophilic amino acid.
  • hydrophobic amino acids are Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids are Asn, Gln, Ser, and Thr.
  • the basic hydrophilic amino acids are Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
  • the CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino and/or carboxyl ends or both the amino and carboxyl end. In general, the peptide does not include more than 40 amino acids.
  • the CPF peptides including the above basic structure preferably have from 1 to 4 additional amino acids at the amino end.
  • R 21 , R 22 and R 25 are as Previously defined.
  • the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.
  • the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
  • X is the hereinabove defined basic peptide structure and Z 20 is
  • R 21 -R 21 -R 24 -R 24 -R 26 -Gln (vi) R 21 -R 21 -R 24 -R 24 -R 26 -Gln; or (vii) R 21 -R 21 -R 24 -R 24 -R 26 -Gln-Gln, wherein R 21 and
  • R 24 are as previously defined, and R 26 is proline or a hydrophobic amino acid.
  • Preferred peptides may be represented by the following structural formula
  • the peptide being purified in accordance with the present invention may include one of the following basic structures X 31 through X 37 wherein:
  • X 31 is -[R 31 -R 32 -R 32 -R 33 -R 31 -R 32 -R 32
  • X 32 is -[R 32 -R 32 -R 33 -R 31 -R 32 -R 32 -R 31
  • X 33 is -[R 32 -R 33 -R 31 -R 32 -R 32 -R 31 -R 32
  • X 34 is -[R 33 -R 31 -R 32 -R 32 -R 31 -R 32 -R 32
  • X 35 is -[R 31 -R 32 -R 32 -R 31 -R 32 -R 32 -R 33
  • X 36 is -[R 32 -R 32 -R 31 -R 32 -R 32 -R 33 -R 31 and
  • X 37 is -[R 32 -R 31 -R 32 -R 32 -R 33 -R 31 -R 32
  • R 31 is a basic hydrophilic amino acid
  • R 32 is a hydrophobic amino acid
  • R 33 is a neutral hydrophilic, basic hydrophilic, or hydrophobic amino acid
  • n is from 2 to 5.
  • the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp and Tyr,norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser and Thr.
  • the peptide when the peptide includes the structure X 31 , the peptide may include the following structure:
  • Y 31 is :
  • R 32 -R 33 -R 31 -R 32 -R 32 (v) R 32 -R 33 -R 31 -R 32 -R 32 ; or (vi) R 32 -R 32 -R 33 -R 31 -R 32 -R 32 , wherein R 31 , R 32 , and R 33 are as hereinabove described
  • the peptide when the peptide includes the structure X 31 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • a is 0 or 1
  • b is 0 or 1.
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 32 , the peptide may include the following
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure:
  • the peptide may include the following structure: (Y 33 ) a - X 33 - (Z 33 ) b , wherein Y 33 and Z 33 are as previously defined, a is 0 or 1, and b is 0 or 1.
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
  • Y 34 is:
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure:
  • Y 35 is: s ( i ) R 33 ;
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • a is 0 or 1
  • b i s 0 or 1.
  • the peptide when the peptide includes the structure X 36 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 36 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • a is 0 or 1
  • b is 0 or 1.
  • the peptide when the peptide includes the structure X 37 , the peptide may includes the structure Y 37 -X 37 , wherein X 37 is as hereinabove described, and Y 37 is:
  • the peptide when the peptide includes the structure X 37 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • n 3
  • the peptide has one of the following structures as given in the accompanying sequence listing:
  • Lys Ile Ala (Lys Ile Ala Gly Lys Ile Ala) 3 (SEQ ID NO: 69)
  • amphiphilic peptide includes the following basic structure X 40 :
  • the peptide may include the following structure:
  • Y 40 -X 40 wherein X 40 is as hereinabove described, and Y 40 is :
  • the peptide may include the following structure:
  • X 40 -Z 40 wherein X 40 is as hereinabove described and Z 40 is:
  • a is 0 or 1
  • b is 0 or 1.
  • the peptide has the following
  • the peptide has the following structural formula as given in the accompanying sequence listing:
  • the peptide has one of the one of the following structural formulae as given in the accompanying sequence listing:
  • the peptide purified in accordance with the present invention may include the following structural formula:
  • n is from 2 to 5.
  • n is 3, and the peptide has the following structural formula:
  • the peptide may include the following structural formula:
  • n is from 2 to 5.
  • n 3
  • the peptide has the following structural formula:
  • the peptide may include the following structural formula:
  • n is from 2 to 5.
  • n is 3, and the peptide has the following structural formula:
  • the peptide may be selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
  • the peptide may be a cecropin or sarcotoxin, or analogue or derivative thereof.
  • cecropins includes any of the natural
  • sarcotoxins includes the basic materials as well as analogues and derivatives thereof.
  • the sarcotoxins and analogues and derivatives thereof are described in
  • amphiphilic peptide may be melittin or an analogue or derivative
  • Melittin is an amphipathic peptide consisting of 26 amino acid residues, and is isolated from honeybee (Apis mellifera) venom. The peptide is known to be cytolytic. See Habermann, et al., Hoppe-Seyler's Zeitschrift Physiol. Chem., Vol. 348, pgs. 37-50 (1987). Melittin has the following structural formula as represented by the
  • apidaecin as used herein includes the basic structure as well as analogues and derivatives thereof. Apidaecins are further described in European Papent Application No. 299,828.
  • amphiphilic peptide purified in accordance with the present invention includes the following basic structure X 50 :
  • R 41 is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid.
  • the peptide includes the basic structure Y 50 -X 50 wherein X 50 is as hereinabove described and Y 50 is:
  • R 41 is leucine. In another embodiment, R 41 is leucine. In another
  • R 42 is lysine.
  • Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
  • amphiphilic peptide includes the following basic structure X 52 :
  • hydrophilic or neutral hydrophilic amino acid hydrophilic or neutral hydrophilic amino acid
  • R 41 is leucine. In another embodiment, R 41 is leucine. In another
  • R 42 is lysine
  • the peptide includes the basic structure Y 52 -X 52 , wherein X 52 is as hereinabove described, and Y 52 is:
  • the peptide may have the following structure
  • the peptide includes the basic structure X 52 - Z 52 , wherein X 52 is as hereinabove
  • the peptide may have the following structure :
  • the peptide may include the structure:
  • the peptide includes the following basic structure X 54 :
  • R 41 and R 42 are as hereinabove described, and R 43 is a netural hydrophilic amino acid.
  • the peptide may have the following structure:
  • the peptide may have the following amino acids:
  • the peptide includes the following basic structure X 56 :
  • R 41 -R 42 -R 41 -R 41 -R 42 -R 42 -R 41 -R 41 -R 42 -R 42 -R 44 wherein R 41 and R 42 are as hereinabove described, and R 44 is a neutral
  • hydrophilic amino acid or proline hydrophilic amino acid or proline.
  • the peptide may include the
  • R 41 and R 42 are as hereinabove described.
  • the peptide may include the
  • the peptide may have one of the following structures:
  • the peptide may have the structure (Y 56 ) a -X 56 -(Z 56 ) b , wherein X 56 , Y 56 , and Z 56 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
  • the peptide includes the following basic structure X 58 :
  • the peptide may include the structure Y 58 -X 58 , wherein X 58 is as hereinabove described, and Y 58 is:
  • the peptide includes the
  • the peptide has the following
  • the peptide may have the structure (Y 58 ) a -X 58 -(Z 58 ) b , wherein X 58 , Y 58 , and Z 58 are as
  • a is 0 or 1
  • b is 0 or 1.
  • the peptide may have the following structure: ( SEQ ID NO : 107 ) .
  • the peptide may include the structure X 60 -Z 60 , wherein X 60 is as hereinabove described, and Z 60 is:
  • the peptide has a structure selected from the group consisting of:
  • the peptide has the structure (a), and a representative example of such a structure is (SEQ ID NO: 108), which is given in the accompanying sequence listing.
  • the peptide has the structure (b), and a representative example of such a structure is (SEQ ID NO: 109), which is given in the accompanying sequence listing.
  • the peptide has the structure (c), and a representative example of such a structure is (SEQ ID NO: 110) as given the accompanying sequence listing.
  • the peptide has the amino acid sequence: (a)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl
  • the peptide has the structure (e), and representative examples of such a structure are (SEQ ID NO: 112) and (SEQ ID NO: 113) as given in the
  • the peptide has the following structural formula:
  • amphiphilic peptide or protein may be an ion channel-forming peptide or protein.
  • Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), or a pore-forming cytotoxin called variously
  • defensins also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), or a pore-forming cytotoxin called variously
  • perforin perforin, cytolysin, or pore-forming protein.
  • Defensins are described in Selsted, et al., J. Clin. Invest., Vol. 76, pgs. 1436-1439 (1985).
  • MBP proteins are described in
  • ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues and derivatives.
  • the method of the present invention may be used to obtain amphiphilic peptides or proteins from any starting material containing amphiphilic peptides or proteins.
  • starting materials include, but are not limited to, solutions of amphiphilic peptides or proteins which were chemically synthesized, were made by recombinant techniques, were isolated from or found in biological tissues, or were isolated from or found in cell lysates.
  • Such starting materials include the at least one amphiphilic peptide or protein and, depending upon the starting material, may also include lipids, glycolipids, polysaccharides, membrane proteins, soluble or cytosolic proteins, RNA and DNA.
  • a starting material such as any of those hereinabove described, which contains at least one amphiphilic peptide or protein, is placed into an aqueous solution containing acetonitrile and trifluoroacetic acid.
  • the at least one amphiphilic peptide or protein is dissolved in the solution to form an extract solution containing the at least one amphiphilic peptide or protein.
  • the extract solution is then separated from any insoluble materials, if present, and evaporated to dryness, leaving a dry composition containing the at least one amphiphilic peptide or protein. Hydrophobic materials, which are extracted by the acetonitrile, are thereby removed from the starting material.
  • a mixture of chloroform and methanol in a chloroform-to-methanol volume ratio of about 2:1 is added to the dry composition in an amount sufficient to form a single liquid phase, to which is then added 0.1M potassium chloride in an amount sufficient to form an upper aqueous phase containing the at least one amphiphilic peptide or protein, and a lower organic phase containing primarily hydrophobic peptides and proteins, lipids, and other
  • aqueous phase is then separated from the boundary layer and the primarily organic phase, and the at least one amphiphilic peptide or protein is recovered from the primarily aqueous phase by means such as those hereinabove described.
  • a starting material such as any of those hereinabove described, which contains at least one amphiphilic peptide or protein, is present as an aqueous solution or suspension from which any insoluble materials, if present, are separated.
  • chloroform and methanol in a volume ratio of 2:1 is added to the aqueous solution in an amount sufficient to form a single miscible phase, to which is added a solution of sodium chloride in water in a concentration of at least 40 mM and in an amount sufficient to form an upper aqueous phase containing the at least one amphiphilic peptide or protein, and a lower organic phase containing primarily hydrophobic peptides and proteins, lipids, and other
  • the aqueous phase is separated from any boundary layer and the primarily organic phase, and the at least one amphiphilic peptide or protein is recovered from the primarily aqueous phase by means such as those hereinabove described.
  • TFA trifluoroacetic acid
  • KCl chloride
  • phase extract 89 Sample 1 (SEQ ID NO: 115) in
  • the (*) sample values are the same for each pair of samples in the data sets with (SEQ ID NO:7)-NH 2 and (SEQ ID NO: 115) since only one 50 uL sample of total acetonitrile/TFA extract was set aside for each extract type.
  • 0.1 M potassium chloride from chloroform:methanol (2:1, vol:vol) further increases the peptide purity by greater than two-fold and is an efficient extraction step.
  • the 0.1 M potassium chloride extraction step has an average efficiency of about 83% for (SEQ ID NO: 7) - NH 2 and an average efficiency of about 95% for (SEQ ID NO:115).
  • Efficiency is determined by dividing the arbitrary area units of the aqueous phase by the total sum of the arbitrary area units of the aqueous and organic phases. Net yield of peptides (SEQ ID NO: 7) - NH 2 and (SEQ ID NO: 115) into the total aqueous and organic phases is greater than that in the parent 0.1% TFA sample, presumably because peptide-containing material has precipitated or is aggregated that is filtered away upon preparation of the total extract HPLC sample. This undetected peptide fraction is probably solubilized upon mixing of the 0.2% TFA sample with 15 volumes of chloroform:methanol (2:1).
  • the fusion protein was evaporated to dryness under vacuum and resuspended in 5.0 mL 0.1 M Tris-HCl, pH 8.0, 6 M guanidine hydrochloride (GuHCl). Cyanogen bromide treatment of solubilized inclusion body protein from this culture releases the amphiphilic peptide (SEQ ID NO: 116) from the fusion protein.
  • the other sample extract volume was increased to 160 ⁇ L with deionized water, 2.4 mL chloroform: methanol (2:1) was added to the tube, the sample was mixed, then 512 ⁇ L 0.1 M potassium chloride (KCl) was added to the solution and the sample was spun 2000 rpm for 5 minutes, 4°C before the aqueous top phase was transferred to a new glass tube. Again the aqueous top phase was evaporated to dryness under vacuum and resuspended in 250 ⁇ L deionized water before adding 3.75 LL chloroform:methanol (2:1). The sample was mixed, and 800 ⁇ L 40 mM KC1 was added.
  • KCl potassium chloride
  • the sample was again mixed, spun 2000 rpm for 5 minutes, 4°C, and the aqueous top phase was again transferred to a new glass test tube.
  • the aqueous top phase was evaporated to dryness again, and the precipitate was resuspended in 520 ⁇ L deionized water before adding 7.8 mL chloroform:methanol (2:1) and mixing the sample.
  • About 1.65 mL of 40 mM KCl then was added, the sample was mixed, and it then was spun 2000 rpm for 5 minutes, 4°C, before the top aqueous phase was transferred to another glass test tube.
  • the volume of this sample was reduced under vacuum from about 3.0 mL to about 1.2 mL before 100 ⁇ L of this sample and the 100 ⁇ L sample set aside earlier were each filtered into an HPLC vial through an 0.45 micron cellulose acetate filter (Millipore).
  • the samples were analyzed by HPLC analysis on a Rainin Dynamax instrument using an Applied Biosystems 2.5 mm ⁇ 50 mm C8 Aquapore reverse phase column ( 300 Angstrom particle size , 20 micron pore size ) with a 2%-60% acetonitrile in 0.1% TFA mobile phase gradient of 30 minute duration.
  • the A ?2n peak material corresponding to (SEQ ID NO: 116) was identified using a chemically synthesized (SEQ ID NO: 116) standard chromatographed under the same conditions.
  • the percent total A 220 peak material present as (SEQ ID NO: 116) was calculated by the Rainin instrument software and gave values of 6.8% for one process purification cycle and 8.8% (mean, two readings) for four process purification cycles.
  • the (SEQ ID NO: 116) chromatographic peak was superimposed on a complex background of A. 220 absorbing material following one process purification cycle, suggesting that the 6.8% value is a substantial overestimate of (SEQ ID NO: 116) purity.
  • Tris-HCl guanidine hydrochloride (GuHCl) extract described in Example 2 Twelve 50 ⁇ L portions of the 0.1 M Tris-HCl guanidine hydrochloride (GuHCl) extract described in Example 2 were placed in glass 17 ⁇ 100 mM test tubes and were mixed with 750 ⁇ L chloroform: methanol (2:1) before adding 0-100 ⁇ L of either 0.1 M NaCl or 0.1 M KCl to each tube. The aqueous phase additions were all standardized at 100 ⁇ L by adding additional deionized water where appropriate. These samples were thoroughly mixed and were spun 2000 rpm for 5 minutes, 4°C, before the top aqueous phases were removed to new test tubes and evaporated to dryness under vacuum.
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan,
  • NAME/KEY Magainin II peptide.
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide

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Abstract

Procédé permettant de purifier au moins un peptide ou une protéine amphiphile selon lequel on met en contact une matière contenant le (ou les) peptide(s) ou protéine(s) amphiphile(s) avec un mélange constitué d'un solvant organique aprotique et d'un alcool qu'on ajoute sous forme de phase liquide unique, dans laquelle on ajoute ensuite une matière aqueuse telle qu'une solution d'eau de sel pour former une première phase aqueuse contenant le (ou les) peptide(s) ou protéine(s) amphiphile(s) et une phase organique immiscible. On récupère ensuite dans la phase aqueuse le (ou les) peptide(s) ou protéine(s) amphiphiles. Dans une forme d'exécution de l'invention avant de mettre en contact ladite matière avec un mélange constitué d'un solvant organique aprotique et d'un alcool, on met en contact une matière contenant au moins un peptide ou une proteine amphiphile avec une solution renfermant un solvant organique aprotique à pH acide pour obtenir un extrait en solution contenant le (ou les) peptide(s) ou protéine(s) amphiphiles et on élimine de l'extrait en solution au moins une partie du solvant organique aprotique pour obtenir une matière résultante qui contient le (ou les) peptide(s) ou protéine(s) amphiphiles. On peut également utiliser ce procédé dans plusieurs applications successives permettant de purifier plus encore le (ou les) peptide(s) ou protéine(s) amphiphile(s). Ces procédés permettent de purifier des peptides ou des protéines biologiquement actifs sans employer de techniques ou d'agents de purification coûteux.
PCT/US1993/007694 1992-08-28 1993-08-13 Purification de composes amphiphiles Ceased WO1994005308A1 (fr)

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US07/936,504 1992-08-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3989819A (en) * 1969-03-05 1976-11-02 American Cyanamid Company Process for separating proteins from central nervous system tissue and treating experimentally induced demyelinating diseases
US4647445A (en) * 1984-03-28 1987-03-03 Massachusetts Institute Of Technology Radiolabelled lipoproteins and method for making same
US5116952A (en) * 1986-07-18 1992-05-26 The University Of Melbourne Protein active in humoral hypercalcemia of malignancy-pthrp

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3989819A (en) * 1969-03-05 1976-11-02 American Cyanamid Company Process for separating proteins from central nervous system tissue and treating experimentally induced demyelinating diseases
US4647445A (en) * 1984-03-28 1987-03-03 Massachusetts Institute Of Technology Radiolabelled lipoproteins and method for making same
US5116952A (en) * 1986-07-18 1992-05-26 The University Of Melbourne Protein active in humoral hypercalcemia of malignancy-pthrp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
METH. ENZYMOL., Vol. 1, issued 1955, R.K. MORTON, "Methods of Extraction of Enzymes from Animal Tissues", pp. 25-50. *
METH. ENZYMOL., Vol. 22, issued 1971, PENEFSKY et al., "Extraction of Water Soluble Enzymes and Proteins from Membranes", pp. 204-219. *

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