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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• • A—HUMAN NECESSITIES
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• Acetylcholinesterase (AChE, EC 3.1.1.7) is preferentially active with acetylcholine and is inhibited by B -284C51 (Koelle, G.B. (1955) J. Pharmacol. Exp. Ther. 114: 167-184; Hol stedt, B. (1957) Acta. Physiol. Scand..40: 322- 330; Holmstedt, B. (1959) Pharmacol. Rev. .11: 567-688; Silver, A. (1973) in Cholinesterases, Academic Press; and Austin, L and Berry, .K. (1953) Biochem. J. 5.: 695-700).
• AChE inhibitors may be used to enhance the nicotinic and muscarinic actions of acetylcholine.
• Some cholinesterase inhibitors are the main ingredient of insecticides used against house pests or in agriculture. Cholinesterases may have use as prophylactic or therapeutic agents in cases of organophosphate poisoning.
• Acetylcholinesterase (AChE) is primarily associated with nerve and muscle, typically localized at synaptic contacts.
• AChE an enzyme which degrades the esters of choline, emerged as a key component in neurotransmission within the autonomic and somatic motor nervous system (Dale, H.H. (1914) J. Pharmacol. Exp. Ther. 6:147-190.1.
• Other cholinesterases e.g. butyrylcholine ⁇ terase (BuChE, EC 3.1.1.8) are located at other sites and have other physiological functions.
• Cholinesterases exist in a variety of molecular forms which differ in size, level of oligomerization, lipid content, glycosylation, collagen content and hydrodynamic properties.
• the catalytic subunits may be associated with a collagen- like or lipid-linked subunit which forms distinct heteromeric species.
• the collagen-associated enzyme consists of tetra ers of catalytic subunits that are linked via disulfide bonds (Cartand, J. , Bon, S. and Massoulie, J. (1978) J. Cell. Biol.22: 315-322; Anglister, L. and Silman, I (1978) J. Mol. Biol. 125: 293-311; Rosenberry, T.L. and Richardson, J.M.
• the lipid containing form of AChE contains covalently attached fatty acids and is approximately 20kD in mass
• AChE is a homomeric form that exists as dimers and tetramers of identical catalytic subunits. This form is referred to as the globular or G form.
• the globular form is subdivided into hydrophilic or hydrophobic G forms. These two forms differ in that a glycophospholipid is associated with hydrophobic G form (Silman, I. and Futerman, A.H. (1987) Eur. J. Biochem. 170: 11-22; Roberts, W.L. , Kim, B.H. and Rosenberry T.L. (1987) Proc. Natl. Acad. Sci. U.S.A. ___!: 7817-7821; and Toutant, J.P., Richards, M.K. Kroll, J.A. and Rosenberry T.L. (1990) Eur. J. Biochem. 187: 31-38) .
• AChE and BuChE are both encoded by single genes, yet extensive polymorphism of the gene products has been observed (Schumacher, M. et. al. (1986) Nature 319: 407-409; Maulet, Y. et. al (1990) Neuron 4.: 289-301; Arpagaus, M. et. al. (1990) Biochemistry 29: 124-131; Prody, C. et. al. (1986) J. Neurosci 16: 25-35; Prody, C. et. al. (1987) Proc. Natl. Acad. Sci.
• AChE and BuChE are glycosylated.
• the amino acid sequence that harbors the signal for glycosylation is Asn-x-Ser/Thr.
• the number of such sites on cholinesterase varies.
• Torpedo AChE contains four
• hBuChE contains nine
• Drosophila AChE contains five potential sites (Toutant, J.P. (1989) Prog. Neurobiol. 32: 423-446).
• the importance of glycosylation for enzyme activity has not yet been elucidated.
• Organophosphate poisoning occurs most frequently among farmers upon exposure to pesticides such as althione and parathione due to improper handling. Treatment of such poisoning calls for administration of anti-muscarinics, anti-convulsants, and oxi e reactivator drugs (Gray, A.P. (1984) Drug Metab Rev. _L£__ 557-589).
• the possibility of chemical warfare and poisoning of high density population centers by organophosphates such as soman emphasize the need to develop an effective prophylactic and therapeutic treatment.
• Soreq et al. Proc. Natl. Acad. Sci. USA £2:9688-9692 (1990) disclose the cloning of AChE cDNA into a transcription vector, production and isolation of mRNA and its translation in microinjected oocytes into acetylcholinesterase.
• Velan et al. Cellular and Molecular Neurobiology, 11:143-156 (1991) disclose the secretion of recombinant human acetylcholinesterase from transiently transfected 293 cells as a soluble globular enzyme.
• the subject invention provides an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the subject invention additionally provides an enzymatically active recombinant monomeric human acetylcholinesterase comprising a polypeptide characterized by an amino acid sequence in which serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase (position 580 in the mature polypeptide) and an enzymatically active recombinant human acetylcholinesterase comprising at least one polypeptide characterized by the presence of a methionine of the N- terminus of the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the subject invention also provides therapeutic and diagnostic methods using the enzymatically active recombinant acetylcholinesterase.
• r-met-AChE is used herein to describe authentic AChE with an additional N-terminal methionine.
• ser/r-met-AChE is used herein to describe a mutant of AChE containing serine at position 611 instead of the naturally- occurring cysteine.
• FIG. l construction of Plasmid pBR-AChE.
• the large fragment isolated from EcoRI digestion of plasmid pGEM- 7Z(f+) was further cleaved with Xhol.
• the 2650 bp fragment was isolated and ligated to the large fragment isolated from EcoRI-Sall digestion of plasmid pBR322. Since Sail and Xhol are complementary, they may be ligated without any difficulty.
• the resulting plasmid designated pBR-AChE contains the DNA sequence encoding authentic AChE, but does not express it since it lacks a promoter and ribosomal binding site.
• Plasmid pAIF-04 Construction of Plasmid pAIF-04. Plasmid pAIF-2 produced as described in Example 1, was digested with Dral and Aatll. The DNA was further digested with the Klenow fragment of DNA polymerase to remove the 3' ends, and then digested with Ndel. The 1879 bp fragment was isolated and ligated to the large fragment isolated from Smal-Ndel digestion of plasmid pMLK-6891 which contains the deo promoter. The resulting expression plasmid designated pAIF- 04 contains authentic AChE DNA under control of the deo P promoter, and the deo ribosomal binding site. However as described in Example 1, it failed to express AChE.
• FIG. 3 Construction of Plasmid pAIF-11.
• the small fragment isolated from Sall-Ndel digestion of plasmid pAIF- 04 was ligated to the large fragment isolated from Sall-Ndel digestion of plasmid pMLK-100 (deposited in E.coli 4300 as ATCC Accession No. 68605) .
• the resulting expression plasmid designated pAIF-11 contained authentic AChE DNA under control of the ⁇ P L promoter (and C ⁇ ribosomal binding site) . However, as described in Example 1, it did not express AChE.
• Figures 4A-E (SEQ ID NO:l) . Sequence of Naturally Occurring Unprocessed Human AChE DNA. This figure shows the nucleotide and corresponding amino acid sequence of AChE, as disclosed by Soreq, H. et al. (1990) Proc. Natl. Acad. Sci. 82:9688. The line below the amino acids shows the amino acid numbering. The nucleotide numbering is found under the second codon from the left of each row beginning with the initiator methionine. Transcription terminates at the transcription termination codon TGA immediately following the leucine residue at position 614.
• Unprocessed acetylcholinesterase contains 614 amino acids.
• the first 31 amino acids constitute a leader (or signal) sequence subsequently cleaved to produce mature naturally- occurring AChE containing 583 amino acids and having as N- terminus Glu 32 encoded by the GAG codon.
• Figure 5. Mutational Changes in AChE cDNA This figure shows the GC to AT base substitutions in the two duplexes described in Example 2.
• the original G and C bases are in boxes in the upper row.
• the corresponding synthetic duplex containing the AT substitutions is recited in the lower row.
• Plasmid pAIF-34 expresses AChE under control of the ⁇ P L promoter and C n ribosomal binding site, and was deposited in E.coli A4255 in the ATCC under Accession No. 68638. Plasmid pAIF-51 expresses AChE under control of the deo P promoter and deo ribosomal binding site.
• Plasmid pAIF-51 was digested with Xhol, filled-in with Klenow and further digested with Bglll and Seal. The large fragment resulting was ligated to the large fragment produced by digestion of plasmid pMF5520 which had been digested with Bglll and Stul.
• Plasmid pMF5520 is an SOD expression plasmid which harbors the Tet R gene sequence; the construction of this plasmid is fully described in applicant's copending patent application, EPO Publication No. 303,972.
• pAIF-52 expresses r-met- AChE under control of the deo P promoter. It is similar to plasmid pAIF-51 ( Figure 6) except that it is Tet R instead of
• Plasmid pMLF-52ser was constructed from pAIF-52 as described in Example 4. It is identical to pAIF-52, except that the cysteine residue of position 611 (see Figures 4A-D and SEQ ID NO:l) was replaced by serine. Plasmid pMLF-52ser was introduced into E.coli S ⁇ 930 and deposited in the ATCC under Accession No. 68637. Plasmid pMLF-52ser is elsewhere designated also as pMFL-52ser.
• FIG. 8 Lineweaver-Burk Plot of ser/r-met-AChE. Enzyme kinetics were determined for the mutant rAChE using acetylthiocholine as substrate. The results were plotted on a Lineweaver-Burk plot and the K m was calculated to be about 1.0X10' 4 M.
• FIG. 9 Beat Inactivation of ser/r-m ⁇ t-AChE. This figure shows the results of heat inactivation of ser/r-met-AChE by incubation at 50°C as described in Example 5. 50% of the activity was lost after 7 minutes, and 90% was lost after 25 minutes.
• FIG. 10 Gel Filtration Chromatography of r-met-AChE. Recombinant mutant AChE was subjected to gel filtration column chromatography as described in section 5 of Example 4. The chromatogram shows that most of the protein is in the form of inactive aggregates which eluted in the void volume of the column. The active enzyme peak eluted in fraction 7 with a specific activity of 10.6 U/mg.
• FIG. 11 Isolation and Purification of Inclusion Bodies. This figure is a flow chart showing the steps performed as described in Example 6 to obtain purified inclusion bodies of ser/r-met-AChE from the E. coli cells obtained by fermentation.
• FIG. 12 Solubilization of Inclusion Bodies and Refolding of ser/r-met-AChE. This figure is a flow chart showing the steps performed as described in Example 6 to solubilize the inclusion bodies and then refold the ser/r- et-AChE to obtain an enzymatically active polypeptide.
• FIG. 13 Purification of Refolded and Enzymatically Active ser/r-met-AChE. This figure is a flow chart showing the steps performed as described in Example 6 to purify the refolded and enzymatically active ser/r-met-AChE.
• FIG. 14 Pharmacokinetics of ser/r-met-AChE in mice and rats. This figure shows the time course of the serum concentration of ser/r-met-AChE in mice and rats, determined as described in Example 7. The half life may be extrapolated from this data and is 39 and 33.6 minutes for mice and rats respectively.
• FIG. 15 Renaturation kinetics of ser/r-met-AChE. This figure shows the time course of refolding of ser/r-met-AChE as described in Example 6.
• the plasmids pMLF-52ser and pAIF-34 were deposited in Escherichia coli pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 68637 and 68638, respectively.
• ATCC American Type Culture Collection
• the subject invention provides an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase or analog thereof comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• Enzymatically active recombinant acetylcholinesterase is defined herein as having the same substrate specificity as and reactivity with molecules as that of natural acetylcholinesterase.
• Analog as defined herein encompasses a polypeptide comprising the sequence of human acetylcholinesterase to which one or more amino acids have been added to either the amino terminal end, the carboxy terminal end or both, and/or to which substitutions and/or deletions to the sequence have been made, and which has the enzymatic activity of human acetylcholinesterase.
• Substantially identical as defined herein encompasses the addition of fewer than four amino acids at the N-terminus of the amino acid sequence of naturally-occurring human acetylcholinesterase. Furthermore, there may be substitutions and/or deletions in the sequence which do not eliminate the enzymatic activity of the polypeptide. Substitutions may encompass up to 10 residues in accordance with the homology groups described in Needleman et al., J. Mol. Biol. 48:443 (1970).
• the subject invention further provides enzymatically active, recombinant human acetylcholinesterase wherein serine is substituted for cys 611 in the sequence of naturally occurring human acetylcholinesterase (position 580 in the mature polypeptide) .
• Human acetylycholinesterase consists of 583 amino acids preceded by a 32 amino acid leader sequence which in vivo is removed by cellular processing enzymes. Cys 611 refers to the numbering of the amino acids from the start of the leader sequence. Upon removal of the leader sequence, position 611 becomes position 580.
• the subject invention provides an enzymatically active recombinant human acetylcholinesterase or analog thereof comprising at least one polypeptide characterized by an amino acid sequence in which serine is substituted for cys 611 in the sequence of naturally-occurring, human acetylcholinesterase (position 580 in the mature polypeptide) .
• the subject invention also provides an enzymatically active recombinant human acetylcholinesterase or analog thereof comprising at least one polypeptide characterized by the presence of a methionine at the N-terminus of the amino acid sequence of naturally-occurring human acetylcholinesterase.
• This enzymatically active human acetylcholinesterase may comprise one polypeptide or more than one identical polypeptide.
• the enzymatically active human acetylcholinesterase comprises a monomer.
• the subject invention further provides an expression vector encoding any of the recombinant acetylcholinesterases described above as well as a host such as a recombinant host comprising the expression vector.
• viruses such as bacterial viruses, e.g., bacteriophages (such as phage lambda), cosmids, plasmids, and other vectors.
• Genes encoding the relevant polypeptides are inserted into appropriate vectors by methods well known in the art. For example, using conventional restriction endonuclease enzyme sites, inserts and vector DNA can both be cleaved to create complementary ends which base pair with each other and are then ligated together with a DNA ligase. Alternatively, synthetic linkers harboring base sequences complementary to a restriction site in the vector DNA can be ligated to the insert DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available.
• Vectors comprising a sequence encoding the polypeptides may be adapted for expression in bacteria, yeast, or mammalian cells which additionally comprise the regulatory elements necessary for expression of the cloned gene in the bacteria, yeast, or mammalian cells so located relative to the nucleic acid encoding the polypeptide as to permit expression thereof.
• Regulatory elements required for expression include promoter sequences to bind RNA polymerase, operator sequences for binding represser molecules, and a ribosomal binding site for ribosome binding.
• a bacterial expression vector may include a promoter-operator sequence such as ⁇ P L 0 L or deo promoters.
• the ⁇ C ⁇ or deo ribosomal binding sites may be used.
• Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art, for example the methods described above for constructing vectors in general.
• the subject invention provides expression plasmids encoding any of the recombinant acetylcholinesterases described above.
• these expression plasmids are plasmids pAIF-34 deposited under ATCC Accession No. 68638 and plasmid pMLF-52ser deposited under ATCC Accession No. 68637.
• plasmids deposited in connection with this application may be readily altered by known techniques (e.g. by site-directed mutagenesis or by insertion of linkers) to encode expression of related polypeptides.
• known techniques e.g. by site-directed mutagenesis or by insertion of linkers.
• Such techniques are described for example in Sambrook, J. , Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.
• the expression plasmids of this invention further comprise suitable regulatory elements positioned within the plasmid relative to the DNA encoding the polypeptide so as.to effect expression of the polypeptide in a suitable host cell, such as promoter and operators, e.g. deo P j P., and ⁇ P L 0 L , ribosom ⁇ al binding sites, e.g. deo and C ⁇ , and repressers.
• suitable regulatory elements positioned within the plasmid relative to the DNA encoding the polypeptide so as.to effect expression of the polypeptide in a suitable host cell, such as promoter and operators, e.g. deo P j P., and ⁇ P L 0 L , ribosom ⁇ al binding sites, e.g. deo and C ⁇ , and repressers.
• the suitable regulatory elements are positioned within the plasmid relative to the DNA encoding the acetylcholinesterase so as to effect expression of the acetylcholinesterase in a suitable host cell.
• the regulatory elements are positioned close to and upstream of the DNA encoding the acetylcholinesterase.
• the expression plasmids of this invention may be introduced into suitable host cells, preferably bacterial host cells.
• suitable host cells preferably bacterial host cells.
• the expression plasmids of this invention may be suitably modified for introduction into fungi, yeast, or eukaryotic cell lines such as CHO, chicken embryo, fibroblast or other known cell lines.
• Preferred bacterial host cells are Escherichia coli cells. Examples of suitable Escherichia coli cells are strains S 930 or A4255, but other Escherichia coli strains and other bacteria can also be used as hosts for the plasmids.
• the bacteria used as hosts may be any strains including auxotrophic (such as A1645) , prototrophic (such as A4255) , and lytic strains; F* and F " strains; strains harboring the cl 857 repressor sequence of the ⁇ prophage (such as A1645 and A4255) ; and strains devoid of the deo repressors and/or the deo gene (see European Patent Application Publication No. 0303972, published February 22, 1989).
• Escherichia coli strain A4255 has been deposited under ATCC Accession No. 53468
• Escherichia coli strain S ⁇ 930 has been deposited under ATCC Accession No. 67706.
• the invention provides a bacterial cell which comprises these expression plasmids.
• the bacterial cell is an Escherichia coli cell.
• the invention provides an Escherichia coli cell containing the plasmid designated pMLF-52ser, deposited in E. coli strain S ⁇ 930 with the ATCC under ATCC Accession No. 68637 and pAIF-34, deposited in E. coli strain A4255 with the ATCC under ATCC Accession No. 68638.
• E. coli host strains described above can be "cured" of the plasmids they harbor by methods well-known in the art, e.g. the ethidiuro bromide method described by R.P. Novick in Bacteriol. Review 33. 210 (1969) .
• the subject invention provides a method of producing an enzymatically active recombinant human acetylcholinesterase or analog thereof not previously taught or suggested by the prior art which comprises culturing the recombinant hosts so as to obtain expression of the recombinant acetylcholinesterase or analog thereof in the host, recovering the recombinant acetylcholinesterase or analog thereof so expressed from the host, and treating the recombinant acetylcholinesterase or analog thereof so recovered so as to obtain the enzymatically active, recombinant human acetylcholinesterase or analog thereof.
• the invention provides a method of producing large amounts of purified, enzymatically active recombinant human acetylcholinesterase or analog thereof comprising isolation of inclusion bodies of the recombinant human acetylcholinesterase or analog thereof from the host cell in which they were produced, dissolving the inclusion bodies so isolated, refolding the enzymatically inactive recombinant human acetylcholinesterase or analog thereof to obtain an enzymatically active recombinant human acetylcholinesterase or analog thereof, and purifying the enzymatically active recombinant human acetylcholinesterase or analog thereof so obtained.
• the subject invention also provides a method of hydrolyzing an ester of choline which comprises contacting the ester with an enzymatically active, nonglycosylated, recombinant human acetylcholinesterase under conditions such that the ester is hydrolyzed.
• the subject invention further provides a method of preventing the toxic effects of an acetylcholinesterase inhibitor which comprises contacting the inhibitor with an amount of an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase bound to a solid support effective to prevent the toxic effects of the acetylcholinesterase inhibitor, wherein the acetylcholinesterase comprises at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the subject invention further provides a protective gas mask comprising an amount of an enzymatically active, recombinant, human acetylcholinesterase effective to prevent the toxic effects of an acetylcholinesterase inhibitor, wherein the acetylcholinesterase comprises at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the enzymatically active, recombinant, human acetylcholinesterase is nonglycosylated.
• the enzymatically active, recombinant, human acetylcholinesterase is bound to a solid support.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase.
• the acetylcholinesterase inhibitor is an insecticide or a nerve gas.
• the subject invention further provides a method of determining whether a molecule is an inhibitor of acetylcholinesterase which comprises determining, in the presence of the molecule, the enzymatic activity of an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurringhumanacetylcholinesteraseand comparing the activity so determined with the activity determined in the absence of the molecule.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally- occurring human acetylcholinesterase.
• the subject invention further provides a method of treating a subject exposed to an inhibitor of acetylcholinesterase which comprises administering to the subject an amount of an enzymatically active, recombinant, human acetylcholinesterase effective to treat the subject, wherein the acetylcholinesterase comprises at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the enzymatically active, recombinant, human acetylcholinesterase is nonglycosylated.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase.
• the inhibitor of acetylcholinesterase is neostigmine or isofluorophate.
• the subject invention further provides a method of treating post-surgery apnea which comprises administering to -the subject an effective amount of an enzymatically active, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the enzymatically active, recombinant, human acetylcholinesterase is nonglycosylated.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcho1inesterase.
• the subject invention further provides a method of treating gastrointestinal disorders which comprises administering to the subject an effective amount of an enzymatically active, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the enzymatically active, recombinant, human acetylcholinesterase is nonglycosylated.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase.
• the subject invention further provides a method of treating central nervous system disorders which comprises administering to the subject an effective amount of an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase.
• the central nervous system disorder may be Parkinson's or Alzheimer's Disease.
• the subject invention further provides a method of using as an aminopeptidase an enzymatically active, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• the enzymatically active, recombinant, human acetylcholinesterase is nonglycosylated.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase.
• the subject invention further provides a method of using in an enzyme immunoassay an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurring human acetylcholinesterase.
• the subject invention further provides a method of detecting cancer which comprises contacting DNA from a tissue sample with a DNA probe to which a marker is attached, wherein the DNA probe is obtained from a nucleic acid producing an enzymatically active, nonglycosylated, recombinant, human acetylcholinesterase comprising at least one polypeptide characterized by an amino acid sequence which is substantially identical to the amino acid sequence of naturally-occurring human acetylcholinesterase, detecting the amount of DNA probe hybridized to the DNA from the tissue sample by detecting the presence of the marker, wherein abnormally high levels of expression of acetylcholinesterase indicate the presence of cancer.
• a methionine may be present at the N-terminus of the sequence.
• a serine is substituted for cys 611 in the sequence of naturally-occurringhuman acetylcholinesterase.
• Plasmid pGEM-7Z(f) (Fig. 1), which was obtained from the U.S. Army, contains a 4kb cDNA fragment flanked by EcoRI restriction sites, and encompasses the entire coding sequence shown in Soreq et al.
• the sequence coding for native acetylcholinesterase i.e.
• Plasmid pGEM-7Z(f + ) was then digested with EcoRI and the resulting 4kb fragment was purified from an agarose electrophoresis gel and was digested with Xhol.
• the 2650 bp fragment isolated from Xhol digestion of the purified 4kb fragment was ligated to the large fragment isolated from EcoRI-Sall digestion of plasmid pBR322. Sail and Xhol sites are complementary and can be ligated. After ligation, presence of the 2650bp sequence in the resulting plasmid designated pBR-AChE was verified by restriction mapping. Plasmid pBR-AChE was then introduced into E. coli MC1061 (ATCC Accession No. 53338) by transformation.
• the cloning vector, plasmid pAIF-2 ( Figure 2) was constructed as follows. Two synthetic oligomers, "A” and “B” were prepared, purified and annealed to generate the duplex shown below:
• Plasmid pBR-AChE was digested with Ndel and Nael. The large fragment was isolated and ligated with the synthetic duplex shown above. After transformation into E. coli MC1061, colonies harboring the synthetic linker were identified by hybridization on nitrocellulose filters to the synthetic radioactively labeled oligonucleotide "B" at 60°C overnight. The filters were then washed at 60°C with lxSSC containing 0.1% SDS, dried, exposed to x-ray film for 3 hours, and developed. Several colonies yielding strong signals were picked and analyzed with restriction endonucleases Ndel and Nael. One of the candidates, designated pAIF-2, was used for further manipulation. This plasmid contains the initiation codon ATG at the 5' end of the AChE gene; however it does not express AChE since it lacks a promoter and ribosomal binding site.
• Plasmid pAIF-2 was first cleaved with Dral and Aatll and the resulting 3* overhanging ends were removed (blunt-ended) by digestion with E.coli DNA polymerase large fragment (Klenow fragment) .
• the plasmid was then digested with Ndel and a 1879 bp fragment was isolated and purified from an agarose electrophoresis gel. This fragment was then ligated to the large fragment isolated from Smal-Ndel digestion of plasmid pMLK-6891. The ligated mix was used to transform E.coli MC1061.
• Verification that the plasmid retained the Ndel site was done by extracting the plasmid according to the method described by Birnboim et al. (Nuc. Acid. Res.2, 1513, 1979) and cleaving with Ndel.
• This mini-prep plasmid extract * was used to transform E.coli strains S ⁇ 732 and S 930 (ATCC Accession Nos. 67362 and 67703, respectively) which are appropriate hosts for optimal expression of plasmids under control of the deo P promoter.
• the resulting plasmid designated pAIF-04 contains the native, naturally occurring DNA sequence encoding AChE under control of the deo promoter.
• Plasmid Construction of a plasmid under control of the ⁇ P L promoter is shown in Figure 3 and was performed as follows.
• the small fragment isolated from Ndel-Sall digestion of plasmid pAIF-04 was ligated with the large fragment isolated from Sall-Ndel digestion of the ⁇ P L vector pMLKlOO (deposited in E.coli A4300 as ATCC Accession No. 68605) .
• the resulting plasmid which was designated pAIF-11 contains the native, naturally occurring DNA sequence encoding AChE under control of the ⁇ P L promoter.
• This plasmid was introduced to E. coli A4255 (ATCC Accession No. 68456) and also to E. coli A4300 (also known as AC4300) which both carry the temperature sensitive repressor ⁇ cl 857 on the bacterial chromosome.
• E. coli S ⁇ 930 (ATCC Accession No. 67706) containing plasmid pAIF-04 was grown in 1 ml of LB + lOO ⁇ g/ml ampicillin at 37°c overnight. The culture was processed for SDS-PAGE by pelleting the cells, and suspending them in lysis buffer containing SDS. Electrophoresis was performed at room temperature for 3 hours on a 10% polyacrylamide gel to evaluate expression.
• E. coli A4255 (ATCC Accession No. 68456) containing plasmid pAIF-11 was grown in LB + 100 ⁇ g/ml ampicillin additionally containing 0.2% glucose to a cell density of OD ⁇ of 0.6-0.8 at 30°C and then induced at 42 ⁇ C for a period of 1_ - 3 hours. Expression was evaluated on SDS-PAGE as described above.
• the cloned acetylcholinesterase gene was expected to produce a polypeptide that consists of 584 amino acids. Taking into account that the average molecular weight of an amino acid is 110, the molecular weight of a monomer of AChE was predicted to be about 62-64kD.
• the electrophoretic pattern observed on 10% SDS-gels revealed a very faint band at a position corresponding to the expected molecular weight. However, this band co-migrated with a band obtained from lysates of control cultures which did not harbor an AChE insert. Although the intensity of the protein band was higher in the lane containing the extract from the expressing clones than in the lane containing the control culture, we suspected that it was not related to the desired protein product.
• Example l disclosed the construction of plasmids for achievement of expression of AChE harboring the authentic sequence as found in natural sources. As disclosed in Example l, these plasmids were not able to express AChE.
• Two synthetic DNA duplexes were prepared such that 24 base pair substitutions of GC to AT were introduced along a stretch of 130 base pairs as shown in Figure 5. These duplexes were joined to produce a synthetic linker flanked by Ndel-Ncol restriction sites which are compatible with sites on plasmids pAIF-11 and pAIF-04. The same procedure was used in order to modify both plasmids.
• the synthetic linker was ligated to the large fragment isolated from Ndel-Ncol digestion of plasmids pAIF-11 or pAIF-04. The ligation of the synthetic linker with plasmid pAIF-11 resulted in plasmid pAIF-34 which expresses AChE under control of the ⁇ P L promoter.
• Plasmids pAIF-34 and pAIF-51 (shown in Figure 6) were introduced into host cells E. coli A4255 (ATCC Accession No. 68456) and E. coli S ⁇ 930 (ATCC Accession No. 67703) respectively. E.coli A4255 harboring plasmid pAIF-34 was deposited in the ATCC under Accession No. 68638.
• plasmids pAIF-34 and pAIF-5l do in fact contain the modified DNA fragment between the Ndel-Ncol restriction sites.
• an 800 bp Scal-SphI fragment spanning a few nucleotides upstream of the ATG initiation codon from plasmid pAIF-34 was inserted into the appropriate M13 phage and then sequenced by the Sanger dideoxy DNA sequencing method. The sequencing data confirmed that no changes other than those intended are present.
• plasmid pAIF-52 was cleaved with Ncol, blunt-ended with Klenow enzyme, and then cleaved with Bglll. This cleavage generated a 700 bp fragment introduced into appropriate M13 DNA that was previously cleaved with BamHI and Smal to generate compatible ends for ligation. Dideoxy DNA sequencing data obtained was identical to that obtained for pAIF-34.
• a plasmid under control of the deo P promoter was constructed containing the gene for tetracycline resistance (Tet R ) instead of ampicillin resistance (Amp R ) .
• the resulting plasmid designated pAIF-52 contains the gene for tetracycline resistance (Tet R ) and modified DNA encoding authentic AChE under control of the deo P promoter (see Figure 7) .
• the advantage of this plasmid is its improved stability as described in section 4 below.
• plasmids pAIF-34, pAIF-51 and pAIF-52 all encode the same authentic AChE amino acid sequence with the additional N- ter inal methionine (hereinafter "r-met-AChE”) .
• E. coli A4255 harboring the ⁇ P L expression plasmid pAIF-34 was grown at 30 ⁇ C to an OD ⁇ of 0.7 in LB medium supplemented with 100 ⁇ g/ml ampicillin and 0.2% glucose.
• the temperature was elevated to 42°C and the culture was grown for an additional 2-3 hours. Samples were removed at 1 hour intervals and adjusted to contain the same cell density. Total cell lysate prepared in SDS-NaOH was applied to SDS-PAGE.
• E. coli S ⁇ 930 harboring the deo P expression plasmid pAIF-51 was grown overnight at 37°C in LB buffered with M9 salts medium (Miller, 1972) and supplemented with 0.1% glucose and 100 ⁇ g/ml ampicillin. The culture was harvested and processed as described above.
• the 62kD protein of rAChE reacted with anti-AChE antiserum prepared with AChE purified from human erythrocytes, thus confirming that the plasmids are producing rAChE, immunologically similar to naturally occurring AChE.
• Plasmid stability was determined for E. coli S ⁇ 930 containing plasmid pAIF-51 by inoculating 200ml buffered LB medium containing 100 ⁇ g/ml ampicillin to contain 20 cell/ml. The culture was grown at 37 ⁇ C for 16-18 hours and reached an OD ⁇ of 4-5. Assuming that 1 OD 660 represents about 5xl0 8 cells/ml the number of generations from seeding to harvest is 22-25. A sample from the first culture was diluted appropriately and a new flask with 200 ml was seeded as described for the first culture. Four such successive transfers account for 100 generations.
• plasmid pAIF-5l which contains the Amp ⁇ gene.
• the rationale for these differences in plasmid stability appears to be based on the biochemical properties of the two antibiotics.
• the jS-lactamase gene confers a picillin-resistance by producing a protein that is able to degrade ampicillin. This rapidly reduces the ampicillin concentration in the medium thus enabling plasmid-less cells to increase.
• the gene conferring tetracycline resistance produces a protein that prevents the antibiotic from entering the cell. Since tetracycline is not destroyed it remains active during the process and those cells that lose the plasmid become tetracycline sensitive and cannot multiply.
• E.coli cells S093O/AIF-51 or S0930/pAIF-52 were resuspended in 20mM Tris-HCl pH 8.0 containing lOmM EDTA, sonicated and then centrifuged. The insoluble pellet was dissolved in SDS and processed for SDS-PAGE. The electrophoretic pattern reveals that most of the r-met-AChE is indeed in the pellet which is composed of inclusion bodies. A minor fraction is apparently soluble and was noted in the supernatant of cell extracts.
• the two E. coli strains described in Example 2 were grown in 2 liter fermentation vessels in medium composed of 10 g/1 yeast extract, 20 g/1 N-Z amine (casein hydrolysate) and 10-15 g/1 glucose, and additionally containing 100 mg/L ampicillin or 12.5 mg/L tetracycline, depending on the strain used.
• Strain S ⁇ 930/pAIF-51 was grown at 37 ⁇ C for 10 hours and harvested at OD g ⁇ - ⁇ 12-15.
• Inclusion bodies were isolated by resuspending 5-10g packed cells in 50 ml of 25% sucrose, 50 mM Tris-HCl pH 8.0, 10 ⁇ M EDTA and 10 ⁇ g/ml lysozyme. The cells were allowed to lyse by incubation on ice for 1-2 hours. The highly viscous extract was then sonicated intermittently for 5 minutes or until more than 90% of the cells were disrupted. The sonicated extract was centrif ⁇ ged at 17K for 30 minutes at 4 ⁇ C and the pellet resuspended in the same buffer. The pellet was washed in 4M urea containing 20mM Tris-HCl pH 8.0 for 30 minutes and spun at 17K. The last wash was in H 2 0 for 10 minutes. The final pellet was kept frozen and aliquots taken for further studies.
• the wash-through containing the r-met-AChE was then concentrated and dialyzed against 20mM Tris-HCl pH 8.0, 2.5mM EDTA and 10% glycerol at 4°C.
• the dialyzed material remained clear and was injected into a rabbit to produce anti-r-met-AChE.
• erythrocyte hAChE purchased from Sigma was also used to produce rabbit anti-AChE antiserum and confirmed the results obtained using anti-r-met-AChE antiserum.
• the partially purified r-met-AChE was subjected to gel- filtration chromatography on FPLC (Pharmacia) using Superose-12 (Pharmacia) both with and without 8M urea.
• the chromatographic data suggest that most of the r-met-AChE in 8M urea is in a form of monomer-dimer while after dialysis only multimeric forms are observed.
• the diluted material was kept at room temperature for 2-3 hours, and then centrifuged to remove precipitates. The clear supernatant was then assayed for enzyme activity.
• Enzymatic activity was determined according to Ellman (Biochem. Pharmacol. 2 88 1961) .
• a radioactive assay using 3 H-acetylcholine iodide as substrate was implemented (Johnson, C. and Russel, R.L. Analytical Chemistry (1975) j54.: 229-238) .
• the r-met-AChE initially produced had no measurable enzymatic activity. We believed that this was due to faulty refolding/oxidation of the molecule. The development of a refolding/oxidation procedure was very arduous. The standard refolding procedures did not produce active enzyme, e.g. use of guanidine produced precipitation of the enzyme. Eventually the refolding procedure described below (in section 3) was developed which produced enzymatically active AChE; however the enzymatic activity was low.
• the hAChE catalytic subunit contains 7 cysteine (Cys) residues (see Figures 4A-D and SEQ ID NO:l), six of which are involved in intrasubunit disulfide linkages. We considered that exchange of the C-terminal cysteine residue
• This synthetic linker containing a serine codon (TCA) instead of the naturally occurring cysteine codon (TGC) was prepared such that it is flanked by a Sad site at the 5' end and an Xbal site at the 3' end.
• the linker was ligated to the large fragment isolated from Sacl-Xbal digestion of plasmid pAIF-52 ( Figure 7) .
• the resulting plasmid encodes serine instead of cysteine at position 611 and was designated pMLF-52ser ( Figure 7) .
• Plasmid pMLF-52ser expresses the ser/r-met-AChE polypeptide under control of the deo P promoter with a selection marker of tetracycline resistance.
• This plasmid was introduced to E. coli host S ⁇ 930 by transformation and deposited in the ATCC under ATCC Accession No. 68637. It expresses ser/r-met-AChE protein at a level of about 10% of total bacterial protein.
• Inclusion bodies were then dissolved in 8M urea or 6M guanidine thiocyanate containing 20mM Tris HCl pH 8.8 for several hours and centrifuged to remove undissolved matter.
• the denatured AChE thus produced has no enzymatic activity.
• the denatured enzyme In order to obtain an enzymatically active polypeptide, the denatured enzyme must be properly refolded.
• AChE activity was assayed by the spectrophotometric method of Ell an using acetylthiocholine as substrate (Ellman, G.L. et al. (1961) Biochem. Pharm.2'- 88-95)).
• Acetylthiocholine hydrolysis generates free thiocholine which reacts with Ellman reagent (DTNB) to produce a yellow chromophore.
• the concentration of the yellow chromophore is determined by the absorption at 412nm and is proportional to the amount of AChE present.
• One enzyme unit (U) is the amount of enzyme which hydrolyses l ⁇ mole of substrate per minute.
• the extinction coefficient of 1M chromophore is 13.6X10 4 .
• the assay solution contained 0.1M HEPES pH 8.0 instead of 0.1M NaP, because we found that spontaneous non-enzymatic degradation of acetylthiocholine in HEPES is much slower than in phosphate buffer.
• Table 1 is an example showing the recovery of active r-met- AChE and ser/r-met-AChE following in vitro refolding. Refolding of both r-met-AChE and ser/r-met-AChE was performed under identical conditions and at similar protein concentrations. It is clear that GSSG enhances the recovery of active enzyme.
• the protein obtained by the method of folding described above had a specific activity of up to 1.3 U/mg.
• the following procedure enables recovery of protein having much higher specific activity.
• Washed inclusion bodies of ser/r-met-AChE obtained as described above were dissolved in 6M guanidine thiocyanate, lOmM Tris HC1 pH 8.3 to a protein concentration of 3-5 mg/mL. Monomers and dimers were then separated from multimers by gel filtratation on Sephacryl-400 (Pharmacia) . 20-35 mg protein were loaded on the Sephacryl-400 column which had been previously equilibrated with 8M urea, lOmM Tris HC1 pH 8.3. Protein fractions corresponding to monomers and dimers were pooled and diluted into refolding chloride) to a final protein concentration of 25-50 ⁇ g/ml.
• AChE activity was eluted from Q-Sepharose in an NaCl gradient at about 0.275-0.375mM NaCl and the fractions pooled. Calculations of yield showed that 64% of the activity was recovered with a specific activity of 117u/mg. After concentration by precipitation with ammonium sulfate at 45% saturation, and dialysis against 20mM HEPES pH 8.0 for 24h, a total of 805 units was obtained. Successive 180- unit aliquots were applied to a MAC-Sepharose 4-B affinity column (1ml bed volume) until the entire 805 unit batch was processed.
• the " active fractions were pooled, concentrated by ammonium sulfate precipitation (45% saturation) , resuspended in ImL 20mM HEPES pH 8.0, 2.5mM EDTA, and dialyzed against 4 liters of the same buffer. A total of 0.29 mg protein containing 661 units of active ser/r-met- AChE was obtained.
• the affinity chromatography step improved purity by 19 fold with an overall recovery of about 84%.
• r-met-AChE and ser/r-met-AChE derived from S ⁇ 930/pAIF-52 and S ⁇ 930/pMLF-52ser respectively, generated a major activity band on the gel.
• the intensity of the ser/r-met-AChE band was considerably greater than that of the r-met-AChE band.
• the positions of the activity bands on the gel are not identical: ser/r-met-AChE migrates more slowly than r-met- AChE.
• the refolded ser/r-met-AChE showed nearly 10 times the activity towards 0.5mM acetylthiocholine, (i.e. 0.047 U/mL) as towards lOmM butyrylthiocholine, (i.e. 0.0044 U/mL) at the same protein concentration (0.15 mg/mL) . Since the butyrylthiocholine was present at a 20-fold higher concentration than acetylthiocholine it is seen that the enzymatic activity towards the butyrylthiocholine is 200- fold less than towards the specific substrate. No activity at all was detected towards the 0.05mM butyrylthiocholine.
• the specific inhibitor showed a strong and dose-dependent effect on the activity of the refolded ser/r- met-AChE towards the specific substrate, progressing from
• Triton X-100 reduced enzyme activity approximately 50%, while lauryl acid sodium salt and quaternary ammonium salts at 0.1% inhibited enzyme activity completely. This is in contrast to erythrocyte AChE, which requires Triton-X 100 for solubilization and maximum activity.
• the column bed volume was 140ml (in a 16mm by 650mm column) .
• the flow rate was 30ml/hr and 8ml fractions were collected.
• Figure 10 shows the gel filtration profile.
• a large amount of highly aggregated material devoid of enzyme activity was recovered in the void volume.
• AChE activity began to be detected in fraction 4 and increased to its highest point in fraction 7.
• the specific activity in fraction 7 was 10.6 U/mg.
• SDS-PAGE analysis of fractions 6 and 7 revealed considerable levels of impurities, which may be removed by other methods such as ion-exchange chromatography.
• Preliminary experiments indicate that the refolded AChE binds to anion exchange columns. Note that prior to refolding, the urea-solubilized inclusion bodies which contain the AChE did not bind to ion-exchange resin.
• r-met-AChE obtained from S ⁇ 930/pAIF-52 electrophoresed on SDS-PAGE was blotted onto PVDF paper and the protein band corresponding to r-met-AChE was isolated.
• N-terminal amino acid sequencing was carried out by the method of automated Edman degradation for the first 11 amino acids and the sequence obtained (shown below) was found to be correct.
• the second row contains the single-letter amino acid notation corresponding to the sequence shown in Figures 4A- D (SEQ ID NO:l) from position 32 et seq (not including the initiator methionine) .
• r-met-AChE expressed by plasmids pAIF-34 and pAIF-51 and ser/r-met-AChE expressed by plasmid pMLF-52ser all have the identical N-terminal sequence.
• the molecular weight of the purified enzymatically active mutant rAChE, ser/r-met-AChE was determined by gel permeation chromatography on Sephacryl 300.
• the column was equilibrated with lOmM L-arginine pH 10.0 and 50mM NaCl.
• Bovine serum albumin containing the 67kD monomer and 135kD dimer was used as an internal control.
• About 50 units of the highly purified recombinant ser/r-met-AChE were mixed with BSA and applied to the column.
• the 135kD BSA dimer peaked at fraction 45 and the 67kD monomer peaked at fraction 50.
• the AChE activity of ser/r-met-AChE peaked in fraction 53.
• Recombinant AChE is not glycosylated due to the lack of glycosylating enzymes in E. coli. Although glycosylation is not essential for catalytic activity, it may have a role in stabilization of the enzyme in vivo or in vitro.
• This example describes methods used to scale up the refolding and purification of recombinant AChE.
• the scale-up consisted of two phases.
• the first phase focused on the development of membrane filtration technology and the recovery of active ser/r-met-AChE after each step. This phase was aimed at obtaining 8-12 mg of highly purified ser/r-met-AChE.
• the second phase focused on development of a 25 fold scale-up of the first phase and enabled the obtaining of 169 mg of purified enzymatically active ser/r- met-AChE from 560 g dry cell weight of E. coli.
• the overall yield of ser/r-met-AChE was 60% of the initial activity obtained following in-vitro refolding. Assumptions and calculations leading to above figures are presented in the following description.
• the step by step purification procedure is shown schematically in the flow charts of Figures 11-13.
• the procedure consists essentially of solubilization of purified inclusion bodies in guanidinethiocyanate (GTC) followed by exchange of the GTC with urea, further dilution into refolding buffer (0.5M arginine pH 10.0, 0.3mM GSSG, 0.3% PEG 4000 (polyethylene glycol) , and 0.2M tetramethylammonium chloride) , separation of the active ser/r-met-AChE from inactive or contaminating proteins by DEAE column chromatography followed by affinity chromatography on MAC- Sepharose-4B.
• GTC guanidinethiocyanate
• refolding buffer 0.5M arginine pH 10.0, 0.3mM GSSG, 0.3% PEG 4000 (polyethylene glycol) , and 0.2M tetramethylammonium chloride
• a total of 1.7 x 10 6 units was measured upon completion of refolding which is equivalent to 283 mg of protein (assuming 6000 units/mg specific activity) .
• the refolded enzyme was then concentrated by ultrafiltration and loaded onto a DEAE column consisting of 2.5L of packed resin in a 15cm x 45cm column. The flow rate of the column was 100-150ml per min.
• the 1.7 x 10 6 units measured above were eluted from the DEAE column in 1856 mg protein which were then loaded onto a MAC- Sepharose 4B column having a bed volume of 80ml packed in a 2.6 x 40cm column. 1560 mg protein comprising mainly impurities did not bind to the resin and washed through.
• the bound protein amounted to 296mg and was further washed with 20mM HEPES pH 8.0 containing 0.275M NaCl to remove additional impurities.
• This wash step eluted 133mg of protein containing impurities and additionally containing 0.4xl0 6 units of activity.
• the ser/r-met-AChE was then eluted with 0.2 M L-arginine pH 10.0, containing 2.5mM EDTA. 169mg active protein were thus obtained.
• the expected amount of protein retained on the column is 163mg (296-133mg) , and therefore the observed value of 169mg is in good agreement with the expected number.
• the overall recovery of active ser/r-met-AChE was 1.01 x 10 6 units which constitutes a yield of about 60%.
• Table 3 shows the amino acid composition of purified ser/r- met-AChE. This data was deduced from amino acid analysis, based either on the average protein content, or on the protein content obtained by adding up the mass of measured amino acids. Excellent agreement with the theoretical composition is obtained by both methods for 16 of the 18 measured amino acids (16 Trp and 6 Cys residues are not recovered, i.e. the total theoretical number of residues is 562) . In the case of Ser, extensive loss (about -16%) noted and was therefore not taken into account when the average protein content was determined. The value for Leu is also about -10% lower than the theoretical value. For all the other amino acids the deviations are in the range of about ⁇ 6%.
• nmol amino acid/average nmol of protein nmol amino acid/ ⁇ nmol of 18 amino acids
• E. coli cells were resuspended (1:10 w/v) in: 50mM Tris-HCl pH 8.0; lOmM EDTA; 10 micrograms/ml lysozyme, and incubated at 4°C for 16-18 hours.
• the cells were disrupted using a Dyno-Mill and centrifuged.
• the pellet containing the inclusion bodies was collected, resuspended in water of the same volume as initially used to resuspend the cells, and stirred for one hour.
• the suspension was centrifuged, the pellet was resuspended in one half the initial volume of 4M urea containing lOmM Tris-HCl pH 8.0, and stirred for 2 hours.
• the solution was centrifuged, the pellet was resuspended in lOmM acetate buffer pH 5.2-5.4, and incubated while stirring at 4°C for 16-18 hours. The suspension was centrifuged and the weight of the resulting pellet determined.
• the solubilization and refolding experiments were performed by initially solubilizing the pellet of inclusion bodies in 6M guanidinethiocynate solution (GTC) , adding 7-10 ml of GTC per gram of pellet. Following solubilization, the pH of the solution was adjusted to 10.0 with 10M NaOH. After stirring the solution for 2 hours, the pH was readjusted to 5.4 with acetic acid. DTT was then added to a final concentration of 5mM, and the solution was stirred for 10-16 hours at room temperature. The solution was diluted 1:20 into 8.5M urea containing lOmM Tris-HCl pH 8.6, and left at room
• the 8.5M urea solution was diluted into cold refolding buffer (6- 10°C) by slow pumping and stirring, then incubated for 48 hours at 6-10°C.
• the volume of the solution was reduced by 75% by ultrafiltration on a 10K membrane, then dialyzed against 5-10mM L-Arginine pH 10.0.
• the solution was left at room temperature for 3-5 days, and the activity increase followed daily according to Ellman.
• the solution was then concentrated to 20L on a 10K membrane.
• the pH of the concentrate resulting from the steps described above was adjusted to 8-8.2 and the concentrate was loaded onto DEAE equilibrated with 20mM Tris-HCl pH 8.0, 2mM EDTA.
• the DEAE was washed with the same buffer to reach a base line at 280 nm, and further washed with 20 mM Tris-HCl pH 8.0, containing 0.2M NaCl.
• Ser/r-met-AchE was eluted from the DEAE with 20mM Tris-HCl, pH 8.0, containing 0.4M NaCl.
• rhAChE was eluted from the DEAE with 20mM Tris-HCl pH 8.0, containing 0.4M NaCl.
• the eluant was concentrated/dialyzed on a 10K membrane with lOmM Tris-HCl pH 8.0, (or HEPES), 2.5mM EDTA, and applied to a MAC-Sepharose affinity column equilibrated with the above buffer. The column was washed with starting buffer, then with buffer containing 0.25M NaCl. rAChE was eluted with 0.2M L-Arginine pH 10.0, 2.5mM EDTA, then concentrated and dialyzed against 20mM HEPES, pH 8.0, 2.5mM EDTA, 50mM NaCl.
• Recombinant acetylcholinesterase (r-met AChE or ser/r-met- AChE or similar polypeptides) produced as described in the previous Examples, may be used for many different purposes. Some of the potential uses are described below. The investigation of these uses of AChE has heretofore been limited or non-existent due to the limited availability of naturally occurring AChE.
• Organophosphorous Compounds are highly toxic agents utilized as insecticides and war nerve gases. As insecticides, OP compounds comprise a significant portion of the $6 billion worldwide insecticide industry and have replaced organochlorine compounds as the predominant insecticidal agents (Bull, David, The Growing Problem: Pesticides and the Third World Poor, Oxford, U.K.,: OXFAM, 38-45 (1982)). Accidental worker-related OP poisoning accounts for a large number of the 500,000 to 1,000,000 annual pesticide-associated poisonings (Bull, David, The Growing Problem: Pesticides and the Third World Poor, Oxford, U.K.,: OXFAM, 38-45 (1982)).
• OP compounds are the most important lethal agents currently available for military application (Kirk-Othmer Encyclopedia of CHEMICAL Technology, Wiley Corporation, Volume 5, (1984)) and have been used as recently as the Iran-Iraq War (Jane's NBC (Nuclear, Biological, Chemical) Protection Equipment, Forecast International, 1988-89) . Consequently, the market potential for both pre-exposure and post-exposure defense against organophosphorous compounds is quite significant. -56-
• This second reaction is termed “aging” because it transforms the inhibited cholinesterase into a form that can no longer be reversed by the commonly used “reactivators,” quaternary oximes such as pralidoxime mesylate which ordinarily dephosphorylate AChE. (Fortunately, aging only occurs after the administration of certain OP agents such as soman, one of the three OP war gases.) The inhibition of AChE leads to an accumulation of acetylcholine which causes convulsions and uncontrolled depolarization of cholinergic neurons
• the conventional therapy of antimuscarinics and oxi es has two significant drawbacks: 1) serious side effects are the norm rather than the exception and 2) oxime reactivation therapy is thwarted by the "aging" reaction.
• Acetylcholinesterase is preferable to current OP antidotes a) because it hydrolyzes acetylcholine directly and thus operates irrespective of the aging reaction, b) because it presents no apparent side effects, and c) because it eliminates the need for multiple drug therapy.
• AChE could be administered to soldiers or civilians before an imminent OP attack. In cases of OP intoxication, AChE would replace the current atropine -58-
• Acetylcholinesterase may be utilized in an enzyme-inhibition detection device for the presence of OP compounds on the battlefield (Goodman, A., and Martens, H. , Studies on the Use of Electric Eel Acetylcholinesterase for Anticholinesterase Agent Detection, Edgewood Arsenal Report No. Ed-TR-74096, Feb. 1975; Goodson, L.H. , Feasibility Studies on Enzyme System for Detector Kits, Edgewood Arsenal Report No. Ed-CR-77019, Dec. 1976; Levin, H.W. , and Erenrich, E.S., Enzyme Immobilization Alternatives for the Enzyme Alarm, Edgewood Arsenal Report No.
• acetylcholinesterase presents a possibly more effective mode of treatment because although it will temporarily lower acetylcholine concentration, it can directly hydrolyze the succinylcholine and thus more quickly restore normal respiratory activity.
• Anticholinergic drugs are often utilized in the treatment of gastric and duodenal ulcers and irritable bowel syndrome (irritable colon, spastic colon and mucous colitis) .
• Oral administration of antimuscarinic agents and quaternary oximes blocks the action of acetylcholine and thus reverses the effects caused by oversecretion of gastric acid by the vagus nerve.
• AChE would be at least as effective (if not more so) than the antimuscarinic and oxime drugs because AChE interacts directly with acetylcholine rather than-indirectly blocking acetylcholine activity.
• human AChE is an authentic human protein, it is likely that under normal circumstances it would not induce toxic or immunologic complications as do the other anticholinergic drugs.
• Acetylcholinesterase drugs such as neostigmine and isofluorophate which block the effects of AChE and BuChE by competing with acetylcholine for attachment to the enzyme active sites, are utilized in the treatment of myasthenia gravis (a neuromuscular disease) and glaucoma.
• the current therapy for overdose or accidental misuse of these agents is atropine alone or atropine with an oxime (Jane's NBC (Nuclear, Biological, Chemical) Protection Equipment, Forecast International, 1988-89) .
• oxime Greene's NBC (Nuclear, Biological, Chemical) Protection Equipment, Forecast International, 1988-89
• acetylcholinesterase could act as a zymogen precursor of a 25 kDa polypeptide that exhibited N-terminal aminopeptidase activity (Small, D.H., Trends in Biochemical Sciences, 15: 213-216) .
• the catalytic esteratic subunit of AChE is a 75 kDa polypeptide but it is tightly bound to this smaller subunit capable- of proteolytic activity.
• researchers have debated whether the smaller subunit is a unique gene product or whether it is simply a fragment from the C-terminus of the AChE catalytic subunit.
• the recombinant AChE protein of the instant invention could be tested for aminopeptidase activity; if present, this might be an additional potential use of the recombinant protein.
• Enzyme immunoassays using acetylcholinesterase have been utilized as simplified replacements of radioimmunoassays. Sensitivity and specificity have been shown to be comparable (Morel, A., Dar on, M. , amd Delaage, M., An Immunoenzymoassay for Histamine, Agents Actions, ____;: 291-293 (1990)) .
• AChE In addition to its presence in the membrane of mature erythrocytes, AChE is intensively produced in developing blood cells and its activity serves as an accepted marker for developing mouse megakaryocytes (European Patent Office -62-
• DNA probes from human cholinesterase genes can be used to detect and identify defective genes which may be responsible for the aforementioned diseases.
• Alzheimer's Disease or Down's Syndrome modification in both the level (Spokes, E.G.S., Brain 103: 179-183 (1980)) and the composition of molecular forms of human brain acetylcholinesterase have been reported (Atack, J.R. , et al., Neurosci. Lett. 40: 199-204 (1983)). Detection of altered level of type of AChE may be achieved by radioimmunoassay using antibodies elicited against human AChE.
• MOLECULE TYPE DNA (genomic)
• GGC ATC CCC TTT GCG GAG CCA CCC ATG GGA CCC CGT CGC TTT CTG CCA

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