US20150166973A1 - Recombinantly produced neutral protease originating from paenibacillus polymyxa - Google Patents

Recombinantly produced neutral protease originating from paenibacillus polymyxa Download PDF

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US20150166973A1
US20150166973A1 US14/592,969 US201514592969A US2015166973A1 US 20150166973 A1 US20150166973 A1 US 20150166973A1 US 201514592969 A US201514592969 A US 201514592969A US 2015166973 A1 US2015166973 A1 US 2015166973A1
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neutral protease
host organism
protease
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sequence
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Thomas Greiner-Stoeffele
Stefan Schoenert
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Roche Diagnostics Operations Inc
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Publication of US20150166973A1 publication Critical patent/US20150166973A1/en
Priority to US15/334,658 priority Critical patent/US10526594B2/en
Priority to US16/355,259 priority patent/US20190203191A1/en
Priority to US16/700,268 priority patent/US10889809B2/en
Priority to US17/386,106 priority patent/US11542489B2/en
Priority to US18/057,800 priority patent/US20240228999A9/en
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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)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24028—Bacillolysin (3.4.24.28)
    • C—CHEMISTRY; METALLURGY
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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  • the present disclosure provides the sequence of a Paenibacillus polymyxa preproenzyme which is the precursor of a neutral protease, expression thereof in a transformed host organism, and methods for production of the neutral protease, by recombinant means. Further, use of the recombinantly produced neutral protease is disclosed in the field of cell biology, particularly for the purpose of tissue dissociation. The disclosure also includes blends with other proteases. Further disclosed are nucleotide sequences encoding the neutral protease, as well as fragments thereof.
  • the present invention is directed to the means for providing a recombinantly expressed and enzymatically active neutral protease from Paenibacillus polymyxa , also known as Dispase®.
  • Paenibacillus polymyxa also known as Dispase®.
  • an amino acid sequence is provided which is suited for large-scale production by way of recombinant expression thereof, specifically and with particular advantage in transformed Bacillus species serving as a recombinant host strain.
  • recombinantly expressed Paenibacillus polymyxa neutral protease is secreted into liquid culture medium and purified therefrom.
  • dissociation of a tissue into cell layers or even suspensions of single cells is frequently performed with the activity of this enzyme, either with Dispase® alone or with Dispase® as a component of blends, i.e. combined other proteolytic enzymes, specifically Collagenases, e.g. as disclosed in U.S. Pat. No. 5,830,741.
  • U.S. Pat. No. 3,930,954 discloses a neutral protease from B. polymyxa strain having the accession number ATCC 21993 (in the document also referred to as FERM-P No. 412).
  • the document particularly describes culturing of the bacterial strain under aerobic conditions in a complex liquid medium (culture broth) containing a carbon source, a nitrogen source and inorganic salts.
  • the proteolytic activity present in the culture broth was monitored during cultivation, indicating the amount of neutral protease secreted by the cells into the liquid supernatant.
  • the culture was harvested and particulate components including bacterial cells were separated from the supernatant by gel filtration, followed by concentration of the filtrate under reduced pressure.
  • protease enrichment taught in U.S. Pat. No. 3,930,954 include salting out with ammonium sulfate and precipitation with methanol, ethanol and acetone, each resulting in a crude preparation. Subsequently, further purification steps were applied, ultimately leading to a purified preparation. By way of ultracentrifugation analysis a molecular weight of 35,900 Daltons (Da) was determined, and a number of other biochemical and biophysical parameters were examined. However, no unequivocal data were supplied clarifying whether the disclosed preparation contained a homogeneously purified single protease or a mixture of different proteins.
  • the Coomassie Blue-stained gel presents a thin major band migrating at 41 kDa, but also at least two faint bands migrating between 30 and 20 kDa, and a further faint band migrating between 20 and 14.4 kDa.
  • the molecular weight of the conceptual mature (processed) secreted protein comprising 304 amino acids was calculated to be 32,477 Da.
  • Neutral protease expressed in E. coli from a genomic B. polymyxa fragment and analyzed from the supernatant of disrupted transformed E. coli cells was found to migrate at about 35 kDa in SDS PAGE gels.
  • B. polymyxa extracellular neutral protease from culture fluid.
  • the N-terminal amino acid sequence of the purified neutral protease was determined.
  • the first three amino acid residues in the B. polymyxa N-terminal sequence of Ala Thr Gly Thr Gly Lys Gly Val Leu Gly Asp Xaa Lys Ser Phe (SEQ ID NO:4) differ from the predicted amino acid sequence comprised in SEQ ID NO:2 at the positions 287-301 which were found to be Asn Glu Ala Thr Gly Lys Gly Val Leu Gly Asp Ser Lys Ser Phe. The reason for this discrepancy remained unclear and was not elucidated further.
  • a first aspect of all embodiments as disclosed herein is a method for recombinantly producing a neutral protease, the method comprising the steps of (a) providing in an expression vector a DNA with a sequence encoding a preproenzyme according to SEQ ID NO:5, and transforming a host organism with the expression vector, thereby obtaining a transformed host organism, wherein the host organism is a gram-positive prokaryotic species; followed by (b) expressing the DNA in the transformed host organism, wherein the transformed host organism secretes the neutral protease; followed by (c) isolating the secreted neutral protease; thereby recombinantly producing the neutral protease.
  • a second aspect of all embodiments as disclosed herein is a neutral protease obtained by performing a method for recombinantly producing a neutral protease as disclosed herein.
  • a third aspect of all embodiments as disclosed herein is a method of isolating living cells from animal tissue in vitro, comprising the steps of (a) providing a recombinantly produced neutral protease obtained by performing a method according to any of the claims 1 to 8 , and (b) incubating the tissue in vitro with the neutral protease of step (a), wherein protein components of the extracellular matrix of the tissue are proteolytically degraded, and wherein a layer of cells or a suspension of individual living cells is obtained, thereby isolating living cells from animal tissue in vitro.
  • a fourth aspect of all embodiments as disclosed herein is the use of a neutral protease obtained by performing a method for recombinantly producing a neutral protease as disclosed herein, the use of the neutral protease being the isolation of living cells from animal tissue in vitro.
  • a fifth aspect of all embodiments as disclosed herein is a kit of parts comprising in a sealed compartment a lyophilizate of a neutral protease obtained by performing a method for recombinantly producing a neutral protease as disclosed herein.
  • a sixth aspect of all embodiments as disclosed herein is a method for making a blend of a plurality of proteases, comprising the steps of (a) providing a recombinantly produced neutral protease obtained by performing a method obtained performing a method for recombinantly producing a neutral protease as disclosed herein, and (b) mixing the neutral protease of step (a) with a further protease.
  • a seventh aspect of all embodiments as disclosed herein is a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of position 289 to position 592 of SEQ ID NO:5, the nucleotide sequence being selected from the group consisting of (a) a nucleotide sequence having the sequence of position 898 to position 1811 in SEQ ID NO:6; (b) nucleotide sequences derived from the nucleotide sequence of position 898 to position 1811 of SEQ ID NO:6 as a result of the degenerated code.
  • An eighth aspect of all embodiments as disclosed herein is a vector containing a nucleotide sequence as disclosed herein.
  • a ninth aspect of all embodiments as disclosed herein is a transformed prokaryotic Gram-positive host organism containing at least one vector as disclosed herein.
  • FIG. 1 Alignment of the published amino acid sequence of Takekawa, S., et al., J. Bacteriology 173 (1991) 6820-6825 (SEQ ID NO:3; “Seq-1” in the Figure) with the amino acid sequence originating from P. polymyxa ATCC 21993 (SEQ ID NO:5; “Seq-2” in the Figure), disclosed herein.
  • Dispase® neutral protease originating from Paenibacillus polymyxa, P. polymyxa
  • P. polymyxa is a metalloenzyme which is classified as an amino-endo peptidase capable of cleaving fibronectin, collagen IV, and collagen I, but the latter apparently to a lesser extent.
  • P. polymyxa neutral protease prepared recombinantly according to the present disclosure is useful to prepare many types of cells for culture.
  • P. polymyxa neutral protease as provided herewith is a rapid, effective, but gentle agent for separating even cell layers, that is to say intact epidermis from the dermis and intact epithelial sheets in culture from the substratum. In both cases, it affects separation by cleaving extracellular matrix proteins in the basement membrane zone region while preserving the viability of the epithelial cells.
  • P. polymyxa neutral protease according to the present disclosure and used as sole protease is useful for detaching epidermal cells as confluent, intact sheets from the surface of culture dishes without dissociating the cells.
  • P. polymyxa neutral protease Such a procedure paves the way for the use for culture and even transplantation of skin epithelial cell sheets detached from the culture substrate by P. polymyxa neutral protease. Also, P. polymyxa neutral protease is useful for the harvest and transfer of normal diploid cells and cell lines. Further applications for tissue dissociation make use of blends of P. polymyxa neutral protease and a further protease such as a collagenase.
  • a method for recombinantly producing a neutral protease comprising the steps of (a) providing in an expression vector a DNA with a sequence encoding a preproenzyme according to SEQ ID NO:5, and transforming a host organism with the expression vector, thereby obtaining a transformed host organism, wherein the host organism is a gram-positive prokaryotic species; followed by (b) expressing the DNA in the transformed host organism, wherein the transformed host organism secretes the neutral protease; followed by (c) isolating the secreted neutral protease; thereby recombinantly producing the neutral protease.
  • the DNA sequence originates from Paenibacillus polymyxa ATCC 21993.
  • the sequence encoding the preproenzyme according to SEQ ID NO:5 can be expressed in any suitable host organism known to the skilled person.
  • a particular host organism is a gram-positive bacterium, specifically a species selected from the group consisting of Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus .
  • a very suitable way of recombinantly producing the neutral protease encoded by SEQ ID NO:5 makes use of the species Bacillus amyloliquefaciens as transformed host organism.
  • the step of expressing the DNA in the transformed host organism is performed by culturing the transformed host organism in a liquid medium, wherein the transformed host organism secretes the neutral protease into the liquid medium. Subsequently, the secreted neutral protease can be isolated from the liquid medium.
  • P. polymyxa neutral protease recombinantly produced according to the present disclosure is provided as a lyophilizate.
  • the lyophilizate is dissolved in a physiologically suited buffer, e.g. in PBS (phosphate buffered saline) which is free of Mg 2+ and Ca 2+ ions.
  • PBS phosphate buffered saline
  • the P. polymyxa neutral protease solution is then sterilized, e.g. by way of filtration through a filter membrane (e.g. 0.22 ⁇ m pore size).
  • a sample of living tissue is obtained, i.e. removed from the animal.
  • tissue a culture vessel with adherent cells or a culture vessel with cell aggegates
  • the cells are also referred to as “tissue” herein.
  • the tissue is fragmented by mechanical means (e.g. using scissors or a scalpel), and the fragments are washed in sterile PBS. Subsequently, the fragments are incubated in pre-warmed P. polymyxa neutral protease solution, whereby the fragments are covered by the solution. Incubation with P. polymyxa neutral protease is typically performed at physiological temperature, particularly at 37° C.
  • the time needed for the desired (i.e. the degree or extent of) tissue dissociation is usually determined empirically, wherein typically P. polymyxa neutral protease concentration in the solution and/or incubation time are varied. Incubation time in P. polymyxa neutral protease solution can be several hours without adverse effects on the cells.
  • the incubated tissue can optionally be agitated gently. If necessary, dispersed cells can be separated from still existing aggergates by way of passing the obtained cell suspension through a sterile mesh or grid. Decanting is also a method to obtain dissociated cells. Further techniques are known to the skilled person, particularly to remove cell layers which are detached from tissue underneath by incubation with P. polymyxa neutral protease. Fresh Dispase solution may be added if further disaggregation is desired.
  • Dissociated cells or cell layers can be pelleted, enzyme solution can be removed by decanting, or the P. polymyxa neutral protease solution is diluted with cell culture medium, in order to inhibit further proteolytic activity. Other methods to do so are possible. Cells obtained by the above workflow can be plated and cultured using standard procedures.
  • the present disclosure further provides a method to isolate living cells from animal tissue in vitro, comprising the steps of (a) providing a recombinantly produced neutral protease obtained by performing a method according to any of the claims 1 to 8 , and (b) incubating the tissue in vitro with the neutral protease of step (a), wherein protein components of the extracellular matrix of the tissue are proteolytically degraded, and wherein a cell layer or a suspension of individual cells is obtained.
  • the animal tissue origins from a vertebrate animal, more specifically from an animal species selected from mouse, guinea pig, hamster, rat, dog, sheep, goat, pig, bovine, horse, a primate species, and human.
  • a method to isolate living cells from animal tissue in vitro comprises the use of a protease blend which includes a P. polymyxa neutral protease recombinantly produced as disclosed herein.
  • the blend may, by way of example comprise a further neutral protease such as thermolysin.
  • blends of P. polymyxa neutral protease with a collagenase provide great advantage for tissue dissociation.
  • P. polymyxa neutral protease recombinantly produced as disclosed herein or a blend of proteases including P. polymyxa neutral protease recombinantly produced as disclosed herein is provided as a lyophilizate, i.e. as a freeze-dried preparation. Such a preparation can be stored for an extended amount of time.
  • kits of parts comprising in a sealed compartment, such as a bottle, a lyophilizate of a neutral protease obtained by performing a method for recombinantly producing a neutral protease, as disclosed herein.
  • the kit may contain in a separate sealed compartment a lyophilized preparation of a collagenase.
  • the kit may also contain in a separate sealed compartment a lyophilized preparation of a thermolysin.
  • kits comprising in a sealed compartment, such as a bottle, a lyophilizate of a neutral protease obtained by performing a method for recombinantly producing a neutral protease, as disclosed herein, wherein the neutral protease is blended with a further protease such as (but not limited to) a collagenase and/or thermolysin.
  • a further protease such as (but not limited to) a collagenase and/or thermolysin.
  • PCR polymerase chain reaction
  • oligonucleotides representing subsequences of the desired coding and non-coding genomic DNA strands
  • artificial gene sequences were generated.
  • partially overlapping pairs of oligonucleotides representing fragments of opposite strands were hybridized with template DNA, and double-stranded DNA molecules were generated by polymerase-mediated strand-extension, and subsequent PCR amplification. Further DNA molecules were created synthetically. All sequences of artificially generated DNAs were verified by sequencing.
  • a first attempt to express P. poymyxa neutral protease was based on the disclosure of Takekawa, S., et al., J. Bacteriology 173 (1991) 6820-6825.
  • the nucleotide sequence of SEQ ID NO:1, specifically the subsequence of CDS (343) . . . (2115) corresponding to the open reading frame encoding SEQ ID NO:2 was adapted by changing the codon usage. While the encoded amino acid sequence remained unchanged, neutral mutations optimizing the open reading frame for expression in Bacillus subtilis were introduced. An artificial DNA with the reading frame encoding SEQ ID NO:2 was created and synthesized. It encoded the P.
  • polymyxa amino acid sequence of the preproenzyme with 590 amino acids i.e. including the signal sequence and the propeptide.
  • a B. subtilis -specific ribosome binding site was introduced upstream of the open reading frame.
  • the DNA construct was cloned in an expression vector which provides a growth phase-specific promoter driving transcription in B. subtilis in the stationary phase of growth in liquid culture.
  • the resulting selectable and replication-competent expression plasmid was pLE2D01nprPp.
  • a derivative was constructed by fusing three Glycines and six Histidines to the C-terminus of the amino acid sequence of the preproenzyme resulting in an encoded polypeptide with 599 amino acids with six terminal Histidines.
  • the resulting selectable and replication-competent expression plasmid was pLE2D01nprHisPp.
  • Transformed B. subtilis strains were generated and expression experiments under standard conditions were made; i.e. conditions were applied in case of other expression targets have shown to be permissive with expression and secretion of detectable quantities of target protein.
  • the promoter in each of the two above plasmids was exchanged by another promoter driving expression dependent on the addition of a specific inductor compound to the culture.
  • the resulting expression plasmids were designated pLE2E01nprPp and pLE2E01nprHisPp. Expression experiments were made including the step of addition of the inductor. As a result, these modifications did not lead to a change. Both further attempts to express and secrete P. polymyxa neutral protease were unsuccessful.
  • FIG. 1 On the amino acid sequence level an alignment with the published sequence of Takekawa, S., et al. (supra) is presented in FIG. 1 .
  • the alignment shows a number of amino acid exchanges and even a deletion and an insertion. 17 of the amino acid exchanges could be of higher-order structural relevance since in these cases the amino acids are not similar (size, charge) but differ significantly.
  • the amino acid sequence determined in the present study contained the N-terminus determined earlier by Takegawa S.et al. (supra).
  • positions 289 to 303 of SEQ ID NO:5 correspond to the previously determined N-terminal sequence of SEQ ID NO:4.
  • the extracellular neutral protease derived from the ATCC 21993 strain is the polypeptide given by the amino acid sequence of SEQ ID NO:5 from position 289 to position 592.
  • the DNA encoding the neutral protease was isolated from Paenibacillus polymyxa strain ATCC 21993 as described in Example 3. Based on the amino acid sequence of SEQ ID NO:5 a DNA sequence for expression in B. subtilis encoding the neutral protease was devised and cloned in different expression vectors, in analogy to Example 2.
  • the DNA sequence of a cloned fragment including the coding sequence of the of the neutral protease (preproenzyme) of said Paenibacillus polymyxa strain ATCC 21993 is presented as SEQ ID NO:6.
  • the DNA construct was cloned in an expression vector which provides a growth phase-specific promoter driving transcription in B. subtilis in the stationary phase of growth in liquid culture.
  • the resulting selectable and replication-competent expression plasmid was pLE2D01DisnatPp.
  • the expression plasmid pLE2D01DisnatPp was transformed into several Bacillus species, including not only Bacillus subtilis but also Bacillus amyloliquefaciens . Control transformations were made with “empty” expression vectors, as described before.
  • Particular transformed Bacillus subtilis host strains used for transformation contained loss-of-function mutations in one or more endogenous genes encoding an extracellular (secreted) protease. Such strains are considered to be advantageous, particularly in the present case when the desired target protein to be recombinantly expressed and secreted is a protease itself. Particularly in the transformed B. subtilis host protease genes selected from AprE, NprE, Epr, and a combination thereof were mutated. In addition, strains were obtained in which all three of these genes were mutated.
  • advantageous mutations in the host strain included the endogenous extracellular protease genes Npr and Apr. Respective transformants out generated including one or both of the two aforementioned protease loss-of-function mutations.
  • the EnzChek® Protease Assay Kits were used (Invitrogen, E6638).
  • the direct fluorescence-based assay detects metallo-, serine, acid and sulfhydryl proteases.
  • the assay kit contains casein derivatives that are labeled with the pH-insensitive greenfluorescent BODIPY® FL (E6638) dye, resulting in almost total quenching of the conjugate's fluorescence.
  • Protease-catalyzed hydrolysis releases fluorescent BODIPY FL dye-labeled peptides.
  • the accompanying increase in fluorescence which can be measured with a spectrofluorometer, minifluorometer or microplate reader, is proportional to protease activity.

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US15/334,658 US10526594B2 (en) 2012-07-09 2016-10-26 Recombinantly produced neutral protease originating from Paenibacillus polymyxa
US16/355,259 US20190203191A1 (en) 2012-07-09 2019-03-15 Methods of recombinantly producing neutral protease originating from paenibacillus polymyxa
US16/700,268 US10889809B2 (en) 2012-07-09 2019-12-02 Methods of recombinantly producing neutral protease originating from Paenibacillus polymyxa
US17/386,106 US11542489B2 (en) 2012-07-09 2021-07-27 Expression vector encoding a Paenibacillus polymxa neutral protease
US18/057,800 US20240228999A9 (en) 2012-07-09 2022-11-22 Methods of recombinantly producing neutral protease originating from paenibacillus polymxa

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