Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
• • 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
  • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates generally to molecular biology and biochemistry, more particularly related to modified ⁇ bosome inactivating proteins from pokeweed plant
• the pokeweed plant is also known as Phytolacca amet icana and the pokeweed ⁇ bosome inactivating protein is also called pokeweed antiviral protein, often abbreviated "PAP"
• PAP pokeweed antiviral protein
• PAP pokeweed antiviral protein
• conjugates of other natural toxins such as ⁇ cm and diphtheria toxin
• other natural toxins such as ⁇ cm and diphtheria toxin
• Natural (also referred to as "native") PAP is isolated fiom the pokeweed plant, and while attempts have been made to utilize natural PAP in a compound-toxin conjugate, such attempts have not proved reliable As would be expected, variability in isoforms, from year to year and batch to batch, proved onerous and unworkable in the context of pharmaceutical quality control Moreover, some isoforms did not conjugate, and different isoforms conjugated differently from each other
• this invention provides compositions comprising recombinant pokeweed antiviral proteins having a free cysteine, preferably a terminal cysteine, more preferably an N-terminal cysteine Also provided aie those rPAP molecules wherein the PAP is a full length rPAP, more preferably a full length rPAP composing a free cysteine, most preferably a full length rPAP comp ⁇ smg a free cysteine and an ammo acid linker Preferred are those rPAP molecules comprising an N-terminal Cys and an amino acid linker, most preferably those which have at least one iepeat of GIy- Gly-Gly-Gly-Ser More preferred are Cys-Gly-Gly-Gly-Gly-Ser-full length rPAP and Cys-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Ser-full length rPAP
• the present invention provides rPAP which does not kill host cells when expressed according to the present methods rPAP utilized in the present compositions and methods is preferably equal to or greater than 29 5 Daltons, more preferably equal to or greater than 30 Daltons, most preferably equal to or greater than 30 5 Daltons
• compositions and methods that utilize full length rPAP having a molecular weight equal to or greater than 31 5, 31 75 and 32 Daltons
• Full length rPAP proteins that which equate to the molecular weight of a natural PAP that has not been post-translationally modified is the preferred material used in the present invention
• nucleic acids comprising the inventive nucleic acids and proteins, with a preferred cell being E coli
• conjugates having the structure
• X-Y-Z wherein X is full length rPAP having a free cysteine, Y is absent or a chemical linker, and Z is a compound
• Preferred methods are those as desc ⁇ bed, wherein said chemical bond is induced via a hetero-bifunctional crosshnker, more preferably those wherein the chemical bond is induced between the free cysteine and a maleimido group on the compound
• the hetero-bifunctional crosshnker is GMBS, and/or the compound is d-lys 6 -gonadotropm releasing hormone
• Also provided are methods to grow cells comprising: incubating cells transformed with nucleic acid comprising full-length rPAP, wherein the rPAP is under the control of a T7 promoter system, and wherein said T7 promoter system has RNA polymerase under the control of an arabinose promoter.
• the full length rPAP is selected from the group consisting of: a chemically-modified rPAP, a natural variant rPAP, and a genetically-engineered rPAP.
• conjugates comprising the PAP compositions herein.
• X-Y-Z wherein X is full length rPAP having N-terminal Cys-Gly-Gly-Gly-Gly-Ser; Y is a chemical linker, and Z is a protein.
• Free cysteine means any cysteine other than one which is bound to another cysteine via a di-sulfhydryl bond.
• free cysteine includes cysteines that are bound to another residue or compound, so long as the cysteine is not bound to another cysteine via a di-sulfhydryl bond.
• Full length rPAP means any recombinant PAP which has toxin activity and has a molecular weight greater than or equal to 29,500 Daltons.
• Figure 1 is a stylized depiction of the structure of a full length pokeweed antiviral protein showing the C-terminal residues that are ordinarily cleaved in the plant during post translational processing, and additional, non-naturally-occurring, N-terminal amino acid residues.
• Figure 2 is a stylized depiction of the structure of: a pokeweed antiviral protein showing C-terminal that are ordinarily cleaved in the plant during post translational processing; and additional, non-naturally-occurring, N-terminal amino acid residues; a N-terminal linker; and an exemplary compound, modified gonadotropin releasing hormone (GnRH).
• GnRH modified gonadotropin releasing hormone
• Figure 3 is a stylized depiction of the structure of a recombinant non- cleavable (abbreviated "nc" and meaning that the linker does not possess a disulfide cleavage site) pokeweed conjugate, with detail at the amino terminus showing a linker and modified gonadotropin releasing hormone.
• nc non- cleavable
• Figure 4 is a stylized depiction of the interaction of gonadotropin releasing hormone and the extracellular domain of the gonadotropin releasing hormone receptor.
• Figure 5 is a graph depicting biological activity of rPAP as measured by inhibition of protein synthesis of a luciferase mRNA in rabbit reticulocyte lysate assay.
• Figure 6 is a graph depicting the results of a competitive radio-immuno receptor binding assay. The curve closest to the X axis reflects the data for the standards. The next curve reflects the data for a d-lys 6 -GnRH-GMBS-PAP, wherein the PAP was purified from plant parts (also known as a natural or native PAP).
• Figure 11 Sequence of expressed rPAP, with internal disulfide bonding cysteines denoted in large bold type: Cys-34 binding to Cys-258 and Cys-84 binding to Cys-105; Bold; engineered linker with N-terminal cysteine; VNTII....: native PAP sequences; Bold and underlined is C-terminus of natural pre-PAP (post-translationally cleaved in the plant).
• the present invention provides a recombinant pokeweed antiviral protein that is expressible at high yields in E. coli, and which has 30 to 40 times greater specific activity (biological activity/unit mass) than any other recombinant PAP. Moreover, the present invention provides methods for producing rPAP in pharmaceutical quantities.
• the present rPAP materials proteins, nucleic acids, constructs, cells, etc. may be used to produce rPAP conjugates having rPAP and a targeting compound bound to them, either via a linker or directly. In one embodiment, the rPAP has a free cysteine, for optional use in linking a linker to another compound.
• the present rPAP proteins provide a convenient N-terminal cysteine for such purposes, although the use of the present rPAP is not limited to N-terminal conjugation.
• the rPAPs of the present invention may be used as a toxin without conjugation or may be conjugated via a free cysteine, at a terminal cysteine, or at an internal cysteine.
• the rPAP molecules described herein are active in the rabbit reticulocyte lysate assay, with or without linker or targeting compounds conjugated to them.
• the present invention includes methods to express, refold, conjugate and purify recombinant PAP. Several obstacles were overcome to achieve successful expression.
• rPAP in non-pokeweed host cells is a toxin and kills the host cells. Attempts were made to express the mature (post-translationally cleaved) PAP in E. coli, using the T7 system. The cells grew poorly, if at all, and showed distress prior to induction of the rPAP protein. Subsequently, attempts to express the full length rPAP (the mature PAP plus the C-terminal portion that is ordinarily cleaved post translationally in the plant) using the T7 inducible promoter system in E. coli were also unsuccessful. The cells also showed distress during the growth phase and prior to induction of the rPAP.
• the protein may optionally be purified by a variety of methods including ion exchange chromatography, hydrophobic interaction chromatography, and hydroxyapetite chromatography, all of which are well-described in the art.
• the preferred method is cation exchange chromatography, particularly as described in Example 5.
• the specific activity (biological activity/unit mass) of the inventive rPAPs are 30-40x more active in inhibiting protein translation in a rabbit reticulocyte lysate than another, reported, rPAP.
• the rPAP concentration was determined by rPAP- specif ⁇ c radioimmune assay, which is very sensitive, and can detect sub-nanomolar amounts of rPAP.
• Recombinant PAP proteins ideally folded so as to retain toxin function, preferably those retaining the natural disulfide bridges of the naturally-occurring cysteines, and preferably those having at least one free cysteine (eg. one that is not present in a naturally-occurring sequence), most preferably a terminal free cysteine capable of selectively binding other compounds, are provided herein.
• any PAP sequence is appropriate for use as a starting material in the present invention. Any known isotype, or any that becomes apparent will be useful for preparing the present invention.
• Full length PAP has the following amino acid sequence at the C-terminus:
• YNQNAMFPQLIMSTYYNYVNLGDLFEGF-COOH This sequence is ordinarily cleaved in the pokeweed plant post-translationally but is retained in preferred embodiments of the present invention.
• Naturally-occurring, post-translationally-cleaved PAP has a molecular weight of 29,308.5 daltons.
• compositions as described above which are selected from the group consisting of SEQ ID NO: 1; a protein which comprises a free cysteine and is at least 90% identical to SEQ ID NO: 1 using the BLAST software version 2.2.21 on default settings; a protein which is encoded by SEQ ID NO: 2; a protein comprising a free cysteine and is encoded by a nucleic acid which is at least 90% identical to SEQ ID NO: 2 using BLAST version 2.2.21 software on default settings.
• sequence identity is selected from the group consisting of: 95%; 96%; 97%; 98%; and 99%.
• nucleic acids selected from the group consisting of SEQ ID NO: 1; a protein which comprises a free cysteine and is at least 90% identical to SEQ ID NO: 1 using the BLAST software version 2.2.21 on default settings.
• nucleic acid which is at least 85% identical to SEQ ID NO 2 using the BLAST software version 2 2 21 on default settings and encodes a protein having a free cysteine
• a nucleic acid which encodes SEQ ID NO 1 a nucleic acid which encodes SEQ ID NO 1
• compositions as above wherein the sequence identity is selected from the group consisting of 90%, 95%, 96%, 97%, 98%, and 99%
• a preferred nucleic acid comprises a nucleic acid which encodes the proteins herein
• a more preferred embodiment of this method is one wherein said non-aqueous condition comprises the steps of solubihzmg GMBS in methanol to create a first non-aqueous solution, solubihzmg d-lys6-gonadotropm releasing hormone in methanol to create a second non-aqueous solution, mixing said first and second non-aqueous solutions at a molar ratio of 1 1 1
• those rPAPs which are at least 90% identical, preferably at least 95% identical, most preferably at least 99% identical to SEQ ID NO 1 are useful m the present methods Those that also comp ⁇ se a free CYS residue are most useful Moreover, conserved sequences should not be changed, and non-conserved sequences are optionally changeable In PAP, the disulfide bonds between naturally occurring cysteines provide the tertiary structure necessary for toxin function, and are ideally conserved m the present inventive molecules and methods Mutations in the C-termmal domain affect processing localization of PAP, and may be altered if altered processing is desired Mutations that affect RNA binding as well as depunnation are known For example, truncation of the first 16 amino acids eliminates PAP cytotoxicity and ability to depu ⁇ nate ⁇ bosomes In addition, ⁇ bosome depunnation decreases as ammo acids are removed from the C-termmus, and is eliminated when a stop codon is introduced
• SEQ ID NO: 2 is preferred.
• the present invention includes nucleic acids which are homologous to using hybridization under stringent conditions, identical to using BLAST, have minor changes not affecting function, such as point mutations not changing the protein sequence, codon changes not changing the protein sequence, etc. with the nucleic acids of the present invention.
• conjugates and methods to conjugate a compound herein are also provided.
• Conjugates are ideally designed to selectively bind a receptor in which cell damage is desired.
• the conjugate is taken up by receptor mediated endocytosis and delivers the conjugate to the cell.
• the rPAP portion of the conjugate binds to the ribosomal RNA by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA.
• Depurinated ribosomes are unable to bind elongation factor 2, and, thus, the translocation step of the elongation cycle is inhibited, resulting in a shutdown of protein synthesis. The cell eventually dies.
• One particular method for conjugating compounds to certain rPAP proteins herein comprises inducing a chemical bond between an N-terminal cysteine and another compound.
• the compound is an antibody, a hormone, a modified hormone releasing factor, or a hormone releasing factor are preferred.
• the hormone releasing factor is GnRH are more preferred, although most preferred is conjugation to a d-lys 6 -modified GnRH.
• Conjugation can take place via any known method, but preferably via creation of a sulfhydryl bond between the targeting compound and the rPAP, whether via a linker or other bridging compound.
• modified gonadotropin releasing hormone "d-lys 6 -GnRH" is conjugated to full length rPAP.
• the d-lys 6 -GnRH is preferably activated with the linker GMBS for ease of binding to a free cysteine on a full length rPAP.
• a one-to-one ratio of rPAP to d-lys 6 -GnRH was also achieved.
• a heterobifunctional crosslinker is preferred, particularly GMBS, but also any heterobifunctional crosslinker that will facilitate the binding to d-lys 6 -GnRH via an NHS ester group located on the linker, or attachment to a free sulfhydryl group on the rPAP via a maleiniide group located on the linker.
• GnRH and d-lys6-GnRH are the substitution at position 6 of a glycine for a D-lysine.
• the ends are blocked.
• the C-terminus is blocked with an ethyl-amide group (ET- NH 2) , thereby replacing the glycine at position 10 of the natural compound.
• the natural GnRH compound is NH 2 Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyCOOH.
• the preferred analog is dK6: Hp-Glu-His-T ⁇ -Ser-Tyr-DZ> «-Leu-Arg-Pro-£ ⁇ NH2.
• amino acid sequence Cys-Gly-Gly-Gly-Gly-Ser is added to the full length rPAP and used to bind targeting compound. Cys-Gly-Gly-Gly- Gly-Ser is not part of the natural PAP sequence. VaI- Asp are the first two amino acids of the natural PAP sequence.
• a most preferred conjugate of the present invention has the following structure:
• X-Y-Z wherein X is d-lys ⁇ -GnRH; Y is a GMBS linker; and Z is a full length rPAP having CGGGGS at the N-terminus.
• Conjugates may be made via the methods described herein, or any method known or developed in the art. Moreover, conjugates may be modified so as to provide any functionality desired, as is known in the art. The examples describe the preferred conjugation methods.
• Any salt, suspension, dispersion, etc. may be used so as to administer the present conjugates.
• Preferred is a 0.7% - 10%, more preferably 0.9%, sodium chloride solution that is sterile and non-pyrogenic, more preferably such a solution that is also 4.5-7 pH.
• any administration method is acceptable, provided that the conjugate provides the proper impact.
• the most preferred embodiment of the present invention is to use a rP AP-GNRH salt, in solution, to inject in animals, for the purpose of reproductive sterilization.
• Sterilization need not be complete, nor reversible; however, the best mode contemplated is a non-reversible rPAP-d-lys 6 -GnRH injectible for use in animals, particularly dogs, cats, horses, livestock for food or other products (cattle, dairy cows, swine, sheep, goats, bison, bison/cattle breeds, etc.), working livestock, zoo animals, and wildlife (particularly deer, elk and other ungulates susceptible to chronic wasting disease).
• the foregoing invention has been described in accordance with the relevant legal standards, thus the description is exemplary rather than limiting in nature. Variations and modifications to disclosed embodiments may become apparent to those skilled in the art and are within the scope of the invention.
• Example 1 Expression of rPAP in E. coli.
• the full length sequence (SEQ ID NO: 2) was obtained by PCR amplification using a forward primer, rPAP-F: 5'-CCCGGG CATATG TGC GGA GGC GGA GGC AGT GTG AAT ACA ATC ATC TAC AAT GTT GGA AGT ACC-3, and a reverse primer, rPAP-R: 5'-GCG CGC AAG CTT TCA GGA TTC TTC AAA TAG ATC ACC AAG ATT AAC C.
• the reaction mix consisted of the following components: 600 mM Tris-
• the PCR reaction was carried out under the following conditions: 94 0 C x 2 min (1 cycle), 94 0 C x 30 sec, 52 0 C x 30 sec, 68 0 C x lmin (15 cycles), 94 0 C x 30 sec, 55 0 C x 30 sec, 68 0 C x lmin (25 cycles).
• the rPAP was refolded by snap dilution Following isolation of the inclusion bodies, the inclusion bodies we solubihzed in 8M urea, 5OmM T ⁇ s HCl, pH 8 5 DTT was added to a final concentration of 1OmM, and the mixture was stirred at room temperature for 90 mm The solubihzed protein was than added dropwise into a solution containing 5OmM T ⁇ s, pH 8 5, 0 4M sucrose, 0 05% polyethylene glycol-3550, 0 9mM oxidized cysteamine (TPEGS), while it was stirring at room temperature. The final concentration of rPAP in the refolding solution was between lOug/ml and 50ug/ml.
• the mixture was stirred for an additional 24 hours at 4 0 C. After 24 hours, the mixture was centrifuged at 16000xg for 15 min, the supernatant was decanted, and following refolding, the protein solution was dialyzed against buffer containing 5OmM Tris, pH 7.0, ImM EDTA. The pH of the buffer had a range of 6.8-8.5. After dialysis, the solution is centrifuged at 16000xg for 15 min., and the supernatant was placed over a cation exchange resin.
• the column is than washed with 5OmM Tris-HCl, pH 7.0, LOmM EDTA, and the protein is eluted with a buffer containing 5OmM Tris, pH 7.0, IM NaCl.
• the eluted protein is dialyzed against conjugation buffer, which contains 5OmM NaPO 4 , pH 7.2, 10OmM NaCl, ImM EDTA.
• conjugation buffer which contains 5OmM NaPO 4 , pH 7.2, 10OmM NaCl, ImM EDTA.
• the protein concentration is adjusted to a concentration of 0.2mg/ml- 1.0mg/ml.
• Example 3 Activation of d-lys ⁇ modified gonadotropin releasing hormone (GnRH) with maleimidobutyryloxy-succinimide ester (GMBS) linker.
• GnRH d-lys ⁇ modified gonadotropin releasing hormone
• GMBS maleimidobutyryloxy-succinimide ester
• D-IyS 6 -GnRH having a molecular weight of 1224 daltons, was prepared by solid-phase synthesis (Anaspec Corp., Fremont, CA). Six milligrams of d-lys 6 -GnRH was mixed with 1.5 ml deionized methanol, and adjusted to a pH of 7.0 using diisopropylethanolamine (DIPEA).
• DIPEA diisopropylethanolamine
• GMBS was purchased from Thermo Fisher Scientific (Rockford, IL). 1.25 mg of GMBS was mixed with 1.5 ml deionized methanol.
• the resulting d-lys 6 -GnRH-GMBS had a molecular weight of approximately 1421 daltons, indicating that one molecule of GMBS was bound to one molecule of d-lys 6 -GnRH. This was confirmed by mass spectroscopy.
• Example 4 Conjugation of rPAP to d-lys 6 -GnrH-GMBS.
• the solution of Example 3 was evaporated with a centrifugal evaporation unit.
• TCEP ⁇ C1 Tris(2-Carboxyethyl) phosphine hydrochloride is added to a final concentration of 0.05mM to the refolded recombinant PAP dissolved in conjugation buffer. The mixture was incubated for l-2hr at room temperature.
• the refolded rPAP dissolved in conjugation buffer, was added directly to the dried down d- IyS 6 -GnRH-GMBS so that the ration of d-lys 6 -GnRH-GMBS to rPAP was 20:1. Tween 20 was added to a final concentration of 0.25%. The pH was adjusted to 7.3, if needed, using 1OmM phosphoric acid, and the reaction was allowed to proceed in the dark, at room temperature (70 0 F) for approximately 2-3 hours.
• Example 5 Purifying d-lys 6 -GnRH-GMBS-rPAP
• d-lys 6 -GnRH-GMBS-rPAP was further subjected to size exclusion chromatography using a 10 ml Bio-Rad Bio-Gel PlO column, to remove excess dK6 remaining after the conjugation reaction.
• the protein solution was dialyzed against buffer containing 5OmM Tris, pH 7.0, ImM EDTA. The pH of the buffer had a range of 6.8-8.5. Following dialysis, the solution was centrifuged at 16000xg for 15 min., and the supernatant was placed over a cation exchange resin. The column was than washed with the same buffer, and the protein was eluted with a buffer containing 5OmM Tris, pH 7.0, IM NaCl.
• the purified, refolded d-lys ⁇ -GnRH-GMBS-rPAP of Example 5 was used in a competitive radio-immuno receptor binding assay.
• Purified pituitary membranes having gonadotropin releasing hormone receptors were flooded with I 1 "-radiolabeled d- LyS 6 -GnRH.
• Different concentrations of the d-lys 6 -GnRH-GMBS-rPAP was subsequently added to the membranes, the membranes washed with ImM Tris-Cl Ph 7.4, ImM CaCl, 1% BSA. The reactions were incubated for 4 hr, diluted with the same buffer.
• AU buffers and solutions were prepared with DEPC-treated H2O.
• Dilution buffer was prepared [0.5ml to 1 ml of a 0.5M stock (DEPC-treated H2O, 0.1M NaCl, dilution buffer (5OmM NaCl 0.5% Fraction V BSA)] for the toxins and/or toxin buffers to be tested.
• a 0.5 nM dilution of the toxins/conjugates was prepared. Then, 100 uL serial dilutions (1 :2.5 for each dilution) of the toxins/conjugates was prepared, using the 0.5nM (50OpM) stock. The following dilutions were prepared: 20OpM; 8OpM; 32pM;
• the lysate was thawed on ice, and 17.5uL of test dilution or control was added to each tube, on ice, and mixed gently with pipette. The lysate/test or control was then pre-incubated on ice for 15 min, and 2.5ul of an nutrient premix was added after the 15 minute pre-incubation period (Amino acids (-hie); 4.2uL; Amino acids (-met); 4.2uL; 2.5M KCl 11.76uL; RNAsin 8.4uL; DEPC H 2 O10.92uL; Luciferase mRNA 2.52uL; total to 42uL). During the 15 minute pre-incubation period, the mRNA is added to the pre-mix. The total volume of each reaction tube was 25uL.
• the pET3a expression plasmid containing a T7 promoter upstream of one of four mature PAP- encoding sequences (each plasmid contains the DNA sequences encoding a mature form of rPAP that is identical to the post-translationally modified form of plant-derived PAP: clones 1-4.1, 1-4.2, 1-4.3, and 1-4.4) were transformed into E. coli BL21 (AI) (Invitrogen Corp. Carlsbad, CA) having T7 RNA polymerase under control of an arabinose promoter (AraD).
• the cells were grown for approximately 12 hours (overnight) at 37°C with shaking, in minimal media containing glucose and ampicillin. The cells were transferred to Luria broth in the morning. The same process was followed for a full length clone (3.2 ). The cells harboring the plasmids were induced after growth for 2 hours by the addition of arabinose to a final concentration of 0.2%, and isopropyl /3-D-l-thiogalactopyranoside to a concentration of ImM The A600 was measured every hour thereafter, for three hours. The results are shown in the table to Example 8. [00081] Table to Example 8.
• a single colony from two different isolates and a control harboring plasmid without a rPAP insert were each inoculated into Luria broth medium containing 100ug/ml ampicillan. The three cultures were then grown for approximately 18 hours (overnight) at 37°C, with shaking. Each grown culture was diluted 1 :25 into fresh Luria broth medium, in the presence of lOOug/ml ampicillan, and grown at 37°C, with shaking, for two hours. [00083] The results were as follows:

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Endocrinology (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• General Chemical & Material Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Gastroenterology & Hepatology (AREA)
• Botany (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Diabetes (AREA)
• Virology (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (2)

Family

ID=41319605

Family Applications (1)

Country Status (6)

Cited By (2)

Families Citing this family (1)

Family Cites Families (5)

• 2009
  • 2009-07-15 US US13/054,187 patent/US8575109B2/en not_active Expired - Fee Related
  • 2009-07-15 AU AU2009270920A patent/AU2009270920A1/en not_active Abandoned
  • 2009-07-15 CA CA 2730933 patent/CA2730933A1/en not_active Abandoned
  • 2009-07-15 EP EP20090790464 patent/EP2318431B1/en not_active Not-in-force
  • 2009-07-15 WO PCT/US2009/050685 patent/WO2010009227A2/en not_active Ceased
  • 2009-07-15 JP JP2011518880A patent/JP2011528230A/ja active Pending
• 2013
  • 2013-10-03 US US14/045,437 patent/US9023596B2/en not_active Expired - Fee Related

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events