EP1131448A2 - Renforcement de la production de peptides cationiques antimicrobiens dans des cellules hotes - Google Patents

Renforcement de la production de peptides cationiques antimicrobiens dans des cellules hotes

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Publication number
EP1131448A2
EP1131448A2 EP99955614A EP99955614A EP1131448A2 EP 1131448 A2 EP1131448 A2 EP 1131448A2 EP 99955614 A EP99955614 A EP 99955614A EP 99955614 A EP99955614 A EP 99955614A EP 1131448 A2 EP1131448 A2 EP 1131448A2
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European Patent Office
Prior art keywords
peptide
cationic
expression cassette
cationic peptide
promoter
Prior art date
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EP99955614A
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German (de)
English (en)
Inventor
Jan Burian
Daniel Bartfeld
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Biowest Therapeutics Inc
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Micrologix Biotech Inc
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Publication of EP1131448A2 publication Critical patent/EP1131448A2/fr
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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates generally to methods for obtaining recombinant peptides and proteins from host cells.
  • the present invention relates to improved processes for producing and purifying cationic peptides from recombinant host cells in which the peptide is expressed in high yield and is easily recovered.
  • Antimicrobial peptides particularly cationic peptides have received increasing attention as a new pharmaceutical substance, because of their broad spectrum of antimicrobial activities and the rapid development of multi-drug-resistant pathogenic microorganisms.
  • Endogenous peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. These endogenous antimicrobial peptides are usually cationic amphipathic molecules that contain 10 to 45 amino acid residues and an excess of lysine and arginine residues, (for a review, see Broekaert et al., Plant Physiol. 705: 1353, 1995; Ganz and Lehrer, Pharmacol. Ther.
  • cationic peptides include rabbit defensin, crab tachyplesin, bovine bactenecin, silk-moth cecropin A, frog magainins, and bovine indolicidin.
  • the main site of action of the peptides is the cytoplasmic membrane of bacteria and other microbes. Due to their amphipathic nature, the peptides disrupt the membrane, causing a loss of potassium ions, membrane depolarization, and a decrease in cytoplasmic ATP.
  • cationic peptides are particularly important in the initial phases of resistance to microbial invasion. Cationic peptides are also effective when administered as therapeutic agents.
  • an ⁇ -helical magainin variant peptide has been shown to be effective against polymicrobic foot ulcer infections in diabetics, and a protegrin-derived peptide was found useful for treatment of oral polymicrobic ulcers in cancer patients (Hancock and Lehrer, TIBTECH 76:82, 1998).
  • a practical drawback in cationic peptide therapy is the lack of a cost effective, mass-production method of the agents.
  • the isolation of cationic peptides from natural sources is not cost-effective, and does not apply to the production of engineered cationic peptide variants which may have increased efficacy.
  • chemical peptide synthesis can be used to manufacture either natural or engineered cationic peptides, this approach is very costly.
  • cationic peptides have been produced in bacteria, such as E. coli or Staphylococcus attreiis, yeast, insect cells, and transgenic mammals (Piers et al., Gene 134:1, 1993, Reichhart et al.. Invertebrate Reprod. Develop. 27: 15, 1992, Hellers et al., Ear. J. Biochem. 199:435, 1991, and Sharma et al., Proc. Nat 'l Acad Sci.
  • coli such as, insulin A and B chain, calcitonin, Beta-globin, myoglobin, and a human growth hormone (Uhlen and Moks, "Gene Fusions for Purposes of Expression, An Introduction” in Methods in Enzymology 755: 129-143 Academic Press, Inc. 1990).
  • recombinant gene expression from a host cell presents a number of technical problems, particularly if it is desired to produce large quantities of a particular protein.
  • the protein is a cationic peptide
  • such peptides are very susceptible to proteolytic degradation, possibly due to their small size or lack of highly ordered tertiary structure.
  • coli as a fusion peptide with a truncated portion of the L-ribulokinase gene product
  • Piers et al.et al. Gene 134.1, 1993
  • expressed fusion proteins in E. coli that comprised glutathione-S- transferase and either defensin (HNP-1) or a synthetic cecropin-melittin hybrid while Hara et al., Biochem. Biophys. Res. C ⁇ mmun. 220 664, 1996, expressed silkworm moricin in £. coli as a fusion protein with a ⁇ -galactosidase or a maltose-binding protein moiety
  • One of the better options to avoid the toxic effects of a bacteriolytic peptide on the host bacterial cells in highly efficient production, and to avoid proteolytic degradation of the peptides, is to utilize the intrinsic bacterial host mechanism of driving heterologous proteins into inclusion bodies as a denatured insoluble form.
  • the present invention provides compositions and methods for expressing large quantities of a selected polypeptide.
  • large quantities of a selected polypeptide can be expressed utilizing a multi-domain fusion protein expression cassette which comprises a promoter operably linked to a nucleic acid molecule which is expressed as an insoluble protein, wherein the nucleic acid molecule encodes a polypeptide comprising the structure (cationic peptide) -[(cleavage site)-(cationic peptide)] court, wherein n is an integer having a value between 1 and 100.
  • a cleavage site may be inserted on either side of the structure, e.g.. (cleavage site)-(cationic peptide) -[(cleavage site)-(catiomc peptide)] register cleavage site, wherein // is an integer having a value between 1 and 100.
  • n is an integer having a value of 2, 3, 5, 10, or, 20 on the lower end, and 10, 15, 20, 30, 40, 50, 75, or 80 on the upper end (e.g., n may be an integer between about 2 and 30, 2 and 40, etc., 5 and 30, 6, or, 7 and 40, etc., up to 10 or 20 to 40, 50, 70 or 80). As an example, within one embodiment n has a value of between 5 and 40 or 10 and 40.
  • the nucleic acid molecule may further comprise a carrier protein.
  • a carrier protein to the extent that a carrier protein is to be expressed by the expression cassette, it can be located at either the N- terminus or the C-terminus of the fusion protein.
  • a wide range of carrier proteins can be utilized, including for example, a cellulose binding domain (CBD), or, a fragment of CBD.
  • CBD cellulose binding domain
  • the carrier protein can be greater than, equal to, or less than 100 amino acids in length.
  • the cleavage sites within the expression cassette can be cleaved by, for example, low pH, or, by a reagent such as cyanogen bromide, 2-(2-nitrophenylsulphenyl)-3-methyl-3'-brornoindolenine, hydroxylamine, o- iodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase, Staphylococcits aitreits V8 protease, endoproteinase Arg-C, or trypsin.
  • a reagent such as cyanogen bromide, 2-(2-nitrophenylsulphenyl)-3-methyl-3'-brornoindolenine, hydroxylamine, o- iodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase, Staphylococcits aitreits V8 protease, end
  • the expression cassette may more specifically be comprised of (a) a carrier protein, (b) an anionic spacer peptide component having at least one peptide with the structure (cleavage site)-(anionic spacer peptide), and (c) a cationic peptide component having at least peptide with the structure (cleavage site)-(cationic peptide) wherein the cleavage site can be on either side of the anionic spacer peptide or cationic peptide, and elements (a), (b), and (c) can be in any order and or number.
  • the expression cassette may be comprised of (a) an anionic spacer peptide component having at least one peptide with the structure (cleavage site)-(anionic spacer peptide), and (b) a cationic peptide component having at least peptide with the structure (cleavage site)-(cationic peptide), wherein the cumulative charge of said anionic spacer peptide component reduces the cumulative charge of said cationic peptide component.
  • an anionic spacer is included, such a spacer may have, 0,
  • cysteine residues there can be more, the same number, or fewer anionic spacers than cationic peptides in the fusion construct.
  • the anionic spacer is smaller in size than the cationic peptide.
  • cleavage sites can be utilized, including for example, a methionine residue.
  • promoters can be utilized, including for example the lacP promoter, tacP promoter. trcP promoter, srpP promoter, SP6 promoter, T7 promoter, araP promoter, trpP promoter, and ⁇ promoter.
  • the present invention also provides methods for producing fusion proteins utilizing the above-described expression cassettes.
  • such methods generally comprise the step of culturing a recombinant host cell containing an expression cassette, under conditions and for a time sufficient to produce the fusion protein.
  • suitable host cells include yeast, fungi, bacteria (e.g., E. coli), insect, and plant cells.
  • the fusion protein may be further purified and isolated. Further, the fusion protein may be cleaved into its respective components (e.g., utilizing low pH, or, a reagent such as cyanogen bromide, 2-(2- nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine, hydroxylamine, oiodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase, Staphylococcus aureiis V8 protease, endoproteinase Arg-C, or trypsin).
  • a reagent such as cyanogen bromide, 2-(2- nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine, hydroxylamine, oiodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase, Staphylococcus aureiis
  • the fusion protein or cleaved cationic peptide may be purified utilizing a chromatographic method (e.g., an anion chromatography column or resin).
  • a chromatographic method e.g., an anion chromatography column or resin.
  • the column can be charged with a base, and washed with water prior to loading the column with said cationic peptide.
  • the column can be equilibrated with water and up to about 8 M urea.
  • the cationic peptide is solubilized in a solution comprising up to about 8 M urea.
  • the cationic peptide is solubilized in a solution comprising a mild organic solvent, such as, for example, acetonitrile, or, an alcohol such as methanol or ethanol.
  • Figure 1 is the maps of ( A) plasmid pET21(+) (B) plasmid pET- CBD180, (C) a PCR fragment containing cbdl 80 and (D) plasmids pET21 CBD180-B and pET21 CBD180-X
  • Figure 2 shows maps of fusion poly cationic peptide genes
  • Figure 3 is an SDS-PAGE analysis showing the expression of different CBD-poly-MBI-1 1 fusion proteins Column ST molecular weight markers 14 4, 21 5, 31, 45, 66 2 and 97 4 kDa, Column 1 whole cell lysate of E. coli MC4100 (pGPl-2) cultivated at 30°C, Column 2 whole cell lysate of E. coli MC4100 (pGPl-2, pET21 CBD96-l 1) cultivated at 30°C, Column 3 whole cell lysate of induced E. coli MC4100 (pGPl -2, pET21CBD96-l 1 ) at 42°C, Column 4 whole cell lysate of E.
  • E. coli MC4100 ( ⁇ GPl -2, pET2l CBD96-2xl 1) cultivated at 30°C, Column 5 whole cell lysate of induced E. coli MC4100 (pGPl-2, pET21CBD96-2xl 1) at 42 U C, Column 6 whole cell lysate of E. coli MC4100 (pGPl-2) cultivated at 30°C, Column 7 whole cell lysate of E. coli MC4100 ( ⁇ GPl-2, pET21CBD96-3xl 1) cultivated at 30°C, Column 8- whole cell lysate of induced E.
  • Figure 5 presents maps of plasmid pET2l CBD96 and one of its inserts
  • Figure 6 shows maps of fusion multi-domain protein genes.
  • Figure 7 is SDS-PAGE analyses showing the results of fermentation of multi-domain clones having five or more MB1-1 1B7 copies
  • the upper panels represent the multidomain clones fused to CBD carrier
  • the lower panels show the multi-domain clones carrier-free
  • the left panels show the whole cell lysates, where the right side panels show the inclusion bodies partitioning step
  • the major band in each lane represents the relevant multidomain protein and the "x" numbers appearing at the bottom of each lane indicate the number of the MB1- peptide copies Numbers appearing along the left edge of the gels represents molecular weight standards (kD)
  • Figure 8 shows maps of portions of plasmids pET21-3s-5xl lB7 and pET21-5s-7xl lB7
  • Figure 9 is a chromatogram of the Q-Sepharose chromatography step for cationic peptide purification, which monitors UV absorbance at 280 nm and conductivity
  • Figure 10 is a schematic drawing that illustrates the construction of plasmids pETZ l CBD-X and pET 1 CBD-B
  • Figure 1 1 is a graph showing the results of reverse-phase analysis of the Q-Sepharose chromatography leading peak, representing pure cationic peptide
  • a C8-column (4 6 x 10, a-Pak. Waters) was equilibrated with 0 1%TFA in water at 1 ml/min flow rate Then 50 ⁇ l of Q-Sepharose chromatography leading peak material, diluted with 50 ⁇ l equilibration solution, was loaded on the column Elution was performed with a 0-45% gradient of solution B ( 0 1% TFA, 99 9% Acetonitrile) at 1% increase B per min, then step to 100% B
  • An approach to overcome the high basicity of the recombinantly- produced multi-domain cationic protein, and also decrease its toxicity, is to include small acidic peptide sequences in the linker sequences that neutralize the positive charge of the cationic peptide.
  • To keep the economic concept of high ratio of the cationic peptide in the multi-domain protein it is important to engineer the acidic peptide to be as small as possible, preferably smaller than the cationic peptide.
  • the natural phenomenon of a multipeptide precursor structure consisting of cationic peptide and anionic spacer has been described (Casteels-Josson et al (1993) EMBO J , vol.
  • apidaecin an antibacterial cationic peptide.
  • insects such as the honeybee (Apis me/liferaj.
  • Apidaecin is generated as a single gene comprising multiple repeated precursor units, each consisting of an apidaecin peptide gene ( 18 amino acids) preceded by an acidic spacer region (6-8 amino acids)
  • an acidic spacer region (6-8 amino acids)
  • Lee et al.. Protein Exp. Ptirif 12:53 expressed in E. coli six copies of the cationic peptide buforin II per fusion protein, which also included as acidic peptide modified magainin intervening sequences that alternated with the cationic peptide sequences.
  • the present inventors used carrier proteins of different sizes to express monomer and polymer forms of cationic peptides.
  • the test carrier protein of these studies were CBD and a fragment of the same derived from Clostridiitm celhilovorans cellulose binding protein A.
  • the chosen carrier protein fulfilled the requirements of high expression and accumulation in E. coli as insoluble forms. This approach was limited by a significant decrease in expression when the number of cationic fused peptide genes exceeded three copies. There was essentially no expression from vectors containing more than four copies of a peptide gene.
  • a new procedure was designed which allowed the multiplication of relevant cationic peptide genes using a specific anionic spacer sequence that encoded a negatively charged peptide.
  • anionic spacer peptide consisted of 1 1 amino acids.
  • Various genes encoding cationic peptide-anionic spacer peptide multi-domain proteins were constructed and fused to the carrier protein. A high level of expression was achieved for all constructs harboring more than thirty copies of the relevant cationic peptide gene.
  • polymers of cationic peptide genes with anionic spacers were liberated from the carrier and expressed directly. These constructs achieved high levels of expression and a high percentage of target cationic peptide in the carrier-free multi-domain protein.
  • a “structural gene” is a nucleotide sequence that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • mRNA messenger RNA
  • ' "nucleic acid” or " nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides. fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acids can be composed of monomers that are naturally-occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • An "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a cationic peptide that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • isolated polypeptide or protein is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, nucleic acid
  • the isolated polypeptide is sufficiently pure for clinical injection at the desired dose.
  • Whether a particular cationic polypeptide preparation contains an isolated cationic polypeptide can be determined utilizing methods such as Urea / acetic acid polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of the gel, reverse phase high pressure liquid chromatography, capillary electrophoresis, nucleic acid detection assays, and the Limulus Amebocyte Lysate test. Utilizing such a method an isolated polypeptide preparation will be at least about 95% pure polypeptide.
  • insoluble polypeptide refers to a polypeptide that, when cells are broken open and cellular debris precipitated by centrifugation (e.g., 10,000 g to 15,000 g), produces substantially no soluble component, as determined by SDS polyacrylamide gel with Coomassie Blue staining.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • a "cloning vector" is a nucleic acid molecule, such as a plasmid. cosmid. or bacteriophage. that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign nucleotide sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector Marker genes typically include genes that provide antibiotic resistance.
  • An "expression vector" is a nucleic acid molecule (plasmid, cosmid, or bacteriophage) encoding a gene that is expressed in a host cell. Typically, gene expression is placed under the control of a promoter, and optionally, under the control of at least one regulatory element. Such a gene is said to be “operably linked to” the promoter. Similarly, a regulatory element and a promoter are operably linked if the regulatory element modulates the activity of the promoter.
  • a “recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • cationic peptide refers to a peptide that possesses an isoelectric point (pi) of 9 and above.
  • a cationic peptide is at least five amino acids in length, and has at least one basic amino acid (e.g., arginine, lysine, histidine).
  • Cationic peptides commonly do not have more than 50 amino acids, and typically contain 10 to
  • a “carrier protein” is an amino acid sequence that can be individually expressed in host cells and, by recombinant fusion to a desired peptide or polypeptide can act as a carrier, enabling the expression of the desired peptide in host cells.
  • anionic spacer peptide domain is a peptide sequence that is sufficiently anionic to decrease the positive charge of an associated cationic peptide. That is, the combination of a cationic peptide and an anionic spacer peptide has a net charge that is essentially slightly positive, negative or neutral.
  • the size of an anionic spacer domain is similar to but preferably smaller than the size of the cationic peptide domain.
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a "multi- domain protein” comprises a combination of preferably more than one "cationic peptide domain,” and an equal, smaller or higher number of "anionic spacer peptide domains” with suitable cleax age sites for separating cationic peptide from the rest of the multi- domain protein.
  • the multi-domain protein can be fused to a earner protein to achieve higher expression and/or stability If stability and expression level of multi-domain protein are satisfactory there is no need to use a carrier protein
  • An anionic spacer peptide component comprises at least one anionic spacer peptide with a cleavage site
  • the cumulative charge of a cationic peptide component refers to the total charge of all cationic peptides that comprise the cationic peptide component Similarly the 'cumulative charge " of an anionic spacer peptide component refers to the total charge of
  • antimicrobial activity refers to the ability to kill or to prevent the growth of a microbe, or to kill or to prevent the growth of microbe-infected cells
  • microbe includes bacteria, fungi, yeast, algae, protozoa, and viruses This term includes but will not be limited to all these interpretive descriptions of the biological activity of the cationic peptide
  • cationic peptide include but are not limited to, naturally occurring cationic peptides and analogs thereof, cecropins, normally made by lepidoptera (Sterner et al , Nature 292 246, 1981) and diptera (Mer ⁇ field et al , Ctba Found S ⁇ mp 186 5, 1994), by porcine intestine (Lee et al., Proc Nat 'l Acad Set USA 56 9159, 1989), by blood cells of a marine protochordate (Zhao et al , FEBS Lett 412 144, 1997), synthetic analogs of cecropin A, melittin, and cecropin-mehttin chimeric peptides (Wade et al , Int J Pept Protein Res 40 429, 1992), cecropin B analogs (Jaynes et al , Plant Sa 89 43, 1993), chi
  • apidaecins produced by honey bee, bumble bee, cicada killer, hornet, yellow jacket, and wasp (Casteels et al. J. Bioi Chem. 269:26101, 1994; Levashina et al, Ettr. J. Biochem. 233:694, 1995), cathelicidins, such as indolicidin from bovine neutrophils (Falla et al., J. Bioi Chem. 2 .19298, 1996), bacteriocins, such as nisin (Delves- Broughton et al., Antonie van Leenuerihoek J.
  • Nucleic acid molecules encoding cationic peptides can be isolated from natural sources or can also be obtained by automated synthesis of nucleic acid molecules or by using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon known nucleotide sequences of cationic peptides. In the latter approach, a cationic peptide gene is synthesized using mutually priming long oligonucleotides (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 rd Edition, pages 8-8 to 8-9 (John Wiley & Sons 1995), "Ausubel (1995)").
  • PCR polymerase chain reaction
  • analogs of natural cationic peptides can also be recombinantly produced by the presently described methods.
  • the presence of a codon may have an adverse effect on expression and therefore a DNA sequence encoding the desired cationic peptide is optimized for a particular host system, in this case E. coli.
  • Amino acid sequences of novel cationic peptides are disclosed, for example, by Falla et ai, WO97/08199, and by Fraser et al, WO 98/07745.
  • One type of cationic peptide analog is a peptide that has one or more conservative amino acid substitutions, compared with the amino acid sequence of a naturally occurring cationic peptide.
  • a cationic peptide analog can be devised that contains one or more amino acid substitutions of a known cationic peptide sequence, in which an alkyl amino acid is substituted for an alkyl amino acid in the natural amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in the natural amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur-containing amino acid in the natural amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in the natural amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in the natural amino acid sequence, a basic amino acid is substituted for a basic amino acid in the natural amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in the natural amino acid sequence.
  • a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1 ) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • Nucleotide sequences encoding such "conservative amino acid” analogs can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
  • the antimicrobial activity of such analogs can be determined using a standard method, such as the assays described herein.
  • a cationic peptide analog can be identified by the ability to specifically bind anti-cationic peptide antibodies.
  • cationic peptide analogs should exhibit at least 50%, and preferably, greater than 70, 80 or 90%, of the activity of the corresponding naturally occurring cationic peptide.
  • a nucleotide sequence encoding a radish cationic peptide may include a codon that is commonly found in radish, but is rare for E. coli. The presence of a rare codon may have an adverse effect on protein levels when the radish cationic peptide is expressed in recombinant E. coli Methods for altering nucleotide sequences to alleviate the codon usage problem are well known to those of skill in the art (see, for example, Kane, Curt: Opin.
  • anionic spacer peptide As described below, an illustrative anionic spacer peptide has the amino acid sequence of HEAEPEAEPIM where the methionine residue can be used as a cleavage site. Similar naturally occurring examples of anionic spacer peptides include EAEPEAEP, EAKPEAEP, EAEPKAEP, EAESEAEP, EAELEAEP, EPEAEP and EAEP (Casteels-Josson, et al. EMBO J., 12: 1569-1578, 1993). Additional anionic spacer peptides are suitable for use in producing cationic peptides such as doubles or other combinations of those illustrated above.
  • the negative charge of the anionic spacer peptide should substantially reduce the positive charge of the cationic peptide in the multi-domain fusion proteins
  • a cleavage point must be present at which the multi-domain protein will be cleaved to give monomers of the desired peptide
  • the anionic spacer peptide is preferably smaller than the cationic peptide.
  • Such fusion proteins can be designed with alternating units of cationic peptide and anionic spacer peptide. Such a configuration, however, is not required. Any sequence of cationic peptide and anionic spacer peptide is acceptable, as long as the cumulative charge of the concatomer in the multidomain protein will not effect its expression in host cells.
  • a cellulose binding domain (CBD) carrier protein was used to illustrate methods for producing cationic peptides.
  • Additional suitable examples of carrier proteins include, but are not limited to, glutathione-S-transferase (GST), Staphylococcus aureus protein A, two synthetic IgG- binding domains (ZZ) of protein A, outer membrane protein F, ⁇ -galactosidase (lacZ), and various products of bacteriophage ⁇ and bacteriophage T7. From the teachings provided herein, it is apparent that many other proteins may be used as carriers. As shown by the use of the CBD fragment, an entire carrier protein need not be used, as long as it is highly expressed in the host cell.
  • suitable carrier proteins should be as small as possible, around 100 amino acid residues or preferably less. In certain cases, it is desirable to use a carrier protein that either lacks cysteine residues or that contains no more than one cysteine residue. It is also desirable to avoid methionine residues except in the cleavage site if CNBr reagent is to be used to release the linked peptide..
  • amino acids susceptible to cleavage can be used to bridge the carrier protein, a cationic peptide moiety, and an anionic spacer peptide moiety in the multi-domain .protein.
  • the determination and design of the amino acid sequence of the cleavage site is highly dependent on the strategy of cleavage and the amino acid sequence of the cationic peptide, anionic spacer peptide and carrier protein.
  • the removal of the cationic peptide can be accomplished through any known chemical or enzymatic cleavages specific for peptide bonds. Chemical cleavages include (R. A. Jue & R. F. Doolittle, Biochemistry, (1985) 24: 162-170; R.L.
  • Enzymatic cleavages which can be performed include, but are not limited to those catalyzed by Factor Xa, Factor Xlla, thrombin, enterokinase, collagenase, Staphylococcus aureus V8 protease (endoproteinase Glu-C), endoproteinase Arg-C, endoproteinase Lys-C, chymotrypsin or trypsin.
  • a nucleic acid molecule encoding the peptide must be operably linked to regulatory sequences that control transcription and translation (expression) in an expression vector and then introduced into a host cell.
  • expression vectors can include a marker gene which is suitable for selection of cells that carry the expression vector.
  • the expression vectors of the present invention comprise nucleic acid molecules encoding multi-domain fusion proteins with more than one copy of a cationic peptide gene.
  • multi-domain fusion proteins having a carrier protein domain, an anionic spacer peptide component, and a cationic peptide component may include from two to more than 30 copies, of a cationic peptide gene.
  • Multi-domain fusion proteins that lack an anionic spacer peptide component, but contain a carrier protein domain and a cationic peptide component include two to four copies of a cationic peptide gene.
  • multi-domain fusion proteins that lack a carrier protein domain, but include both anionic spacer peptide and cationic peptide components may include from five to more than 20 copies of a cationic peptide gene.
  • cationic peptides are produced in prokaryotic host cells.
  • Suitable promoters that can be used to express polypeptides in a prokaryotic host are well-known to those of skill in the art and for example include T4, T3, SP6 and T7 promoters recognized by specific phage RNA polymerases, the / ' /// , P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUl'5, tac, Ipp-lacSpr, phoA, lacP, tacP, trcP, srpP. araP, and lacZ promoters of E. coli, promoters of B.
  • siibtilis the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the bla promoter of the cat promoter.
  • Prokaryotic promoters have been reviewed by Glick, J. lnd. Microbi ⁇ i 7:277, 1987, Watson et ai, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995
  • Preferred prokaryotic hosts include E. coli, Bacillus subtilus, and Staphylococcits aureits.
  • Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH41, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH1 1 S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (Ed.), Molecular Biology Labfax (Academic Press 1991)).
  • Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA Cloning: A Practical Approach, Glover (Ed.) (IRL Press 1985)).
  • An illustrative strain of Staphylococcits aureus is RN4220 (Kreiswirth et al, Nature 305:709, 1983). The present invention does not require the use of bacterial strains that are protease deficient.
  • An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, microprojectile-mediated delivery, electroporation, and the like. Methods for introducing expression vectors into bacterial cells are provided by Ausubel (1995). Methods for expressing proteins in prokaryotic hosts are well-known to those of skill in the art (see, for example, Williams et a , "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
  • Cationic peptides can also be expressed in recombinant yeast cells. Promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available These vectors include Ylp-based vectors, such as YIp5.
  • YRp vectors such as YRpl7
  • YEp vectors such as YEp l3
  • YCp vectors such as YCp 19
  • the baculovirus system provides an efficient means to express cationic peptide genes in insect cells.
  • Suitable expression vectors are based upon the Autographa calif rnica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, 0 Autographa calif ornica nuclear polyhedrosis virus immediate-early gene promoter (ie- 1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • hsp Drosophila heat shock protein
  • Suitable insect host cells include cell lines derived from IPLB-S/-21, a Spodoptera frugiperda pupal ovarian cell line, such as S/9 (ATCC CRL 171 1), S/21AE, and S 21 (Invitrogen Corporation; San Diego, CA), as well as 5 Drosophila Schneider-2 cells.
  • S/9 ATCC CRL 171 1
  • S/21AE S/21AE
  • S 21 Invitrogen Corporation; San Diego, CA
  • 5 Drosophila Schneider-2 cells 5 Drosophila Schneider-2 cells.
  • Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et ai, "Manipulation of Baculovirus Vectors," in Methods in Molecular Biology, Volume ⁇ : Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168 (The Humana Press, Inc.
  • the recombinant host cells are cultured according to means known in the art to achieve optimal cell growth.
  • the bacteria are introduced into a suitable culture medium containing nutrient materials that meet growth requirements. After inoculation, the bacteria continue to divide and grow until reaching a concentration, saturation density. For example, shake flask fermentation may require 15-17 hours at 30°C to reach this point. Then the bacteria are diluted 1 :3 in fresh medium and allowed to grow to mid or late exponential phase, at which time synthesis of the cationic peptide is induced.
  • concentration, saturation density For example, shake flask fermentation may require 15-17 hours at 30°C to reach this point.
  • the bacteria are diluted 1 :3 in fresh medium and allowed to grow to mid or late exponential phase, at which time synthesis of the cationic peptide is induced.
  • the induction is obtained by raising the temperature to 42°C and maintaining it from about 1 to about 5 hours at a preferred pH range of 6.5-7.2.
  • the expression of the desired gene reaches optimum levels the bacteria are harvested and the cells are either frozen or continue through the recovery process.
  • Plasmid pET-CBD180 (see Shpigel et al., Biotech. Bioeng. 65: 17-23, 1999) was used as the source for the gene encoding the cellulose binding domain (CBD) carrier protein ( Figure IB).
  • a PCR reaction was designed to amplify a fragment containing the CBD 180 gene from pET-CBD180 as a 646 bp fragment ( Figure IC).
  • a BamWl restriction site (GGATCC) was incorporated at the 3'-end of the CBD180 PCR fragment.
  • the Bgf or Xbal sites of pET-CBD180 and BamHI were used to cleave the PCR fragment and two fragments were separately ligated into pET21a(+) resulting in two plasmids pET21CBD-B and pET21CBD-X, respectively ( Figure ID).
  • Plasmid pET21CBD-X contains lacO from pET21a(+), which improves the regulation of the T7 expression system.
  • Both plasmids contain a stop codon downstream of BamHI to allow expression of CBD 180 protein.
  • a T7 expression system was prepared in E.
  • coli MC4100 based on pGPl-2, which carries the T7 RNA polymerase gene under a ⁇ R promoter controlled by cI857 thermo-sensitive repressor. CBD 180 protein was expressed at high levels in both systems. Plasmid pET21CBD-X was used for subsequent studies.
  • Indolicidin is a natural 13-amino acid antimicrobial cationic peptide present in the cytoplasmic granules of bovine neutrophils and has a unique composition consisting of 39% tryptophan and 23% proline.
  • Initial studies used two cationic peptides derived from modifications of indolicidin, MBI-11 peptide (I L K K W P W W P W R R K) and MBI-1 1B7 peptide (I L R W P W W P W R R K), as described by Falla et ai, WO 97/08199, and by Fraser et ai, WO 97/07745.
  • a gene encoding the indolicidin-type cationic peptide MBI-11 was synthesized with BamrU and Hind ⁇ ll cloning sites, fused to CBD 180 carrier protein and expressed. The level of expression was high and equal to that of CBD 180 alone.
  • a tandem of two MBI-1 1 genes (2x1 1) was fused to CBD180, and again high expression was achieved.
  • the 177 amino acids of CBD180 were truncated to 96 amino acids and this v ersion of the carrier protein designated CBD96.
  • the DNA fragment carrying CBD96 was prepared by PCR using pET-CBD180 as a template, and cloned into pET21 a(-r) resulting in plasmid pET2lCBD96 Both single and double copies of the MBI-1 1 gene were fused to CBD96 and expressed at high levels Then poly genes containing up to ten MBI-1 1 units were prepared However expression was only achieved with a fusion protein containing four MBI-1 1 genes in tandem A dramatic decrease in expression was encountered when the number of genes exceeded three ( Figures 2 and 3)
  • vectors were constructed comprising multi-domain fusion proteins with small anionic peptide spacers between the cationic peptide domains This method of construction of multi-domain genes allows the polymerization of any cationic peptide gene without changing its amino acid sequence
  • the MBI-1 1B7 cationic peptide was used
  • One series of the multi-domain proteins comprises // times MBI-1 1B7 peptides and n-2 anionic spacer peptides.
  • // 5. the molecular weight of the multi-domain protein equals 13.46 kDa, which should be sufficiently large for expression in E. coli.
  • DNA fragments containing multi-domain genes of approximately this size were excised from relevant plasmids using restriction endonucleases Nde ⁇ and H/ ' /rdlll and fused into plasmid containing specifically designed leader 1 1 B7 domain.
  • the first methionine in all proteins is translated as formyl-methionine which cannot be cleaved by CNBr.
  • the carrier-free multi-domain proteins were modified in such a way that the first domain begins with M-T-M amino acids, allowing CNBr to cleave the first peptide at the second methionine and release authentic peptide.
  • the relevant portions of plasmids pET21 -3 S-5x 1 1 B7 and pET21 -5 S-7x 1 1 B7 are shown in Figures 8. All of the carrier-free multi-domain constructs containing from 5 to 14 copies of MBI-1 1B7 genes were expressed at high levels as shown in Figure 7. In the same way, constructs were prepared containing an equal number of 1 1B7 and anionic spacer domains with a spacer sequence at the end. They were also expressed at high levels. The theoretical yield of the MBI-11B7 peptide, within experimentally obtained multi-domain proteins, can be seen in Table 2.
  • the invention also provides an additional example of another antimicrobial cationic peptide (MBI-26), twice the size of the peptide described above (MBI-1 1B7), consisting of 26 amino acids, where seven of them are basic amino acids.
  • This peptide was artificially designed by a fusion between selected sequences of the natural antimicrobial cationic peptides cecropin and melittin.
  • the last amino acid serine at the carboxy end was replaced with a methionine residue, which was used for release of the peptide from the multi-domain protein.
  • the production of this peptide was obtained by recombinant synthesis in host cells, using the multi-domain protein method, as described above for MBI-1 1B7 peptide. Details are provided in Example 8.
  • Recombinant Host Cells General techniques for recovering protein produced by a recombinant host cell are provided, for example, by Grisshammer et al., "Purification of overproduced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 59-92 (Oxford University Press 1995), Georgiou, "Expression of Proteins in Bacteria," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc. 1996), Richardson (ed.), Baculovirus Expression Protocols (The Humana Press, Inc.
  • the present invention provides a novel, scaleable, cost-effective purification process for recombinant production of cationic peptides in host cells.
  • the multi-domain fused polypeptide forms an insoluble complex in E. coli called the inclusion body. After the bacteria are mechanically disrupted, these inclusion bodies can be separated from the soluble components of the cell, according to means known in the art such as filtration or precipitation Host impurities can be removed using solvents such as detergent (Triton X- 100), enzvme (hsozvme. DNAse) and salt
  • Cationic peptides can be released from anionic spacer peptides and carrier protein (such as truncated CBD) using standard techniques If methionine residues have been included at desired clea age points, for example, chemical cleavage with cyanogen bromide (CNBr) reagent in an acidic environment can be used
  • the reaction can be performed in 70% formic acid or 70% formic acid and 0 1 N HC1 or 70% TFA (t ⁇ fluoroacetic acid)
  • the reaction mixture is diluted in water, preferably 15 times the volume of the reaction mixture, and then dried At this stage, the carboxyl terminus of the cationic peptide is present as homose ⁇ ne lactone
  • the isoelectric point of polycationic peptides is very high ( 10 5-12 5) which enables the development of a very unique purification process using an anion exchange chromatography column under unusual conditions Almost any anion exchange resin coupled to weak or strong cation ligand, particularly those used for industrial purpose to purify proteins, peptides, carbohydrates and nucleic acids, can be used in the following purification process for cationic peptides
  • This procedure requires the use of only one chromatography step to obtain 95% purification
  • the advantages of this chromatography are that it is short, fast, does not require high pressure equipment and can be performed without organic solvents
  • the preferred procedure relies on dissolving the dried cleavage materials in 7- 8 M urea (alternatively in 50%) ethanol or water) and loading them onto an anion exchange column At this stage, the pH of the loading sample is acidic (pH 2-3)
  • the column is previously washed with two column volumes of 0 5-1 M NaOH and one short wash in water to a conduct
  • the flow through purified peptide will be in urea solution, which can be separated and further purified by high-throughput reverse phase chromatography using the perfusive supports Poros 20 or 50 R-2 resin (PerSeptive Biosystems Inc.). For mass production. Poros 50 is preferred due to better flow and the fact that the use of high pressure equipment is avoided..
  • the recombinant cationic peptide MBI- 1 1B7 was obtained from a multi-domain construct.
  • CNBr cleavage causes the formation of a homoserine lactone residue at the carboxy end which may be easily converted to homoserine by raising the pH.
  • This carboxy terminus is different from the bactericidal amidated chemical synthetic MBI- 1 1B7CN.
  • the antimicrobial activity was compared between chemically and recombinantly synthesized cationic peptide.
  • a cationic peptide there are various in vitro methods for determining the activity of a cationic peptide, including an agarose dilution MIC assay, a broth dilution, time-kill assay, or equivalent methods (see, for example, Shafer (ed.), Antibacterial Peptide Protocols (Humana Press, Inc. 1997)).
  • Antibiotic activity is typically measured as inhibition of growth or killing of a microorganism or a microorganism-infected cell.
  • a cationic peptide is first dissolved in Mueller Hinton broth supplemented with calcium and magnesium, and then this solution is mixed with molten agarose. Other broth and agars may be used as long as the peptide can freely diffuse through the medium.
  • the agarose is poured into petri dishes or wells and allowed to solidify, and a test strain is applied to the agarose plate. The test strain is chosen, in part, based on the intended application of the peptide. Plates are incubated overnight and inspected visually for bacterial growth.
  • a minimum inhibitory concentration (MIC) of a cationic peptide is the lowest concentration of peptide that completely inhibits growth of the organism.
  • Peptides that exhibit acceptable activity against the test strain, or group of strains, typically having an MIC of less than or equal to 16 ⁇ g/ml, can be subjected to further testing.
  • time kill curves can be used to determine the differences in colony counts over a set time period, typically 24 hours. Briefly, a suspension of organisms of known concentration is prepared and a cationic peptide is added. Aliquots of the suspension are removed at set times, diluted, plated on medium, incubated, and counted. MIC is measured as the lowest concentration of peptide that completely inhibits growth of the organism
  • Cationic peptides may also be tested for their toxicity to normal mammalian cells.
  • An exemplary assay is a red blood cell (RBC) (erythrocyte) hemolysis assay. Briefly, in this assay, red blood ceils are isolated from whole blood, typically by centrifugation, and washed free of plasma components. A 5% (v/v) suspension of erythrocytes in isotonic saline is incubated with different concentrations of cationic peptide. After incubation for approximately one hour at 37°C, the cells are centrifuged, and the absorbance of the supernatant at 540 nm is determined.
  • RBC red blood cell
  • a relative measure of lysis is determined by comparison to absorbance after complete lysis of erythrocytes using NH 4 C1 or equivalent (establishing a 100% value).
  • a peptide with less than 10% lysis at 100 g/ml is suitable.
  • the cationic peptide induces less than 5% lysis at 100 g/ml.
  • Cationic peptides that are not lytic, or are only moderately lytic, are desirable and suitable for further screening. In vitro toxicity may also be assessed by measurement of toxicity towards cultured mammalian cells.
  • Additional in vitro assays may be carried out to assess the therapeutic potential of a cationic peptide.
  • assays include peptide solubility in formulations, pharmacology in blood or plasma, serum protein binding, analysis of secondary structure, for example by circular dichroism, liposome permeabilization, and bacterial inner membrane permeabilization.
  • MBI-11B7CN MBI- 11B7HSL (homoserine lactone form)
  • MBI-1 1B7HS homoserine form
  • Cationic peptides can also be tested in vivo for efficacy, toxicity and the like.
  • the antibiotic activity of selected peptides may be assessed in vivo for their ability to ameliorate microbial infections using a variety of animal models.
  • a cationic peptide is considered to be therapeutically useful if inhibition of microorganism growth, compared to inhibition with vehicle alone, is statistically significant.
  • This measurement can be made directly from cultures isolated from body fluids or sites, or indirectly, bv assessing surv iv al rates of infected animals
  • animal models such as acute infection models including those in which (a) normal mice receive a lethal dose of microorganisms, (b) neutropenic mice receive a lethal dose of microorganisms, or (c) rabbits receive an inoculum in the heart, and chronic infection models
  • acute infection models including those in which (a) normal mice receive a lethal dose of microorganisms, (b) neutropenic mice receive a lethal dose of microorganisms, or (c) rabbits receive an inoculum in the heart, and chronic infection models
  • the model selected will depend in part on the intended clinical indication of the cationic peptide
  • mice are inoculated ⁇ or IV with a lethal dose of bacteria Tvpically, the dose is such that 90-100%) of animals die within two days
  • the choice of a microorganism strain for this assay depends, in part, upon the intended application of the cationic peptide Briefly, shortly before or after inoculation (generally within 60 minutes), cationic peptide is injected in a suitable formulation buffer Multiple injections of cationic peptide may be administered Animals are observed for up to eight days post-infection and the survival of animals is recorded Successful treatment either rescues animals from death or delays death to a statistically significant level, as compared with non-treatment control animals
  • ⁇ ⁇ vo toxicity of a peptide can be measured by administration of a range of doses to animals, typically mice, by a route defined in part by the intended clinical use The survival of the animals is recorded and LD, 0 , LD 90 10J , and maximum tolerated dose (MTD) can be calculated to enable comparison of cationic peptides Low lmmunogenicity of the cationic peptide is also a preferred characteristic for in ⁇ ⁇ vo use
  • peptides are injected into normal animals, generally rabbits
  • serum is obtained and tested for antibody reactivity to the peptide analogue
  • Antibodies to peptides may be identified by ELISA, immunoprecipitation assays, Western blots, and other methods (see, generally, Harlow and Lane, Antibodies A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 1988))
  • Expression vectors comprising the multi-domain fusion proteins described herein can be used to produce multi-domain fusion protein representing more than 25%o of the total protein of a recombinant host cell Since the multi-domain fusion proteins comprise multiple copies of a cationic peptide gene, the cationic peptide component of a fusion protein can be practically attained as more than 50%> of the fusion protein
  • Plasmid vector pET21 a(+) (Novagen Corporation, USA), a T7 expression plasmid, was used as the core plasmid for all expression systems ( Figures 1 A and 10).
  • the cellulose binding domain (CBD) from Clostridium cellulovorans was selected as a carrier protein for expression of antibacterial cationic peptides.
  • Plasmid pET-CBD180 (Shpigel et al., supra) was used as the starting material ( Figures IB and 10). Restriction enzymes except I ' spl and N.v/ ' l (Promega Corporation, USA), T4 D ⁇ A ligase and Taq polymerase were purchased from Pharmacia Biotech.
  • CBD The relevant part of CBD, including the T7 promoter of pET-CBD180, was amplified by PCR using 25 pmol each of each of the primers GCGT CCGG CGTA GAGG ATCG (SEQ ID ⁇ O: l) and CCGG GATC CAAT GTTG CAGA AGT AG (SEQ ID NO:2), 2 U of Taq DNA polymerase, corresponding reaction buffer ( lOx PCR reaction buffer: 500 mM KC1. 15 mM MgCL, 100 mM Tris-HCl, pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP, Pharmacia Biotech) and 20 ng of heat-denatured pET-CBD180.
  • PCR was performed in MJ-Research PTC- 100 Thermo-cycler in 50 ⁇ l reaction volume and 30 cycles of 94°C, 30 sec; 55°C, 30 sec. and 72°C, 30 sec.
  • a Ti mHI restriction site (GGATCC) was incorporated at the 3'-end of the cbd gene to allow it to be cloned into pET21a(+).
  • the Bgl ⁇ (AGATCT) or Xbal (TCTAGA) sites already present on pET- CBD180 were used to cut the 5'-end of the PCR fragment.
  • PCR product was digested in a 100 ⁇ l reaction containing 1.5 x OPA (Pharmacia Biotech assay buffer One-Phor-All is supplied at lOx concentration: 100 mM Tris-acetate, pH 7.5; 100 M magnesium acetate and 500 M potassium acetate) and 10 U of BamHI and 10 U of Hind ⁇ ll.
  • Plasmid pET21a(+) was digested in the same way, in 2 x 50 ⁇ l reactions each containing 0.25 ⁇ g of plasmid DNA, 1.5x OPA and 2 U of BamHI and Hindlll each. Reactions were stopped by phenol/CHCl, extraction and ethanol precipitation.
  • TB media Maniatis, et ai, Molecular Cloning: A Laboratory Manual 2 nd Ed., (Cold Spring Harbor Laboratory Press 1989) was added to the cell suspension and bacteria were incubated for 1 hour at 37°C with rigorous shaking Then 10, 50 and 100 ⁇ l of cell suspension were plated on MacKonkey agar (BBL, Becton Dickinson and Company, USA) plates with 100 ⁇ g/ml of Ampicillin and incubated overnight at 37°C. The next day, several colonies were transferred to 2 ml of TB and cultivated at 37°C with vigorous shaking overnight. Then plasmid DNA was isolated and analyzed, including DNA sequencing by methods known to those skilled in the art.
  • Plasmid pET21CBD-X contains lacO, which improves the regulation of the T7 expression system. Both plasmids contain a stop codon downstream oi BamHI to allow expression of CBD180 protein.
  • a T7 expression system was prepared in E. coli MC4100F (Strain MC4100F was prepared by mating E. coli XL 1 Blue and E.
  • coli MC4100 ATCC Number 35695
  • pGPl-2 which carries the T7 RNA polymerase gene under a ⁇ R promoter controlled by cI857 thermo-sensitive repressor (Tabor and Richardson, Biochemistry 52:1074, 1985).
  • CBD180 protein was expressed at high levels in both systems. Plasmid pET21CBD-X was used for subsequent work.
  • Plasmid pET21CBD96 ( Figure 5) was prepared using the same PCR conditions and cloning procedures. In this experiment the carrier protein CBD180 was truncated to about 96 amino acids. Therefore a pair of PCR primers GCGT CCGG CGTA GAGG ATCG (SEQ ID NO:3) and AT AT GGAT CCAG AT AT GTAT CATA GGTT GATG TTGG GC (SEQ ID NO:4) was used to prepare the relevant DNA fragment encoding cbd96 ( Figure 5), which was then cloned into pET21a(+). Then again a T7 expression system was prepared in E. coli MC4100F based on plasmids pET21CBD96 and pGPl-2 and protein CBD96 was expressed at high levels. pET21CBD96 was used for most of the subsequent work.
  • Sequences encoding all cationic peptides were designed from modified indolicidin, a natural anti-microbial peptide. Plasmids pET21CBD-X and pET21CBD96 (0.25 ⁇ g each) were digested with 2 U of BamHI and 2 U oi Hind ⁇ . in 1.5x OPA in 50 ⁇ l reactions at 37°C for 1 hour. In the same way, a fragment encoding MBI-11 was digested (Example 4) using about 1 ⁇ g of DNA and 25U of BamHI and Hindlll each in a 100 ⁇ l reaction. Both reactions were stopped by phenol/CHCK (Sigma-Aldrich Canada Ltd. ) extraction and ethanol precipitation.
  • coli MC4100F harboring plasmids pGPl -2 and pET21 CBD-l 1 or pET21CBD96-l 1 respectively were prepared by electroporation. Final strains were incubated overnight in 2 ml TB at 30°C with rigorous shaking and the next day 1 ml of cell suspension was diluted with the equal volume of fresh TB and cultivation temperature was increased to 42°C for a minimum of 2 hours. Samples of preinduced and induced cells were analyzed by SDS- PAGE. The level of expression of the fusion protein caring MBI- 1 lor 2x MBI- 1 1 gene was high and equal to expression of CBD 180 or CBD96 alone.
  • This experiment was designed to test how many peptide genes in tandem can be fused to a carrier protein and expressed. It was necessary to create two DNA fragments encoding MBI-1 1, one for polymerization by DNA cloning and another one as the last gene in the tandem. Therefore, the original DNA fragment encoding MBI- 11 peptide with COOH end was modified in order to create the last gene in tandem (Example 4) and a new gene was designed for a specific cloning procedure, which allowed construction of multiple tandem peptide genes fused to CBD 180 or CBD96 carrier proteins genes (Example 4). The cloning procedure resulted in addition of an extra isoleucine to the MBI-1 1 tandem sequences.
  • CBD 180 and CBD96 fused peptide polygenes of up to 10 units in tandem were prepared. However good expression was only achieved with a fusion containing two and three MBI- 1 1 domains and practically stopped when the number of peptide genes exceeded four. DNA synthesis and construction of plasmids containing MBI-1 1 polymers is described in Example 4.
  • DNA suitable for DNA cloning DNA suitable for DNA cloning.
  • TGAA AAAA TGG (SEQ ID NO:5) CCGT GGTG GCCG TGGC GTCG TAAA TAAG CTTG ATAT CTTG GTAC CTGC G (SEQ ID NO:6) was synthesized and used as a template for PCR using primers TTTA ACGG GGAT CCG TCTC ATAT G (SEQ ID NO:7) and TAAG CTTG ATAT CTTG GTAC CTGC G (SEQ ID NO:8).
  • the PCR was performed in MJ-Research PTC- 100 Thermo-cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C, 30 sec; 50°C, 30 sec.
  • PCR was used to modify the original DNA fragment encoding MBI-1 1 for use as the last gene in the tandem polypeptide gene.
  • the original oligonucleotide (A) was used as a template.
  • the sense primer TTTA ACGG GGAT CCGT CTCA TATG (SEQ ID NO:9) was identical to that used in the synthesis PCR reaction, but a new antisense primer CGCG AAGC TTAA TAAT ACAT AATT TTAC GACG CCAC GGCC ACCA CGGC (SEQ ID NO: 10) was designed to modify the end of the MBI-1 1 gene (for explanation, see Example 3).
  • the PCR was performed in MJ-Research PTC- 100 Thermo-cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C 30 sec, 51°C, 30 sec. and 72°C, 30 sec, 2 U of Taq DNA polymerase, corresponding reaction buffer (lOx PCR reaction buffer: 500 mM KC1, 15 mM MgC , 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol of the template oligonucleotide.
  • the PCR product was then cloned as a BamHl-HindUl fragment into pBCKS(+) (Stratagene, USA) resulting in plasmid pBCKS-1 1. Modification was verified by DNA sequencing.
  • oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATG ATCC TGAA AAAA TGGC CGTG GTGG CCGT GGCG TCGT AAAA TTAA TTGA ATTC GTCA TAGC TGTT TCCT GTGT GA (SEQ ID NO: 1 1) was synthesized and used as a template for PCR using primers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO: 12) and TCAC ACAG GAAA CAGC TATG AC (SEQ ID NO: 13).
  • Thermo-cycler was performed in MJ-Research PTC-100 Thermo-cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C, 30 sec, 51°C, 30 sec. and 72°C, 30 sec, 2U of Taq DNA polymerase, corresponding reaction buffer (lOx PCR reaction buffer: 500 mM KC1, 15 mM MgCL, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol of template oligonucleotide resulting in 114 bp dsDNA MBI-11 -BE fragment. This fragment was cloned as a BamHI - EcoRl insert into vector pBCKS(+) resulting in pBCKS-HBE.
  • MBI-1 1 designed for the polymerization cloning procedure was cloned into pET21CBD96-l l resulting in pET2lCBD96-2xl l .
  • pBCKS-HBE was digested with 2 U of BamHI and Vsp ⁇ in 2x OPA in 50 ⁇ l reactions at 37°C for 1 hour and pET21 CBD96- l 1 was digested w ith 2 U of BamHI and Ndel in 2x OPA in a 50 ⁇ l reaction at 37°C for 1 hour. Reactions were stopped by phenol/CHCl, extraction and ethanol precipitation.
  • the resulting DNA pellets were dissolved in 8 ⁇ l of water each and mixed, then 2 ⁇ l of 10 mM ATP. 2 ⁇ l of lOx OPA and 2 U of T4 DNA ligase were added and reactions were incubated at 10°C for 1 hour. Then 2 ⁇ l of the ligation mixture was used to electroporate 40 ⁇ l of E. coli XL1 Blue using Gene Pulser cuvettes (0.2 cm electrode gap) and Gene Pulser (Bio-Rad Laboratories) set to 2.5 kV, 200 ohms and 250 ⁇ F. After an electroporation pulse, 1 ml of TB media was added to the cell suspension and bacteria were incubated 1 hour at 37°C with rigorous shaking.
  • the PCR was performed in MJ-Research PTC- 100 Thermo-cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C, 30 sec; 55°C, 30 sec. and 72°C, 30 sec. 2 U of Taq DNA polymerase, corresponding reaction buffer ( l Ox PCR reaction buffer 500 mM KC1, 15 M MgCL 100 M Tris-HCl pH 9), 0 2 mM dNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol of template oligonucleotide resulting in 151 bp dsDNA MBI- 1 1 fragment.
  • reaction buffer l Ox PCR reaction buffer 500 mM KC1, 15 M MgCL 100 M Tris-HCl pH 9
  • 0 2 mM dNTPs dATP, dGTP, dTTP and dCTP
  • the PCR product was purified by phenol/CHCL extraction and ethanol precipitation
  • the resulting DNA was dissolved in 100 ⁇ l lx OPA , 20U of BamHI and 20U of Hm ' dWl and the reaction was incubated at 37°C for 2 hours.
  • the vector pBCKS(+) (0.25 ⁇ g) was digested in the same way. Both reactions were stopped by phenol/CHCl- extraction and ethanol precipitation.
  • oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATG ATTC TGCG TTGG CCGT GGTG GCCG TGGC GTCG CAAA ATGC ATAA GCTT CGAT CCTC TACG CCGG ACGC (SEQ ID NO: 17) was synthesized and used as a template for PCR using primers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO: 18) and GCGT CCGG CGTA GAGG ATCG (SEQ ID NO: 19).
  • the PCR was performed in MJ-Research PTC- 100 Thermo-cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C, 30 se ; 55°C, 30 sec. and 72°C, 30 sec, 2 U of Taq DNA polymerase, corresponding reaction buffer (lOx PCR reaction buffer: 500 mM KC1, 15 mM MgCL, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol of template oligonucleotide resulting in a 1 12 bp dsDNA MBI-1 1 fragment.
  • oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATG ACTA TGAT TCTG CGTT GGCC GTGG TGGC CGTG GCGT CGCA AAAT GCAT AAGC TTCG ATCC TCTA CGCC GGAC GC (SEQ ID NO.23) was synthesized and used as a template for PCR using primers CGCC AGGG TT TT CCCA GTCA CGAC (SEQ ID NO:24) and GCGT CCGG CGTA GAGG ATCG (SEQ ID NO:25).
  • the PCR was performed in MJ-Research PTC- 100 Thermo- cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C, 30 se ; 55°C, 30 sec. and 72° C, 30 sec, 2 U of Taq DNA polymerase, corresponding reaction buffer (lOx PCR reaction buffer: 500 mM KC1, 15 mM MgCL, 100 mM Tris-HCl, pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol of template oligonucleotide resulting in a 1 14 bp dsDNA MBI-1 1 fragment.
  • Plasmid pBCKS-V was prepared from pBCKS(+). The goal was to eliminate all Vspl restriction sites from the original plasmid and use the resulting plasmid for cloning of some of DNA cassettes. About 1 ⁇ g of pBCKS(+) was digested with V pl (Promega) in 50 ⁇ l reaction using l x OPA. The reaction was stopped by phenol/CHCL, extraction and ethanol precipitation. The resulting DNA was dissolved in 50 ⁇ l of lx OPA, 0.2 mM dNTPs and 1 U of Klenow polymerase. The reaction was incubated at 30°C, for 30 min. and then stopped by phenol/CHCL extraction and ethanol precipitation.
  • DNA was then dissolved in 50 ⁇ l of lx OPA, 0.5 mM ATP and 15U of T4 DNA ligase and the reaction was incubated at 10°C and after 4 hours stopped by incubation at 65°C for 30 min. Then 20 U of Vspl was added to the reaction to digest any remaining pBCKS(+) molecules and after 3 hours incubation at 37°C, 2 ⁇ l of the ligation mixture were used to electroporate 40 ⁇ l of E. coli MC4100F using a sterile Gene Pulser cuvette (0.2 cm electrode gap) and Gene Pulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 ⁇ F.
  • Plasmids pET21CBD96 (0.25 ⁇ g) and pBCKS-2xl lB7 (2.5 ⁇ g) were digested with BamHI and Hindl ⁇ l in 1.5x OPA in a 50 ⁇ l reaction at 37°C for 1 hour using 2 U of each restriction enzyme and 20U of each enzyme respectively. Both reactions were stopped by phenol/CHCL, extraction and ethanol precipitation. The resulting DNAs were dissolved in 8 ⁇ l of water and mixed, then 2 ⁇ l of 10 mM ATP, 2 ⁇ l of lOx OPA and 2 U of T4 DNA ligase were added and the ligation reaction was incubated at 10°C for 1 hour.
  • Positive clones pET21CBD96-2xl 1B7 contained tandem MBI-1 1 s ge k nes fused to cbd96.
  • serial cloning procedure The idea of the serial cloning procedure is that the insertion of the BamHI- MBI-1 l l-?-Vspl cassette into the BamHI - Ndel sites of pET21CBD96- 2x1 1B7 and subsequent multi-domain clones always eliminates the original Ndel site by NdellVspl ligation and a new Ndel site is introduced with each insertion, which together with BamHI is used for the next cycle of cloning.
  • Plasmid pET21CBD96-2xl lB7 (0.25 ⁇ g) was digested with 2 U of BamHI and Ndel in 2x OPA in 50 ⁇ l reaction at 37°C for 1 hour.
  • Plasmid pBCKS-V- 1 1B7S (2.5 ⁇ g) was digested in a 100 ⁇ l reaction with 20 U of BamHI and Vspl in 2x OPA at 37°C for 1 hour. Both reactions were stopped by phenol/CHCL extraction and ethanol precipitation.
  • the resulting DNAs were dissolved in 8 ⁇ l of water and mixed, then 2 ⁇ l of 10 mM ATP, 2 ⁇ l of lOx OPA and 2 U of T4 DNA ligase were added and the ligation reaction was incubated at 10°C for 1 hour. Then 2 ⁇ l of the ligation mixture were used to electroporate 40 ⁇ l of E. coli XLl Blue using a sterile Gene Pulser cuvette (0.2 cm electrode gap) and Gene Pulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 ⁇ F. After an electroporation pulse, 1 ml of TB media was added to the cell suspension and bacteria were incubated 1 hour at 37°C with rigorous shaking.
  • pBCKS-V- 5xl lB7S was prepared and each cloning cycle would add five 1 1 B7S domains.
  • 1 1B7S insert of pBCKS-V-1 1B7S was recloned into pTZ18R, resulting in pTZ18R- 1 1B7S.
  • this plasmid was used as the donor of the 1 1B7S domain for the serial cloning into pBCKS-V-1 1B7S using the BamHI- NdeW.spl strategy.
  • the serial cloning procedure was repeated four times resulting in pBCKS-V-5S-5xl 1B7S.
  • the 5S- 5x1 1B7 cassette was then used for construction of CBD96-fused systems containing more than fifteen 1 1 B7 domains and also CBD96-fused multidomain systems with equal numbers of 1 1B7 and anionic spacer domains (Table 2).
  • the cassette 5S-5xl lB7 of pBCKS-V-5S-5xl 1B7 with anionic spacer domain at the end was cloned into pET21CBD96 using BamHI and Kpnl restriction enzymes resulting in pET21 CBD96-5S-5xl 1B7.
  • the same cassette was ligated as BamHl-V.spl fragment of pBCKS-V-5xl 1B7S into BamH -Ndel sites of pET21CBD96-5S-5xl lB7 resulting in pET21CBD96-10S-10xl 1B7. This can be repeated several times to receive constructs with 15, 20, 25 etc. 11B7 domains and equal numbers of anionic spacer domains. Conditions for restriction enzymes, ligation, electroporation and analysis of recombinant plasmids are described above.
  • the first amino acid in all proteins is f-methionine.
  • this amino acid is not cleaved by CNBr, which means that one peptide domain released from a multi-domain protein would start with f-methionine.
  • the solution was to create a modified MBI-11 cassette encoding f-methionine and methionine in tandem at the beginning of the peptide, so the second one would be cleaved by CNBr.
  • the result was the synthesis of the special first domain in multi-domain genes, cassette MBI-1 1B7F, encoding MTM amino acids at the beginning. This domain was fused to the spacer domain in pBCKS-V-S resulting in plasmid pBCKS-V-1 1B7S-F.
  • Plasmid pBCKS-V-1 1B7S-F and the relevant pET21CBD96-multi- domain-11B7 plasmids were used for construction of unfused multi-domain MBI-11B7 genes.
  • Multi-domain genes were liberated from cbd96 by Ndel - Xhol digestion and cloned into the spl - Xliol sites of pBCKS-V- 1 1 B7S-F downstream of the 1 1 B7S insert. This created a line of unfused multi-domain 1 1 B7 genes in plasmid pBCKS-V. These genes were then recloned as Ndel - Xho fragments into pET21 a(+) resulting in a series of pET plasmids capable of expression of multi-domain proteins using the T7 promoter system.
  • Plasmid pBCKS-V- 1 1 B7S-F (0.25 ⁇ g) was digested with 2 U of Ndel and Xhol in 2x OPA in several 50 ⁇ l reactions at 37°C for 1 hour.
  • Relevant plasmids pET21CBD96-multidomain- l 1B7 (2.5 ⁇ g) were digested in 100 ⁇ l reactions with 20 U of Ndel and Xhol in 2x OPA at 37°C for 1 hour. All reactions were stopped by phenol/CHCL, extraction and ethanol precipitation.
  • the resultant vector and insert D ⁇ As were dissolved in 8 ⁇ l of water and mixed, then 2 ⁇ l of 10 mM ATP, 2 ⁇ l of lOx OPA and 2 U of T4 D ⁇ A ligase were added and ligation reactions were incubated at 10°C for 1 hour. Then 2 ⁇ l of each ligation mixture was used to electroporate 40 ⁇ l of E. coli XLl Blue using a sterile Gene Pulser cuvette (0.2 cm electrode gap) and Gene Pulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 ⁇ F.
  • plasmid D ⁇ A was isolated and analyzed, including D ⁇ A sequencing by methods known to those skilled in the art. Positive clones contained pET21 -multidomain- 1 1 B7 plasmids containing 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 and 21 MBI-1 1B7 domains.
  • constructs were prepared containing equal numbers of 1 1B7 and anionic spacer domains.
  • pET21CBD96-5S-5xl lB7 was digested with BamHI and Xhol (or H///dIII) and fragment 5S-5xl lB7 was ligated into BamHl-XhoI (or wdlll) of P BCKS-V-1 1B7S-F resulting in pBCKS-V-6S- 6x1 1B7.
  • CCGT CTCA TATG ACCA TGAA ATGG AAAT CTTT CATC AAAA AACT GACC TCTG CTGC TAAA AAAG TTGT TACC ACCG CTAA ACCG CTGA TCTC TATG CATG CTTA AGCT TCGA TCCT CTAC GCCG GACG C (SEQ ID NO: 26) was synthesized and used as a template for PCR using primers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO: 18) and GCGT CCGG CGTA GAGG ATCG (SEQ ID NO: 19).
  • PCR was performed in an MJ-Research PTC- 100 Thermo-cycler in a 50 ⁇ l reaction volume with 30 cycles of 94°C, 30 sec; 55°C, 30 sec. and 72°C, 30 sec, 2 U of Taq DNA polymerase, corresponding reaction buffer (lOx PCR reaction buffer: 500 mM KC1, 15 mM MgCL, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol of template oligonucleotide resulting in a 1 12 bp dsDNA MBI26 fragment.
  • reaction buffer 500 mM KC1, 15 mM MgCL, 100 mM Tris-HCl pH 9
  • 0.2 mM dNTPs dATP, dGTP, dTTP and dCTP
  • the resulting DNA fragment was cloned into pTZ18R as a BamHl-Hindlll fragment as described in Example 2, paragraph (A) resulting in plasmid pTZ18R-26GT.
  • the 7f ⁇ /??HI-H//?dIII mbi26 fragment was recloned into pBCKS(+) resulting in pBCKS-26GT.
  • the first step in construction was a direct fusion of the mbi26 cassette to cbd96 in pET21CBD96.
  • Plasmids pET21CBD96 (0.25 ⁇ g) and pBCKS-26GT (2.5 ⁇ g) were digested with BamHI and Hindlll in 1.5x OPA (50 ⁇ l reaction volume) at 37°C for 1 hour using 2 U of each restriction enzyme and 20U of each enzyme respectively. Both reactions were stopped by phenol/CHCl, extraction and ethanol precipitation.
  • Each resulting DNA was dissolved in 8 ⁇ l of water; the two were mixed together with 2 ⁇ l of 10 mM ATP, 2 ⁇ l of lOx OPA and 2 U of T4 DNA ligase and the ligation reaction was incubated at 10°C for 1 hour.
  • 2 ⁇ l of the ligation mixture was used to electroporate 40 ⁇ l of E. coli XL l Blue using a sterile Gene Pulser cuvette (0.2 cm electrode gap) and Gene Pulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 ⁇ F. After an electroporation pulse, 1 ml of TB media was added to the cell suspension and bacteria were incubated 1 hour at 37°C with rigorous shaking.
  • the second step was preparation of a cassette for the serial cloning procedure.
  • the mbi26 fragment of pTZ18R-26GT was cloned into pBCKS-V-S as a BamHl-Nsil fragment basically as described in Example 5 (A), resulting in plasmid pBCKS-V-26S with an mbi26 domain fused to the anionic spacer-encoding sequence.
  • the only exception was that 2x OPA vvas used in the restriction enzyme digest reaction.
  • the insert was then cloned into pTZ18R resulting in pTZ18R-26S. This allowed the cloning of the BamHl-26S-Vspl insert into the BamHl-Nde sites of pBCKS-V-26S, resulting in pBCKS-V-2S-2x26.
  • the third step was the actual serial cloning procedure (for details see Example 6B). Briefly, pBCKS-V-26S was digested with BamHI and Vspl resulting in fragment BamHl-26S-Vspl, which was ligated into plasmid ⁇ ET21 CBD96-26GT digested with BamHI and Ndel. Positive clones pET21CBD96-ls-2x26 contained two MBI-26 units with one spacer fused to cbd96. This was the first cycle of the serial cloning. In the next cycle pET21CBD96-ls-2x26 and pBCKS-V-26S could be used to prepare pET21CBD96-2s-3x26 and so on.
  • Plasmid pET21CBD96-lS-3xl lB7 was used as a vector for serial cloning of the mbi26S domain of pBCKS-V-26S. Briefly, pBCKS-V-26S was digested with BamHI and Vspl restriction endonucleases resulting in fragment BamHl-26S-Vspl, which was ligated into plasmid pET21CBD96-lS-3xl lB7 digested with BamHI and Ndel. Positive clones pET21CBD96-2S-26-3xl lB7 contained an MBI-26 unit with one spacer fused to three 1 1B7 units with one spacer.
  • TB broth which is prepared as follows: 12 g of Trypticase Peptone (BBL), 24 g of yeast Extract (BBL) and 4 ml of glycerol (Fisher) is added to 900 ml of Milli-Q water. The material is allowed to dissolve and 100 ml of 0.17 M KH 2 PO 4 (BDH), 0.72 M K : HPO 4 (Fisher) is added . The broth is autoclaved at 121°C for 20 minutes. The resulting pH is 7.4.
  • the culture was then transferred to a 2.0 L flask with 330 ml fresh TB medium (no antibiotics), preincubated at 30°C. After dilution, protein expression was induced by raising the culture temperature to 42°C and shaking at 300 rpm for another 5 to 7 hours. The pH was kept between 6.7 and 7.1 using 30%> ammonium hydroxide. Bacteria were fed at least twice during induction with 0.5 g glucose per flask. Cells were harvested by centrifugation (Sorvall® RC-5B) at 15,000x for 15 minutes and cell pellets were stored at -70°C prior to cell lysis,
  • the bacteria produce the multi-domain proteins as insoluble inclusion bodies.
  • the harvested cells were suspended in 200 ml buffer (50 mM Tris-HCl. 10 mM EDTA, pH 8 0) and lysed ' by sonication (Vibra-CellTM, Sonic and material Inc.) five times for 45 seconds, . on ice, then centrifuged (Sorvall® RC-5B) at 21,875 ⁇ # for 15 min at 4°C. The pellet was homogenized (PolyScience, Niles, IL USA) in 160 ml of lysis buffer (20 M Tris-HCl, 100 ⁇ g/ml lysozyme. pH 8.0) and incubated at room temperature for 45 min.
  • Triton X-100 was added ( 1% v/v), and the mixture was homogenized thoroughly and centrifuged at 21,875 ⁇ for 15 min at 4°C.
  • the inclusion bodies pellet was resuspended in 200 ml of 0.1 M NaCl, homogenized, and precipitated by centrifugation as described above, then resuspended in 200 ml water and precipitated again by centrifugation. At this stage, the inclusion bodies contained greater than 70%> fusion protein.
  • the isolated inclusion bodies were dissolved in 70%> formic acid (100 mg wet weight IB per ml), then CNBr was added to a final concentration of 0.1 to 0.15 M.
  • the cleavage reaction which allowed the release of cationic peptide from the fusion protein and spacer was performed under nitrogen and with stirring, in the dark, for 4 hr.
  • the reaction mixture was diluted with 15 volumes of Milli-Q water and dried in a rotovap machine (Rotovapore, R-124VP, BUCHI Switzerland). The dried pellet was then dissolved in 10 ml of 7-8. M urea and insoluble materials were separated by centrifugation at 21,875 ⁇ g for 15 min.
  • the soluble materials at acidic pH (2-3.3) and low conductivity (1-5 mS), contain the homoserine lactone form of the cationic peptide. This material was further purified using a chromatography procedure.
  • the purification of the homoserine lactone form of MBI-1 1B7 peptide was performed on a BioSysTM 2000 chromatography work station (Beckman Instruments, Inc.), using Fast Flow Q-Sepharose anion exchange resin (Pharmacia Biotech AB) packed in an XK column (1.6 x 11 cm).
  • the column was equilibrated with 2 column volumes (CV) of 1 M NaOH at a flow rate of 9 ml/min, followed by a water wash. Conductivity, pH and absorbency at 280 nm were monitored. When the conductivity dropped below 5 mS, the dried cleavage materials, dissolved in 7-8 M urea, were loaded onto the column and washed with 4 M urea.
  • the flow-through peak was collected and pooled and the pH was adjusted to 7.0-7.5 with 0.2 N HC1.
  • the sample was analyzed for purity by reverse phase HPLC ( Figure 1 1), using a C8 column (4.6x10, Nova-Pak, Waters) and by acid- urea gel electrophoresis (West and Bonner, Biochemistry 79:3238, 1980).
  • the identity of the MBI-1 1B7 peptide was confirmed by mass spectrometry to show that the flow through peak represents the homoserine form of the MBI-11B7 peptide.
  • the separation of the urea from the purified peptide utilized a high- throughput reverse phase chromatography technique by using the BioCADTM (PerSeptive Biosystems Inc.) perfusion chromatography workstation and Poros® R-Il 20 column, 4.6 x 100 mm (PerSeptive Biosystems Inc.). About 10 mg of the peptide were applied on the column at 5 ml/min, followed by equilibration of the column with 0.1% TFA. The peptide was eluted from the column by a gradient of increasing acetonitrile from 0 to 50% for 10 minutes at a flow rate of 5 ml/min. The peak of the further purified and urea free peptide was collected and lyophilized.
  • the antimicrobial activities of the chemically synthesized MBI-11B7CN peptide and recombinant DNA synthesized MBI-11B7HSL (homoserine lactone form) and MBI-11B7HS (homoserine form) peptides were tested against various gram- negative and positive strains of bacteria, including antibiotic resistant strains.
  • the Agarose Dilution Assay was performed as described in "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically-Fourth Edition; Approved Standard" NCCLS document M7-A4 ( ISBN 1-56238-309-4) Vol. 17, No 2 (1977).
  • the agarose dilution assay measures antimicrobial activity of peptides and peptide analogues, which is expressed as the minimum inhibitory concentration (MIC) of the peptides.
  • the bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard (PML Microbiological) and then diluted 1 : 10 before application on to the agarose plate.
  • the final inoculum applied to the agarose is approximately 10 4 CFU in a 5 - 8 mm diameter spot.
  • the agarose plates are incubated at 35°C - 37°C for 16 to 20 hours.
  • the MIC is recorded as the lowest concentration of peptide that completely inhibits growth of the organism as determined by visual inspection.
  • Representative MlCs for the cationic peptides against various bacterial strains are shown in Table 3.

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Abstract

La présente invention concerne des procédés permettant de produire avec efficacité des peptides cationiques à partir de cellules hôtes de recombinaison. En effet, chez les mammifères, les oiseaux, les amphibiens, les insectes et les végétaux, les peptides antimicrobiens cationiques endogènes constituent les composants ubiquistes des défenses contre les hôtes, et l'administration de ces peptides cationiques donne également de bons résultats thérapeutiques. Néanmoins, le coût de production de ces agents constitue un frein à l'utilisation thérapeutique de ces peptides cationiques. L'invention propose ainsi d'utiliser la chromatographie à échange anionique pour accélérer l'extraction, dans les protéines des cellules hôtes, de ces peptides cationiques produits par recombinaison.
EP99955614A 1998-11-20 1999-11-19 Renforcement de la production de peptides cationiques antimicrobiens dans des cellules hotes Withdrawn EP1131448A2 (fr)

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WO2010139736A2 (fr) * 2009-06-03 2010-12-09 Basf Se Préparation de peptides de manière recombinée
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