EP0366662A1 - Aminopeptidase servant a extraire de la methionine a terminaison n a partir de proteines, et proteines preparees a partir de cette aminopeptidase - Google Patents

Aminopeptidase servant a extraire de la methionine a terminaison n a partir de proteines, et proteines preparees a partir de cette aminopeptidase

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Publication number
EP0366662A1
EP0366662A1 EP88904209A EP88904209A EP0366662A1 EP 0366662 A1 EP0366662 A1 EP 0366662A1 EP 88904209 A EP88904209 A EP 88904209A EP 88904209 A EP88904209 A EP 88904209A EP 0366662 A1 EP0366662 A1 EP 0366662A1
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EP
European Patent Office
Prior art keywords
peptidase
met
polypeptide
enzyme
terminal methionine
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EP88904209A
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German (de)
English (en)
Inventor
Charles G. Miller
Rao Movva
Paul Wingfield
Gonzola J. Mazzei
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Biogen Inc
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Biogen Inc
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Publication of EP0366662A1 publication Critical patent/EP0366662A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • This invention relates to enzymes, organisms producing such enzymes, methods for obtaining, culturing and using the enzymes and organisms, and polypeptides prepared using such enzymes. More specifically, this invention relates to enzymes capable of specifically removing an N-terminal methionine from a polypeptide and to polypeptides prepared using these enzymes.
  • AUG is the universal
  • RNA translation start codon It codes for the amino acid methionine (Met).
  • Met methionine
  • N-terminal amino acids of the proteins are usually alanine, serine or threonine (S. S. Sarimo et al., "Taxonomic Comparison of the Amino Termini of
  • N-terminal methionine There are two mechanisms which are believed to "mature" the nascent protein by removing the N-terminal methionine.
  • the initial N-terminal methionine is removed as part of the signal peptide during secretion.
  • the N-terminal methionines of pre- ⁇ -interferon, pre-growth hormone and pre-proinsulin are removed as part of the signal peptide during secretion.
  • the N-terminal methionine of the nascent protein is usually deformylated (J. M. Adams, "On The Release Of The Formyl Group From Nascent Protein", J. Mol. Biol., 33, pp. 571-89 (1968)) and then the N-terminal methionine is removed in vivo by one or several enzymes present in the cell during protein production.
  • proteins began to be produced or overproduced in transformed unicellular hosts that had not previously produced them. These recombinant proteins, of course, had to be produced with an N-terminal methionine.
  • the removal of that methionine to produce a protein identical in amino acid sequence to the corresponding or desired mature protein proved problematical .
  • proteins that were not usually secreted from the cell in which they were made either the level of in vivo enzymes or their activity toward those overproduced, and usually foreign, proteins proved too low to be effective in producing high levels of substantially methionine-free proteins.
  • these proteins could often only be obtained as mixtures of protein molecules some with N-terminal methionine and others with the N-terminal methionine removed. Such mixtures were difficult if not impossible to resolve into single components in commercial purification processes.
  • Similar results were also observed when proteins that were normally secreted were prepared without signal sequences, e.g., recombinant met-interferon, recombinant met-human growth hormone and recombinant met-proinsulin.
  • Non-secreted mature proteins tend to have the following N-terminal amino acids: Ala, Gly,
  • Microorganisms typically contain many enzymes that remove N-terminal amino acids from peptides. Most of these enzymes, however, are broad specificity enzymes, and they may cleave amino acids other than methionine from the N-terminus of their substrates. Such peptidases will not only cleave the N-terminal amino acids from their substrates but will also continue to cleave additional amino acids. This makes them useless for the specific rgmoval of a single methionine from the N-terminus of a polypeptide. For example, at least four such enzymes are present in crude extracts of Salmonella typhimurium and E.coli (C. G. Miller et al., "Peptidase Mutants of Salmonella typhimurium", J.
  • N-terminal Met aminopeptidase that is specific for N-terminal methionine has not been isolated previously. While certain enzymes capable of cleaving methionine from various substituents have been isolated (V. M. Vogt, "Purification and Properties of an Aminopeptidase from Escherichia coli", J. Biol. Chem., 245, 4760-69 (1970)), these enzymes do not specifically remove N-terminal methionine from immature polypeptides. Rather, these enzymes are either nonspecific, removing many N-terminal amino acids other than methionine from their peptide substrates (Vogt, supra), or they are limited to dipeptides having N-terminal methionines (J. L.
  • Peptidase M is an enzyme capable of removing N-terminal methionine from polypeptides containing an N-terminal methionine. Peptidase M does not remove other N-terminal amino acids, nor does peptidase M hydrolyze methionine amino acids in polypeptides at locations other than the N-terminus.
  • the present invention provides mutant strains of microorganisms that overproduce peptidase M so that large amounts of the enzyme may be obtained from culturing the strains.
  • the enzyme may then be isolated and used in vitro.
  • These microorganisms may also be used as hosts for the production of recombinant proteins.
  • eht enzyme acts in vivo to remove the N-terminal methionine of the co-produced recombinant protein.
  • This invention also provides for the cloning of the DNA sequence coding for peptidase M, or active fragments thereof, in host cells that produce a desired recombinant protein, to effect the removal of the N-terminal methionine of the recombinant protein in vivo.
  • the enzyme may be produced in unicellular hosts transformed with that DNA sequence, and the enzyme may then be isolated and used in vitro.
  • the present invention provides mature polypeptides produced using peptidase M, and especially the preferred peptidase M found in certain strains of Salmonella typhimurium. Still other aspects and advantages of the invention will be apparent from the specification.
  • Figure 1 displays a 12.5% acrylamide SDS gel prepared by the method of U.K. Laemmli, Nature (London), 227, 680-85 (1970).
  • Wells 2 and 3 each contained 30 ⁇ g protein from extracts of S .typhimurium strain TN2529 (well 3) and TN2547 (well 2).
  • Well 1 contained purified pepidase M from the active peak of the chromatofocusing column.
  • the peptidase M band in the pepM100 extract (well 2) (a strain that overproduces peptidase M) appears much darker than in the wild-type peptidase M extract (well 3).
  • DNA Sequence A linear array of deoxy nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses .
  • Codon A DNA sequence of three nucleotides (a triplet) that encodes, through its mRNA, an amino acid, a translation start signal, or a translation termination signal.
  • the four DNA bases are adenine ("A"), guanine ("G”), cytosine ("C”), and thymine (“T”).
  • the four RNA bases are A, G, C and uracil ("U”).
  • A indicates either of the purines (A or G)
  • Q indicates either of the pyrimidines (C or T)
  • N indicates any of the four bases (A, G, C, or T).
  • RNA For RNA, "P,” “Q” and “N” have the same meanings except that "U” is substituted for "T.”
  • the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine ("Leu”); TAG, TAA and TGA are translation stop signals; and ATG is a translation start signal in DNA that also codes for methionine. Since these are more possible triplet combinations of nucleotides (64) than amino acids (26), the genetic code is said to be “degenerate”, i.e., several different triplets may encode for the same amino acid. Two DNA sequences are "degenerate” when they encode for the same amino acid sequence though using different codons.
  • Polypeptide A linear array of amino acids connected one to another by peptide bonds between the ⁇ -amino and carboxy groups of adjacent amino acids.
  • Genome The entire DNA of a cell or a virus. It includes, inter alia, the DNA coding for the polypetides of the cell and operator, promoter, and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno sequences for each of the those coding sequences.
  • Gene A DNA sequence that. encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.
  • mRNA messenger RNA
  • Expression The process undergone by a gene to produce a polypeptide. It includes transcription of the DNA sequence to an mRNA sequence and translation of the mRNA sequence into a polypeptide.
  • the DNA sequence For a DNA sequence coding for a polypeptide to be expressed, the DNA sequence must be operatively linked to an expression control sequence that regulates the expression process.
  • Protein A polypeptide of 50 or more amino acids.
  • Preprotein A polypeptide or protein having extra amino acids with respect to a mature (i.e., active) protein.
  • Cloning The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
  • Recombinant DNA Molecule or Hybrid DNA A molecule, comprising segments of DNA from different genomes joined end-to-end outside of living cells, that may be maintained in living cells.
  • Cloning vehicle A plasmid, phage DNA, or other DNA sequence that is able to replicate in a host cell.
  • a cloning vehicle is also known as a recombinant vector.
  • the enzyme of the present invention is specific for removing the N-terminal methionine from polypeptides. While the examples below demonstrate the invention with peptidase M as found in Salmonella typhimurium, it will be apparent that other microorganisms, like E.coli, may also be used as sources of a peptidase M or an analog.
  • An "analog" of peptidase M as used herein, means an enzyme that cleaves N-terminal methionine from a polypeptide, without cleaving other N-terminal amino acids, even though the enzyme may not have an amino acid sequence identical to peptidase M.
  • each such organism would be expected to have at least one enzyme capable of specifically removing an N-terminal methionine. And, therefore, such enzymes may be isolated, produced and used as described herein.
  • the preferred microorganisms for producing the enzyme of the invention are Salmonella typhimurium and E.coli, due to their ease of handling and ready availability. Salmonella typhimurium is especially preferred. Consequently, peptidase M as produced by S.typhimurium and E.coli is preferred and peptidase M from S.typhimurium is especially preferred.
  • the first step in isolating peptidase M or an analog is to obtain mutant strains of the chosen microorganism that do not substantially produce any broad specificity enzymes capable of cleaving N-terminal methionione.
  • the availability of such strains allows the selection of mutants that over- produce the methionine-specific aminopeptidase.
  • the absence of these broad specificity enzymes also aids purification by removing in advance other enzymes which would, if present in the purified preparation, render it useless for specifically removing methionine without causing any other modification in the peptide chain.
  • Strains lacking these peptidases were isolated by a series of steps each one designed to generate a particular mutation leading to the loss of one of the peptidases. (See C.G.
  • This selection produced mutants with elevated levels of peptidase M.
  • One such mutant strain carrying the pepM100 mutation showed a 20-30 fold elevation in the level of Met-Ala-Ser hydrolyzing activity, and it was concluded that this strain overproduced peptidase M.
  • the specificity of the overproduced peptidase in strains carrying the pepM100 is demonstrated by the peptide use pattern shown in Table 2. This strain does not gain the ability to grow on nonmethionine peptides.
  • the selection described requires the use of multiply peptidase deficient strains that carry stable, non-reverting alleles of mutations in peptidase in N, A, B, D, and T as described in Strauch, et al. (cited above).
  • Analogs of peptidase M may have different levels of activity with respect to different polypeptides having N-terminal methionine.
  • the technique for producing a mutant strain that overproduces an analog of peptidase M will require met-polypeptide having a hydrolysis rate low enough to prevent growth by non-overproducing strains. In the case of
  • the met-polypeptide was observed to be Met-Gly-Gly, and that tripeptide is the preferred tripeptide for selecting overproducing strains of S.typhimurium, but other tripeptides may be more preferable for selecting peptidase M analogs in other strains.
  • the enzyme itself may be isolated.
  • the cells obtained from culturing are washed in an isotonic medium and broken using means known in the art.
  • the particulate material from breaking is removed by known means, such as centrifugation and filtering.
  • the supernatant may be applied to a chromatography column and peptidase M concentrated.
  • the preferred microorganism S.typhimurium
  • S.typhimurium we obtained an overproducing mutant strain, TN2270, using the procedure described above.
  • the strain is grown in minimal glucose medium containing 0.4 mM Leu and 0.4 mM Met.
  • the resulting culture is pelleted and suspended in 0.01M potassium phosphate buffer (pH 7.5), and the cells are disrupted by sonication.
  • the disrupted cells are centrifuged.
  • the supernatant is fractionated by chromatography.
  • the active fractions i.e., those fractions that remove N-terminal methionine from met-polypeptides, are combined and concentrated.
  • the chromatography column is preferably a DEAE-cellulose (Whatman DE-52) column equilibrated with potassium phosphate buffer (pH 7.5). The column is eluted in the same potassium phosphate buffer. The active fractions are preferably concentrated over an ultrafiltration membrane (YM-10, Amicon Corp.).
  • the active fractions from the chromatographic separation are determined by adding each fraction to a mixture containing: 0.6 ⁇ mol substrate (.preferably Met-Ala-Ser); 0.03 ⁇ mol CoCl 2 ; and 6 ⁇ mol potassium phosphate buffer (pH 7.5); to make a total volume of 30 ⁇ l.
  • the mixture is incubated for 30 minutes at 37°C and the reaction is then stopped by adding 3 ⁇ l 50% trichloroacetic acid. Precipitated polypeptides are removed by centrifugation.
  • the active fraction concentrated over the ultrafiltration membrane is passed through an Ultrogel AcA54 column (LKB) equilibrated in 0.05 M potassium phosphate buffer (pH 7.5).
  • LLB Ultrogel AcA54 column
  • Met-Ala-Ser hydrolyzing fractions from the procedure described above were again combined and concentrated over an ultrafiltration membrane.
  • This second concentrate was further purified by chromatofocusing using a Pharmacia PBE94 column in 0.025 M imidazole-HCl (pH 7.4, pi 5.2) and eluted with polybuffer 74-HCl (pH 4.0).
  • Example 4 An alternative technique for isolating the enzyme is discussed below in Example 4.
  • Cells from strain TN2270 are broken using the French Pressure Cell technique and centrifuged. The supernatant is then filtered. The supernatant may then be fractionated using chromatography and chromatofocusing.
  • this alternate technique produces highly purified peptidase M.
  • the isolated peptidase M may be used to remove or "clip" N-terminal methionine from polypeptides in vitro. As disclosed in the examples below, peptidase M may simply be mixed with the desired met-polypeptide to produce mature polypeptides.
  • the preferred method of producing mature polypeptides is by exposing the met-polypeptide to purified or partially purified peptidase M, even crude extracts of broken cells that contain peptidase M have activity with respect to removing the N-terminal methionine from met-polypeptides.
  • crude extracts of strains not lacking broad specificity peptidases would not be useful in this invention because the broad specificity peptidases would remove other N-terminal amino acids.
  • Purified peptidase M may be added to the met-polypeptide in a ratio of about 1:100. Under ordinary conditions, no more than two hours are generally necessary to complete the removal of the N-terminal methionine from the polypeptide. Ordinary conditions are defined as approximately room temperature and pressure and a pH of about 7.0. Table 3 shows that a crude cell extract of
  • S.typhimurium strain TN2624 (a strain lacking the broad-specificity peptidases, peptidases N, A, B and T, and carrying the pepM100 mutation) hydrolyzed all of the N-terminal Met tripeptides that support growth as Met sources. In every case only one peptide bond was hydrolyzed, yielding Met as the only single amino acid product. These extracts did not contain detectable activity towards Met-Leu-Gly, Met-Met-Ala, or Met-Met-Met, but did hydrolyze Met-Gly-Gly at about 0.1 the rate of Met-Ala-Ser or Met-Ala-Met.
  • incubation of met-polypeptide with substantially purified or isolated peptidase M is the preferred method for in vitro use.
  • the incubation time of substrate will vary with the ratio of substrate to peptidase M in the reaction mixture. Less time will generally be required to complete removal of N-terminal methionine from a substrate when there is more peptidase M present in the reaction mixture per unit of substrate.
  • Peptidase M may also be used in vivo using a strain in which the enzyme is overproduced as a host for the production of a desired recombinant protein or by isolating the DNA sequence coding for peptidase M, and inserting that DNA sequence, a DNA sequence degenerate with that DNA sequence, or a DNA sequence coding for an active fragment of peptidase M, into a recombinant host that so produces the polypeptides from which met-removal is desired.
  • Recombinant hosts that produce a desired protein often produce the associated met-protein in large amounts, but not all of the met-protein is matured by the host's natural peptidases.
  • Insertion of the DNA sequence coding for peptidase M into such a host so as to permit its expression as that host thus is a great advantage for recombinant systems, because enhanced levels of peptidase M assist in removing N-terminal methionine from preproteins during expression of the protein in vivo. In this way, large amounts of the desired Met- _ protein be produced by the recombinant host.
  • the DNA sequence coding for peptidase M from S.typhimurium has been isolated and inserted into a phage. The peptidase M DNA from that phage was then inserted into a plasmid which, in turn, was placed into E.coli.
  • This recombinant host produced peptidase M E.coli is known as a host capable of producing recombinant proteins. Accordingly, one possible and preferred method of using peptidase M in vivo is to insert the DNA sequence coding for peptidase M into a host so as to allow the peptidase M expressed in the recombinant host to remove N-terminal methionine from a natural or recombinant protein also produced by the host. Preferably, the host will overproduce both the desired protein and peptidase M. Alternative means of using peptidase M are also available. Strains that overproduce peptidase M, shown in more detail below, may themselves serve as recombinant hosts. On the other hand, the DNA sequence coding for peptidase M may be operatively linked to an expression control sequence and inserted into a strain that already overproduces the desired protein using a recombinant vector.
  • the present invention also includes the in vitro use of peptidase M produced by recombinant means.
  • Peptidase M may be overproduced by a recombinant host, and that peptidase may remove N-terminal methionine from any polypeptide either in vitro or in vivo, whether or not that polypeptide is produced by recombinant means.
  • This invention also includes polypeptides that have N-terminal methionine removed by peptidase M.
  • the polypeptides may act as the substrate for peptidase M in vitro or in vivo.
  • Peptidase M may exist in vivo or in vitro, hence any combination of a met-polypeptide and peptidase M that results in removal of the N-teminal methionine from the polypeptide is within the scope of this invention.
  • This invention also includes a method for isolating strains of a microorganism that produce enzymes capable of removing an N-terminal methionine from a polypeptide which would not ordinarily be removed in vivo.
  • N-terminal methionine is apparently not removed from a non-secreted polypeptide when the second amino acid is Arg, Asn, Asp, Glu, Gin, lie, Leu, Lys or Met.
  • Enzymes capable of removing an N-terminal methionine from a polypeptide having one of these amino acids as its second polypeptide may be produced by mutating a peptidase M producting strain (that does not have broad specificity amino peptidases) and growing the mutated strains in a medium containing the polypeptide as the only source of methionine. Strains that grow in such an environment have an enzyme capable of removing N-terminal methionine.
  • Enzymes act upon many different substrates, though with differing degrees of activity.
  • tripeptides of Met-gly-gly are not hydrolyzed as rapidly as some other tripeptides.
  • This lower activity provides a technique for isolating strains that overproduce an enzyme having more than one substrate. Overproducing strains grow in media containing the slower reacting substrate as the only source of an essential nutrient, while strains that do not overproduce the enzyme do not grow in such media. An overproducing strain may then be isolated by culturing mutated microorganisms in such a medium.
  • This technique may be used along with known mutation techniques to produce enzymes in strains, for example, that can cleave N-terminal methionine from any polypeptide, even from polypeptides having a second amino acid that does not normally allow methionine cleavage in vivo.
  • Met-Gly-Gly does not support growth in strains lacking broad specificity peptidases but is hydrolyzed by extracts at a slower rate than substrates with alanine as the second amino acid suggested a method for isolating strains that overproduce peptidase M.
  • a Met-requiring strain carrying nonreverting mutations in the genes specifying the broad-specificity aminopeptidases described above was plated on medium containing Met-Gly-Gly as a Met source, mutants capable of using this peptide were obtained (K. L. Strauch et al., "Overproduction of Salmonella typhimurium Peptidase T," J. Bacteriol., 156, 743-51 (1983)).
  • Mutant strains lacking peptidases N, A, B, D, P, Q and T and dipeptidyl carboxypeptidase were obtained using the procedure of K. L. Strauch et al., "Isolation and Characterization Salmonella typhimurium Mutants Lacking a Tripeptidase (Peptidase T)," J. Bacteriol., 154, 763-71 (1983). These strains were cultured in E medium supplemented by 0.4% glucose and 0.4 mM L-amino acids. Mutagenesis of the strains lacking the broad-specificity peptidases was carried out with diethylsulfate to increase the frequency of mutation. Such mutagenesis was not necessary, however, and spontaneous mutants could be found at a detectable frequency.
  • Mutants able to use Met-Gly-Gly as a methionine source were selected by plating 0.1 ml of a minimal overnight culture of TN2183 on an appropriately supplemented minimal plate containing Met-Gly-Gly (0.1 mM) as a Met source.
  • Met-Gly-Gly 0.1 mM
  • Several of these mutants were purified and characterized. Although these mutant strains grew well on Met-Gly-Gly, they did not use Met-Leu-Gly, Met-Met-Ala, or Met-Met-Met, nor did they grow on any of several N-terminal leucine peptides as Leu sources.
  • Assays of peptide hyrolysis in an extract of one mutant, pepM100 showed a 20-30 fold increase in Met-Ala-Ser hydrolyzing activity. This strain was chosen for further characterization.
  • the peptide use profile of a strain carrying pepM100 is compared to its parent in Table 2.
  • Table 3 shows that, in an extract of the mutant strain, the levels of activity toward N-terminal Met peptides with Ala, Thr, or Gly in the second position all show an approximately 20 fold increase relative to the activity in a wild-type peptidase M-containing strain. Met-Leu-Gly, Met-Met-Met, Met-Met-Ala and several other peptides with N-termini other than methionine are not hydrolyzed by either the mutant or parental extracts. The relative rates of hydrolysis for all substrate peptides are the same in the two extracts. The level of a single peptidase is, therefore, increased by the pepM100 mutation. This peptidase is specific for
  • N-terminal methionine is affected by the peptide's second amino acid.
  • N-terminal Met from Met-Gly-Met-Met shows that the enzyme is capable of hydrolyzing tetrapeptides and is not limited in specificity to tripeptides.
  • the mutant strain did not grow on any of several N-terminal Met dipeptides (for example, Met-Gly and Met-Ala), and extracts did not hydrolyze these dipeptides.
  • Strain TN2270 was grown in minimal glucose medium containing 0.4 mM Leu and 0.4 mM Met. A cell pellet from the culture was suspended in 0.01 M potassium phosphate buffer (pH 7.5) and disrupted by sonication. After centrifugation, the supernatant was applied to a DEAE-cellulose (Whatman DE-52 ) column equilibrated with the phosphate buffer and eluted with a linear gradient of KCl to 0.4M in the same buffer. The active fractions were combined, concentrated over an ultrafiltration membrane (YM-10, Amicon Corp.), and passed through an Ultrogel AcA54 column (LKB) equilibrated in 0.05 M potassium phosphate buffer (pH 7.5).
  • the Met-Ala-Ser hydrolyzing fractions were combined and concentrated as above.
  • the specific activity of this material was approximately 13 fold higher than the starting extract and approximately 290 fold higher than that of a wild-type extract.
  • This material was further purified by chromatofocusing (Phamacia PBE 94 in 0.025 M imidazole-HCl pH 7.4, pi 5.2 eluted with Polybuffer 74-HCl pH 4.0). The purified material from the peak fraction of this column was used for the experiment shown in Fig. 1.
  • the final supernatant was filtered with a 0.45 ⁇ nitrocellulose membrane and applied to a DEAE-Sepharose column (14 x 5 cm diameter) equilibrated with 100mM sodium phosphate, pH 7.0, 1mM sodium azide.
  • the peptidase M that bound to the column was eluted early during the application of 1.5L gradiant of 0 - 0.3M NaCl.
  • the Peptidase M was pooled on the basis of activity (about 300ml total volume was obtained), and concentrated to 20ml using a Diaflo PM10 ultrafiltration membrane.
  • Chromatofocusing was carried out by placing partially purified Peptidase M, as obtained above, in 50 mM sodium- phosphate, pH 7.0 (0.5 to 1.5ml at about 4.0-6.0mg/ml) and applying it to a FPLC Mono P HR5/20 column (Pharmacia) equilibrated with 25mM bis-Tris-HCl, pH 7.4. The column was eluted at lml/min at room temperature with 49ml of Polybuffer 74-HC1 (Pharmacia) diluted 1:10 with water and adjusted to pH 4.0 with HCl. Fractions (0.5ml) were collected into tubes containing an equal volume of 200mM sodium phosphate, pH 7.0.
  • a strain containing a duplication of the pepM locus was constructed by the method of Anderson and Roth, "Gene Duplication in Bacteria: Alteration of Gene Dosage by Sister-chromosome Exchanges", Cold Spring Harbor Symp. Quant. Biol., 43, 1083-87 (1978)), using a Tn10 insertion in the pyrA gene.
  • This duplication strain was used for dominance testing and to NOT TO BE TAKEN INTO CONSIDERATION FOR THE PURPOSES OF INTERNATIONAL PROCESSING (See Section 309 (c) (ii) OF THE ADMINISTRATIVE INSTRUCTIONS)
  • cobalt ions may be added to increase the activity of peptidase M. See Table 4.
  • overproducer mutations The basis for selection of overproducer mutations is the observation that Met-Gly-Gly, although a substrate for the enzyme, is apparently not hydroloyzed sufficiently rapidly to allow its use as a Met source.
  • the availability of an overproducer of peptidase M provides a convenient source for purification of the enzyme, but those of skill in the art will recognize that preparation of overproducing strains is not essential for preparing and isolating peptidase M. Overproduction of peptidase M is not expected to be deleterious to cell growth since strains containing the pepM100 overproducer mutation grow normally under all conditions that we have tested.
  • the enzyme has a very pronounced specificity for the second amino acid so that overproduction does not result in removal of Met from proteins that normally remain unmodified.
  • Strains that overproduce peptidase M will be useful for removing N-terminal Met from cloned proteins that are expressed at levels that are too high for efficient N-terminal modification in wild-type cells.
  • the pepM gene can be engineered to be expressed in high quantities, only when needed.
  • IL-1 ⁇ recombinant-derived IL-1 ⁇ (Biogen S.A.) dissolved in 25mM imidazole acetate pH 7.6 (column buffer) was applied either to a FPLC Mono P HR5/20 column (Pharmacia; 2mg was applied at a protein concentration of 2mg/ml) or to a column (11cm x 1.5cm dia.) containing Polybuffer previously equilibrated with column buffer.
  • the Mono P column was eluted at Iml/min at room temperature with 57ml of a Polybuffer 96/74 mixture (20:1 v/v) diluted 1:15 with water and adjusted to pH 6.0 with acetic acid. (Polybuffers from Pharmacia).
  • the Polybuffer exchanger column was eluted at 50ml/h at 4°C with 200 ml of Polybuffer 96 diluted 1:13 with water and adjusted to pH 6.0 with acetic acid. Two distinct fractions were pooled based on their absorbance at 280mm. In order to remove Polybuffer from pooled fractions, solid ammonium sulphate was added to 82% of saturation. The precipitated protein was collected by centrifugation and dissolved in 20mM NH 4 HCO 3 . The clear solution was dialysed at 4°C against several changes of this buffer and then freeze dried.
  • the standard reaction mixture contained, in a final volume of 0.2 ml, 40 ⁇ mol of sodium phosphate, pH 7.5, 40 ⁇ mol of NaCl, and 0.1 ⁇ mol of CoCl 2 , 60-100 ⁇ g of Met-Gly-Gly or Leu-Gly-Gly as the substrate.
  • the reaction was started by the addition of enzyme and was carried out for 15-30 min at 30°C.
  • the amount of free amino acid was determined spectrophotometrically at 45 mm by adding 50 ⁇ l containing 50 ⁇ g L-amino oxidase, 0.5 ⁇ mol of MnCl 2 , 100 ⁇ g horseradish peroxidase and 50 ⁇ g o-dianisidine (this premix allows the production of H 2 O 2 on the oxidation of the free amino acids. H 2 O 2 is then utilized as a substrate by the peroxidase which oxidizes the o-dianisidine that produces a change in color).
  • Leucine aminopeptidase from hog kidney was used as a control aminopeptidase. Similar conditions were used with the control except 0.2 ⁇ mol of MnCl 2 was used as a metal activator.
  • IL-1 ⁇ , recombinant methionylated and non-methionylated IL-1 ⁇ were separated by chromatofocusing as described above.
  • Electrofocusing was carried out on thin-layer polyacrylamide gels (LKB Ampholine PAG plates pH 3.5-9.5). SDS-PAGE was carried out on 13% acrylaraide slab gels using a discontinous buffer system. The purified methionine aminopeptidase was able to hydrolyze the N-terminal amino acid of Met-Gly-Gly and Leu-Gly-Gly at a rate of 10.33 and 0.4 units/mg of enzyme, respectively.
  • the purified IL-1 ⁇ (100 ⁇ g), containing 20% N-terminal methionine, was incubated with peptidase M for different times (the ratio was 1:100 of enzyme to substrate).
  • the N-terminal methionyl residue of the methionylated IL-1 ⁇ was removed within 15 to 30 min. incubation without degradating the non-methionylated IL-1 ⁇ .
  • N-terminal methionylated IL-1 ⁇ form was separated from non-methionylated IL-1 ⁇ in a mixture by chromatofocusing.
  • Peptidase M (0.68 ⁇ g) completely removed the n-terminal methionine of the purified methionylated IL-1 ⁇ (68 ⁇ g) after incubation for 1 to 2 hrs without degradating the non-methionylated form.
  • Leucine aminopeptidase did not remove the N-terminal methionine of the methionylated IL-1 ⁇ as observed on IEF.
  • peptidase M or its analog may be used to treat preproteins in vitro.
  • peptidase M or its analog may be used to treat preproteins in vitro.
  • Those of ordinary skill in the art will also appreciate the advantage of overproducer strains of the microorganisms of the present invention used as hosts for recombinant DNA.
  • the gene coding for peptidase M may also be inserted into a host to assist in the production of mature recombinant or overproduced proteins.
  • S.typhimurium strain TN2529 wild type was digested using Sau3A, and the 9-22 Kb fragment obtained was inserted into the DNA for phage ⁇ EMBL3 and the BamHI site.
  • S.typhimurium strain TN2529 has been deposited with the Deutsche Sammlung Von Mikroorganismen, West Germany, and has been assigned DSM No. 3969.
  • DNA from strain TN2547 (peptidase M overproducer) was also partially digested and the 9-22 Kb fraction was inserted into ⁇ EMBL3 at the BamHI site.
  • Strain TN2547 was also deposited in the Deutsche Sammlung Von Mikroorganismen as DSM No. 3970. 1000 plaques in each phage were then screened using the procedure described by K.
  • Peptidase M was isolated from a crude extract of strain TN2547 using the AcA54 chromatography column described in Example 4. The concentrated peptidase was then further purified by isoelectric focusing. The pi 5.2 band was resulting from isoelectric focusing was peptidase M. Approximately the first 30 amino acids at the N-terminal end of peptidase M were then sequenced in a protein sequencer. The first thirty amino acids of peptidase M are listed in Table 7.
  • the sequence was used to make two oligonucleotide probes, 32/1a and 32/1b, that were
  • probe 32/1a gave positives and 32/1b gave negatives.
  • the probe-attached DNA was washed in 3.2M tetramethyl NH 4 Cl/1% SDS solution at 52°C for 90 minutes. The DNA was then washed in six times SSC and 0.1% SDS at room temperature for 10 minutes. Clones were identified by autoradiography and five positive plaques were initially selected for each strain's DNA sequence. These phages were purified; the DNA was isolated; and one sequence from each strain was selected for further study.
  • the DNA sequence originally from strain TN2529 (the wild type strain) was inserted into phage ⁇ 10A and the DNA sequence originally from strain TN2547 (the overproducer strain) was inserted into phage ⁇ 3B.
  • the phage ⁇ 10A was also hybridized with two probes that were larger than the probes discussed above (the probes are shown in Table 8) to confirm that the inserted DNA sequence was the one coding for peptidase M. These longer probes are also shown in Table 8. The results confirmed that the proper DNA sequence was present.
  • the phage ⁇ 10A DNA sequence was hybridized with the 32/1a probes using the Southern blot techinque to confirm that the peptidase M DNA sequence was present.
  • DNA from phage ⁇ 10A containing the DNA sequence for peptidase M from strain TN2529 was separately cut with XhoI and SalI to give DNA fragments of aproximately 3.5Kb and 4.5Kb.
  • TN2624 TN2626 (pepM + ) (pepM100)
  • the hydrolysis rates for each extract are compared to the hydrolysis rate for Met-Ala-Ser for that extract.
  • the specific activity for Met-Ala-Ser was .029 units/mg protein for TN2624 extract, and 0.53 units/mg protein for TN2626 extract.
  • the assay contained either 140mg protein (TN2624 extract) or 14mg protein (TN2626 extract).
  • panC:Tn10 zae1615 panC:Tn10 zae1615::
  • Probe N52-1B and Probe N52-2' are complementary between the marked bases. These oligonucleotides are annealed to each other and extended with radioactive nucleotide triphosphates with the help of Klenow enzyme, the unincorporated triphosphates of oligonucleotides are separated from the mixture of oligonucleotides. This mixture of oligonucleotides was used as a probe to identify the DNA corresponding to pepM gene.

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Abstract

Une aminopeptidase, appelée ici peptidase M qui extrait uniquement de la méthionine à terminaison N, des souches de microorganismes qui produisent cette aminopeptidase, et des polypeptides traités avec cette aminopeptidase sont décrits. La peptidase M peut être utilisée pour le clivage de protéines de polypeptides in vitro, ou l'ADN des souches qui produisent la peptidase M peut être utilisé pour transformer un organisme hôte recombinant de façon à préparer des protéines matures in vivo. Les souches qui produisent la peptidase M peuvent elles-mêmes être utilisées comme organismes hôtes recombinants. Des procédés servant à isoler la peptidase M et des procédés permettant d'isoler des souches qui produisent en excès de la peptidase M sont également décrits.
EP88904209A 1987-02-10 1988-02-09 Aminopeptidase servant a extraire de la methionine a terminaison n a partir de proteines, et proteines preparees a partir de cette aminopeptidase Withdrawn EP0366662A1 (fr)

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US5013662A (en) * 1985-09-20 1991-05-07 Cetus Corporation Bacterial methionine n-terminal peptidase
JPH04500155A (ja) * 1988-08-26 1992-01-16 ザ・ジェネラル・ホスピタル・コーポレイション メチオニン特異的アミノペプチダーゼ：ｍａｓ　ｉおよびｍａｓ　ｘの単離、精製および特性確認
WO1990002815A1 (fr) * 1988-09-13 1990-03-22 The General Hospital Corporation Isolation, purification et caracterisation des aminopeptidases mas ii et mas iii
AU4221089A (en) * 1988-09-13 1990-04-02 General Hospital Corporation, The Isolation, purification, and characterization of the aminopeptidases: ap2, ap1, and apx
AU4213489A (en) * 1988-09-13 1990-04-02 General Hospital Corporation, The Isolation, purification, and characterization of the aminopeptidases: ap1 and ap122
KR970010135B1 (ko) * 1993-06-17 1997-06-21 주식회사 엘지화학 신규 아미노펩티다제

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WO1986001229A1 (fr) * 1984-08-16 1986-02-27 Bio-Technology General Corp. Procede d'elimination des acides amines residuels a terminal n d'analogues polypeptides eucaryotes et polypeptides obtenus par ce procede
WO1986007380A1 (fr) * 1985-06-04 1986-12-18 Takeda Chemical Industries, Ltd. Procede de preparation de polypeptides
US4865974A (en) * 1985-09-20 1989-09-12 Cetus Corporation Bacterial methionine N-terminal peptidase

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