WO2004011482A2 - Procede d'activation de la clostripaine - Google Patents

Procede d'activation de la clostripaine Download PDF

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WO2004011482A2
WO2004011482A2 PCT/US2003/016646 US0316646W WO2004011482A2 WO 2004011482 A2 WO2004011482 A2 WO 2004011482A2 US 0316646 W US0316646 W US 0316646W WO 2004011482 A2 WO2004011482 A2 WO 2004011482A2
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clostripain
urea
solution
inclusion bodies
concentration
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WO2004011482A3 (fr
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Mary J. Bossard
Edwin H. Merrifield
Daniel Strydom
Yuannan Xia
Fred W. Wagner
Barton Holmquist
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Restoragen Inc
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Restoragen Inc
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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

Definitions

  • the invention relates to an improved method for activating recombinantly produced clostripain.
  • the invention provides clostripain of a high purity, high specific activity, and high concentration.
  • Clostripain is a two-chain proteinase that can be isolated from Clostridium histolyticum. It is highly specific for the carboxyl peptide bond of arginine. To maintain the catalytic activity of clostripain, its sulfhydryl groups must be reduced, which can be achieved by reducing reagents such as dithiothreitol and cysteine. The presence of calcium ions is essential for such activity. The enzyme is inhibited by oxidizing agents, sulfhydryl-modifying agents, and by Co 2+ , Cu 2+ , Cd 2+ , and other metal ions. Citrate, borate, and Tris anions are weakly inhibitory. Clostripain is assayed using a method that measures reaction velocity as an increase in absorbance at 253 nm resulting from the hydrolysis of N-benzoyl-L-arginine ethyl ester.
  • Clostripain may be expressed recombinantly in E. coli as a chimeric protein in the form of an inclusion body. Proteins isolated from E. coli expressed inclusion bodies may be contaminated with varying amounts of bacterial cells, bacterial cell products including pigments, nucleic acids, E. coli derived proteins and lipids.
  • the signal sequence associated with the expressed clostripain caused secretion of soluble prepro enzyme from B. subtilis.
  • Inclusion bodies form convenient tools for peptide and protein purification. They are believed to be aggregates of partially or incorrectly folded proteins, and such states of proteins are generally accepted to be influenced by solvent structure, and thereby by the nature and concentrations of denaturants, chaotropes, detergents, and the like. When solubilized in a strong denaturant (chaotrope), the aggregates are dissociated and dissolved. The denaturing agent is typically removed, or its concentration lowered, to allow for the correct folding of the protein.
  • Witte et al. treated their pelletized fraction from E. coli expression with 4M urea.
  • Witte et al. found that only a minor portion of the core protein was converted into the correctly processed form. They explained that a major portion of the core protein was degraded due to the proteolytic action of the processed clostripain. Consequently, Witte et al. indicate that B. subtilis is the organism of choice for clostripain expression. That expression involves soluble clostripain.
  • the instant invention provides a process for producing gram scale amounts of clostripain having high specific activity, high purity and high concentration.
  • the process is highly cost- effective, robust, environmentally safe, and provides product for immediate or long-term consumption.
  • highly active clostripain is produced by (a) solubilizing in a urea solution clostripain inclusion bodies which have been harvested from a fermentation mixture comprising recombinant host cells which have expressed clostripain; and (b) activating the clostripain by diluting the urea solution of step (a) into a final urea concentration of less than 3 M. Calcium and a reducing agent are preferred as the activation buffer in the urea solution to develop highly preferred activity.
  • the specific activity, purity and concentration of the activated clostripain can be increased in accordance with the instant invention by (a) fractionating the clostripain in the diluted urea solution over an equilibrated anion exchange resin to form a purified clostripain solution; and (b) further purifying and concentrating the clostripain by diafiltration.
  • Solubilization of clostripain in dilute urea concentrations of less than 3 M in accordance with the instant invention produces clostripain solutions that have a higher activity and higher yields than those otherwise obtained by activating clostripain through solubilization in more concentrated urea solutions. Without intending any limitation to the scope of the invention disclosed and claimed herein, it is theorized that this substantial improvement in clostripain activation and yield results in part from stabilized character of the clostripain at the lower urea concentration.
  • FIGURE 1 illustrates optimum pH values for the urea dilution reaction mixture used in the instant invention.
  • FIGURE 2A-2C shows the open reading frame of the cloned prepro- clostripain (1-526; SEQ D NO:12 and SEQ LD NO:13).
  • the pre-sequence, pro- sequence, light-chain, linker, and heavy-chain are indicated by bracketed lines. Restriction enzyme recognition sites are indicated by enzyme name.
  • the stop codon is indicated by a star.
  • FIGURE 3 shows the N-terminal sequence of the cloned T7tag- clostripain (51-526; SEQ LD NO:14 and SEQ LD NO: 15).
  • the T7tag, light- chain, linker, and portion of a heavy-chain are indicated by a bracket line. Restriction enzyme recognition sites are indicated by enzyme name.
  • FIGURE 4 illustrates an SDS-PAGE (4-20 % Tris-Glycine gel) analysis of recombinant clostripain core proteins.
  • the clostripain position is indicated by an arrow.
  • FIGURE 5 shows a plasmid map of a representative expression construct encoding the mature clostripain.
  • FIGURE 6 illustrates an SDS-PAGE (4-20 % Tris-Glycine gel) analysis of in vitro processing of clostripain core protein T7tag-clost(51 -526). The position of clostripain, and clostripain subunits is indicated by arrows.
  • FIGURE 7 shows a comparison of in vitro processing of clostripain proenzyme, core protein, and core protein mutant containing linker mutations.
  • the positions of clostripain subunits is indicated by arrows.
  • FIGURE 8 illustrates HPLC analysis of the clostripain-containing protein solution before and after DEAE column filtration.
  • FIGURE 9 illustrates HPLC analysis of the clostripain-containing protein solution after DEAE column filtration.
  • FIGURE 10 illustrates HPLC analysis of the retentate from diafiltration of the clostripain-containing protein solution.
  • FIGURE 11 illustrates HPLC analysis of the permeate from diafiltration of the clostripain-containing protein solution.
  • FIGURE 12 illustrates SDS PAGE analysis of the activation of T7tag- Clost(51-526) and MRI inclusion bodies in various urea concentrations.
  • the positions of the pre-pro-clostripain, the clostripain heavy chain, and the clostripain light chain are indicated by arrows.
  • FIGURE 13 illustrates SDS PAGE analysis of the activation of MRI inclusion bodies in various urea concentrations.
  • the positions of the pre-pro- clostripain, the clostripain heavy chain, and the clostripain light chain are indicated by arrows.
  • the present invention achieves production of shelf stable, active clostripain through high yield expression from a microbial host such as E. coli.
  • the yield and activity are surprising in light of earlier teaching that microbial expression in a host such as E coli is disfavored because yields are low and activity is poor or practically non-existent.
  • the present invention achieves these results through astute use of processing conditions and attention to the self digestion aspects of clostripain.
  • Clostripain chimeric proteins employed in the instant invention may be expressed in a microbial host cell using known techniques of recombinant DNA production.
  • E. coli is a preferred host cell.
  • the host cell contains an expression vector, which encodes the chimeric protein under the control of a regulatory sequence that is capable of directing its expression in the host, as well as an origin of replication that is functional in the host cell.
  • the vector may contain other DNA sequences conventionally employed in recombinant DNA technology such as sequences encoding selectable markers. Methods for expressing a foreign gene in a host organism also are well known in the art (see, e.g., Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd ed., 1989).
  • the gene encoding a particular polypeptide can be constructed by chemically synthesizing the entire nucleotide sequence, by amplification, such as by the polymerase chain reaction (PCR), or by cloning the gene of interest. The gene is then subcloned into an appropriate expression vector.
  • Cloning vectors, expression vectors, plasmids, and viral vectors are well known in the art Maniatis et al., supra, and (see, e.g., Goedell, Methods in Enzymology, Vol. 185 (Academic Press 1990)). Examples 1-7 provide a detailed description of the preparation of a T7 -based expression system useful for high-level expression of mammalian proteins in E. coli.
  • the host cell containing the expression vector is grown and the chimeric protein expressed under appropriate conditions.
  • the conditions for growth of the host cell and expression of the chimeric protein will vary depending on various factors such as the host cell employed, the promoter, and the particular chimeric protein being expressed. Those skilled in the art are capable of determining the appropriate conditions for the particular host/vector system employed. Methods for expressing a foreign gene in a host organism also are well known in the art (see, e.g., Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd ed., 1989).
  • the gene encoding a particular polypeptide can be constructed by chemically synthesizing the entire nucleotide sequence, by amplification, such as by the polymerase chain reaction (PCR), or by cloning the gene of interest. The gene is then subcloned into an appropriate expression vector.
  • Cloning vectors, expression vectors, plasmids, and viral vectors are well known in the art (see, e.g., Maniatis et al., supra, and Goedell, supra). Examples 1-7 provide a detailed description of the preparation of a T7-based expression system useful for high-level expression of clostripain in E. coli. According to the invention, the clostripain may be conveniently packaged and isolated by inclusion bodies from E.
  • inclusion bodies may be treated with a high concentration of urea solution and then activated by dilution to at least slightly less than 3M urea. This dilution factor provides the appropriate balance between denaturization, proper refolding and autocatalytic cleavage to produce stable active clostripain in solution.
  • clostripain may be recombinantly expressed in E. coli host cells and recovered from such cells in the form of inclusion bodies ("IB's").
  • the host cells may be harvested and opened to separate the IP's, such as by centrifugation, resuspension in a lysis medium, and treated to lyse, such as by mechanical lysing at 12,000 psi.
  • the LB's can be collected to obtain isolated LB's sucha s by washing with water and collecting by centrifugation to form an IB paste.
  • the collected LB's may be solubilized in a concentrated urea solution to obtain solubilized inactive clostripain.
  • an LB paste may be solubilized by adding 8 M urea solution at a ratio of about 50 mL per g of LB, followed by centrifugation (for example at 6-8 °C at 15,000-17,000 x g) to pelletize cell debris.
  • the centrifugation supernatant will contain solubilized inactive clostripain.
  • the inactive clostripain may be activated by dilution treatment according to the invention. Accordingly, the inactive solubilized clostripain, concentrated urea solution may be diluted to at least slightly less than 3M urea to activate the clostripain.
  • the medium with the inactive solubilized clostripain may also contain a concentration of metal ion and oxidation inhibitor, preferably a combination thereof.
  • 100-325 mg of urea-solubilized inactive clostripain solution may be clarified by centrifugation or by filtration through 0.45 micron filter membranes.
  • the clarified, solubilized clostripain can be activated by dilution into reaction mixtures containing a calcium buffer at a concentration of 0.1 mM -5 mM to a final urea concentration of 1 to less than 3 M urea.
  • a calcium buffer at a concentration of 0.1 mM -5 mM to a final urea concentration of 1 to less than 3 M urea.
  • about 850 000 to 880 000 units of the solubilized clostripain can be activated in a bench-top activation process with little or no subsequent need to stabilize or repurify.
  • the diluted reaction mixture is composed of from about 1 to about less than 3 M urea and 0.1 to 5 mM of an alkali or alkaline earth metal or transition metal ion as the activation buffer.
  • a calcium ion activation buffer is preferred. While activation can be performed over a wide pH range, a pH range of 7 to 8 is preferred, as described in detail hereinafter in Example 8, and as illustrated in Figure 1.
  • a variety of buffers such as Tris, HEPES, MES, or phosphate can also be employed to control the pH during activation.
  • An activation buffer comprised of 25 mM HEPES and 5 mM CaCl 2 at pH 7.6 is a particularly preferred buffer.
  • This buffer does not inhibit clostripain or form insoluble complexes with calcium.
  • NaCl was added to the activation reaction subsequent to activation to facilitate fractionation, as explained hereinafter.
  • an oxidation inhibitor can optionally be added to the activated clostripain medium. Any oxidation inhibitor that is compatible with proteases and proteins may be used in this capacity.
  • mercaptans containing SH groups such as mercaptoethanol (0.001-5 M), dithiothreitol (DTT) (0.0001-0.25 M) or cysteine (0.001-0.25 M) can be added to ensure full reduction of the clostripain.
  • 0.001-0.025 M DTT is added to the dilution mixture.
  • a comparison of the activity values listed in Tables 1 and 2 (Example 8) shows the increase in specific activity achieved by use of DTT. Activation can occur over a wide temperature range; at room temperature, maximum activity is usually reached within one to two hours, hi terms of enzyme units ('BAEE'-units, as described below in Example 11, method 2) the yield of active clostripain was in the range of 50 to 120 Units per mL of solution (i.e. 100 to 240 Units per mg of protein).
  • the solubilized protein is diluted into an activation buffer at a final concentration of 0.05-5 mg/mL in the diluted solution, hi a preferred embodiment the final concentration of protein will be 0.4-0.6 mg/mL.
  • the final concentration of urea is less than 3 M.
  • the final concentration of urea is between about 1-3 M.
  • the increase in enzyme activity is monitored by dilution into the standard BAEE kinetic assay as described in Example 11. The activity reaches a maximum level after about 1 to 2 hours at room temperature.
  • the activated clostripain solution can be adjusted to a concentration of 0.05-0.15 M NaCl and filtered through an anion exchange resin to separate pigment, DNA, and other compounds from the clostripain.
  • the activated clostripain solution can be adjusted to a concentration of 0.1 M NaCl and filtered through DEAE-Sepharose FF.
  • Reducing agent such as DTT (5 - 25 mM)
  • Other stabilizing agents such as glycerol can be employed.
  • clostripain may be precipitated by agents such as, but not limited to, ammonium sulfate, ethanol, methanol, or sodium sulfate, with or without subsequent lyophilization.
  • agents such as, but not limited to, ammonium sulfate, ethanol, methanol, or sodium sulfate, with or without subsequent lyophilization.
  • precipitation was achieved with about 60 % ethanol.
  • An ethanol slurry prepared in this way was stable for greater than three months at
  • Diafiltration ca be employed to concentrate the enzyme solution and remove small molecular weight impurities.
  • diafiltration can be employed in order to reduce the volume, and purify the DEAE-breakthrough fraction while mamtaining a sufficient quantity of clostripain to prepare a commercial-size batch of recombinant glucagon-like polypeptide-1 (e.g., 100-130 g).
  • the resulting activated, purified clostripain solutions have been maintained under refrigeration at 5°C for 50 days without any measurable loss in activity (90% -110%).
  • Diafiltration is a mode of operating an ultrafiltration system in which the retentate (that portion of a sample that does not pass through the ultrafiltration membrane) is continuously recycled and diluted with fresh wash solution to replace that removed as permeate.
  • Ultrafiltration maybe defined as separation of particles (e.g., macromolecules) by filtration through membranes based on molecular weight.
  • Ultrafiltration membranes typically have a molecular weight cut-off (MWCO) in the range of 1,000 to 1,000,000 daltons.
  • the MWCO typically is defined as the molecular weight of the globular solute which is 90 % retained by that membrane. See Filtron Catalog, 1995/96, p.5.
  • the actual molecular weight of particles that pass through or are retained by a membrane will depend on the size as well as the conformation and charge of a given molecule.
  • the native clostripain molecule is a heterodimer consisting of a lighter chain of 12 kDa and a heavier chain of 43 kDa (Gilles, Imhoff, and Keil, Journal of Biological Chemistrv, 254: 1462-1468 (1979)); clostripain was found to be retained by a Filtron 10K membrane. While regenerated cellulose membranes are a preferred diafiltration membrane, membranes made of other materials that otherwise meet the retention criteria specified above may be used.
  • the vectors pBNl 15 and pBN121 are E. coli high yield nucleic acid constructs constructed through use of a high copy number vector that is stably maintained within a host cell.
  • the vectors contain an expression cassette having a strong promoter that is operably linked to an open reading frame that encodes clostripain.
  • These vectors were constructed through use of the larger DNA fragment produced from restriction enzyme digestion of pGEX2T (Amersham Pharmacia Biotech, Piscataway, NJ) with Fspl-Sma ⁇ . This fragment contains the replication origin of pMBl for high copy number maintenance, the Laclq gene for promoter suppression, the GST terminator for transcription termination, and the bla gene for ampicillin resistance.
  • Primer 2 5' TCA AAG ATC TTA TCG ACT GCA CGG 3' (SEQ ID NO: 2).
  • the upstream BgHl restriction endonuclease binding site sequence (A GATCT) and the downstream Xbal (T/CTAGA) binding site sequence are underlined with a single line, the -35 and -10 promoter consensus sequences are bolded and underlined with dots, and the downstream transcriptional start A residue (within the lac operator gene sequence) is bolded and underlined with a solid line.
  • the lac operator sequence is enclosed within brackets.
  • the PCR product of the Tac promoter fragment was ligated into the larger Fspl-Smal fragment from pGEX2T. The ligation mixture was transformed into high efficiency E.
  • coli competent cells by heat shock at 42°C for 45 seconds and streaked on LB + 50 ⁇ g/mL AmpiciUin Agar plates. Vectors from cultures of single colonies were prepared. A correct vector was identified by restriction enzyme digestion. The Xbal-Xhol fragment from p ⁇ T23a plasmid (Novagen, Madison, WI), which contained the T7 gene 10 ribosome binding site and the T7tag initiation sequence, was inserted into the correct vector identified above at the Xbal-Smal site of the nucleic acid construct. The resulting vector was named pBNl 15 (Tac).
  • the plasmid pBNl 15 (Tac) was digested with modified Aatll-Fspl to remove the 0.7 kb ampiciUin resistance gene.
  • a 1.1 kb PCR product containing the aminophosphotransferase gene that encodes kanamycin resistance, was then cloned into the pBNl 15(Tac) vector at the Aatll-Fspl sites.
  • the kanamycin resistance gene was produced through selective PCR amplification of the pCT-Blunt plasmid (Invitrogen, Carlsbad, C A) using the following primers:
  • KANXYl (5 '-CCT GAC GTC CCG GAT GAA TGT CAG CTA CTG GGC-3') (Aatll site underlined) (SEQ ID NO:4),
  • KANXY2 (5'-GGC TGC GCA AAG GAG AAA ATA CCG CAT CAG GAA-3') (Fspl site underlined) (SEQ ID NO:5).
  • the resultant plasmid was designated as pBN121(Tac), and E. coli that were transformed with this plasmid could be selected in LB + 25 ⁇ g/mL kanamycin media.
  • pBN121 contains unique Nhel and Xhol restriction sites for inserting the foreign gene sequence to be expressed, such as the gene sequence that encodes clostripain.
  • the prepro-Clost (1-526) gene was PCR amplified from the C. histolyticum genome by using Pfu DNA polymerase (Stratagene, La Jolla, CA) and the following primers :
  • 0925CLA 5'-AGA GCT CAT ATG TTA AGA AGA AAA GTA TCA ACA CTA TTA ATG-3' (Ndel site underlined) (SEQ LD NO:6) and
  • 0925CLB 5'-TTG CTC GAG TTA CCA TTG GTA ATG ATT AAC TCC TCC AGT-3' (Xhol site underlined) (SEQ LD NO:7).
  • the genomic DNA template was prepared by repeated phenol- chloroform extraction and ethanol precipitation of C. histolyticum collagenase (Worthington, Lakewood, NJ).
  • the PCR product was blunt-end inserted into linearized pCR-Blunt vector using the Zero Blunt cloning kit (Invitrogen), and produced the pCR-Blunt-preproClost(l-526) plasmid.
  • the cloned clostripain gene was confirmed by DNA sequencing.
  • Figure 2 provides the open reading frame of the cloned prepro-Clost(l-526) gene with some restriction enzyme sites indicated.
  • the Nde -Xhol fragment of the prepro-Clost(l-526) gene from the above ⁇ CR-Blunt-preproClost( 1 -526) plasmid was cloned into the pET23 a expresson vector (Novagen) at Ndel-Xliol sites to produce a nucleic acid construct.
  • the resulting nucleic acid construct pET23a-preproClost(l-526) was transformed into E. coli BL21(DE3) cells, and the colonies were grown in LB + 50 ⁇ g/mL ampiciUin media.
  • the correct nucleic acid construct was identified by restriction enzyme digestion and DNA sequencing. Glycerol stocks of the E. coli host cells harboring the construct were stored at -80 °C or below with 15 % glycerol.
  • the amplified fragment had the ATG start codon at the N-terminus of the pro-clostripain (28-526).
  • the PCR product was cleaved with the restriction enzymes Ndel-BamHl and inserted using the same enzymes into the pET23a- preproClost(l-526) nucleic acid construct (Example 2) to produce the ⁇ ET23a- proClost(28-526) nucleic acid construct.
  • the nucleic acid construct was transformed into E. coli HM174(DE3) and BL21(DE3).
  • the correct nucleic acid construct was selected in LB + 50 ⁇ g/mL ampiciUin media. Glycerol stocks of the construct were stored as in Example 2.
  • the amplified fragment contained the ATG start codon at the N-terminus of clostripain (51-526).
  • the PCR product was cleaved with the restriction enzymes Ndel-BamH and inserted using the same enzymes into the pET23a- preproClost(l-526) nucleic acid construct (Example 2) to produce the pET23a-
  • Clost(51-526) nucleic acid construct The pET23a-Clost(51-526) nucleic acid construct was transformed into E. coli BL21(DE3) or BL21(DE3)pLysS cells.
  • the correct construct was selected in LB + 50 ⁇ g/mL ampiciUin media.
  • Glycerol stocks of the construct were stored as in Example 2.
  • the pET24a-Clost(51-526) nucleic acid construct was transformed into E. coli
  • BL21(DE3) or BL21(DE3)pLysS cells The correct construct was selected in LB + 25 ⁇ g/mL kanamycin media and glycerol stocks of the construct were stored, as in Example 2.
  • the Ndel-Xhol fragment from pET23a-Clost(51-526) was also inserted into the pBN121(Tac) plasmid (Example 1) at the Ndel-Xhol site to produce the pBN121(Tac)-Clost(51-526) nucleic acid construct.
  • This construct was transformed into E. coli BL21 cells. The correct construct was selected in LB +
  • T7tag 8 aa peptide carrying a strong translation initiation signal from a very efficiently expressed gene, T7 gene 10
  • T7tag a short (8 aa) peptide carrying a strong translation initiation signal from a very efficiently expressed gene, T7 gene 10
  • the DNA fragment coding the T7tag-clostri ⁇ ain (51-526) was PCR-amplified using the pCR-Blunt- preproClost( 1-526) plasmid as the template and the following primers:
  • CLOSPR 2 (5'-CCT AGG ATC CCC CAT GTT AGC TTC ATA TTT ACT-3 ' ; BamHI site underlined) (SEQ LD NO:9). (same as in Example 3) and CLOSPPJM1 (5'-ATA CAT ATG GCT AGC ATG ACT GGT GGA CAG AAC AAA AAT CAA AAA GTA ACT ATT ATG-3'; (SEQ ED NO: 11); Ntiel and Nhel sites underlined).
  • the amplified fragment contained the T7tag sequence at the ⁇ -terminus of clostripain (51-526) light chain ( Figure 3).
  • the PCR product was cleaved with restriction enzymes Nde -BamHl and inserted using the same enzymes into pET24a-Clost(51-526) nucleic acid construct (Example 4) to produce the pET24a-T7tag-Clost(51-526) nucleic acid construct.
  • This construct was transformed into E. coli BL21(DE3) cells. Transformants were selected in LB + 25 ⁇ g/mL kanamycin media and glycerol stocks of cells containing the correct construct were stored as in Example 2.
  • This construct was transformed into E. coli BL21 cells. Transformants were selected in LB + 50 ⁇ g/mL ampiciUin media and glycerol stocks of cells containing the correct construct were stored as in Example 2.
  • the construct was further modified by the method described in Example 1.
  • the resulting construct was designated pB ⁇ 121(Tac)- T7tag-Clost(51-526) ( Figure 3).
  • This construct was transformed into E. coli BL21 cells. Transformants were selected in LB + 25 ⁇ g/mL kanamycin media and glycerol stocks of cells containing the correct construct were stored as in Example 2.
  • LBA media (LB + ampiciUin) were used when expressing the pET23a or pBN115 derived nucleic acid constructs.
  • LBK media (LB + kanamycin) were used when expressing the ⁇ ET24a or pBN121(Tac) derived constructs.
  • Shaking flask cultures of 5 mL LBA or LBK media were started from single colonies of the transformed cells.
  • Shake flask cultures in 5 mL to 500 L LBA or LBK media (inoculated by 100 ⁇ L to 10 mL overnight culture) were grown at 37 °C and 220 rpm to an A 6 oo of 0.5-1.0. Polypeptide expression was induced by addition of LPTG (1 mM final concentration).
  • Cultures were induced for 2 to 8 hours. Samples were taken from cells having the same A 60 o of pre- and post- induced cells. Cells were pelleted and then lysed in distilled water or 10 mM Tris, pH 8, by sonication. The lysate was then centrifuged to separate insoluble and soluble proteins.
  • the supernatant (soluble protein) from the cell lysate was mixed 1 :1 with 2x SDS-PAGE sample buffer.
  • the pellets were resuspended directly in lx SDS- PAGE sample buffer. These samples were resolved by SDS-PAGE (Invitrogen) according to the manufacturer's instructions and stained with Coomassie Brilliant Blue.
  • E. coli BL21 cells transformed with pBNl 21 (Tac)-T7tag-Clost(51-526) produced an insoluble protein of 60 kDa upon LPTG induction, corresponding to the calculated size of the 484 amino acid encoded by the T7tag-Clost(51 -526) construct.
  • BL21 cells transformed with pBN121(Tac)-Clost(51-526) also produced an insoluble protein that was 59 kDa. This protein corresponded in size to the 476 amino acid clostripain ( 1-526) encoded by the construct.
  • the T7tag sequence markedly increased the expression yield of the recombinant clostripain (51 -526) ( Figure 4).
  • Figure 4 illustrates an SDS-PAGE (4-20 % Tris- Glycine gel) analysis of recombinant clostripain core proteins T7tag-Clost(51- 526) and Clost(51-526) expressed in E. coli strain BL21.
  • Bacterial cells harboring pBN121(Tac)-T7-Clost(51-526) or pBN121(Tac)-Clost(51-526) were induced and harvested 4 hr after induction. Cells were lysed and the inclusion bodies, isolated by centrifugation, were boiled in SDS sample buffer.
  • Lane M molecular weight markers, as indicated (kDa).
  • Lanes 1, 2 clost(51-526) from two different isolates.
  • FIG. 1 is a plasmid map of the pBN121(Tac)- T7tag-Clost(51-526) expression vector.
  • T7tag-clostripain by E. coli BL21 cells transformed with the pBN121(Tac)-T7tag-Clost(51-526) nucleic acid construct was evaluated in 5 L or larger fermentations.
  • a 100 ⁇ L glycerol stock of the construct was used to inoculate 200 mL LB + 25 ⁇ g/mL Kanamycin media in a shaking flask.
  • the shake culture was grown in a rotary shaker at 37°C until the A 54 o reached 1.5 ⁇ 0.5.
  • the contents of the shaking flask culture were then used to inoculate a 5 L fermentation tank containing a defined minimal media. Glucose served as the carbon source and was maintained between 1-8 g/L.
  • kanamycin About 10 ⁇ g/mL kanamycin was used in the fermentation. Dissolved oxygen was controlled at 40% by cascading agitation and aeration with additional oxygen. Ammonium hydroxide solution was fed to control the pH at about 6.9 and to supply additional nitrogen. After the A 5 0 reached 50-75, the cells were induced with LPTG at a final concentration of 0.1-1 mM for 4-10 hours. After the induction was complete, the cells were cooled and harvested by centrifugation. The cell sediments were stored at a temperature below -20°C or were lysed immediately for use.
  • LBs inclusion bodies
  • LBs were washed with water until the conductivity dropped to below 100 ⁇ S/cm. Washed LBs were centrifuged to form a paste for storage at -20°C. The polypeptide sediments were dissolved in 8 M urea for further treatment.
  • Figure 6 illustrates a SDS-PAGE (4-20 % Tris-Glycine gel) analysis of vitro processing of clostripain core protein T7tag-Clost(51-526).
  • Lane M molecular weight markers, as indicated (kDa).
  • Lanes 1, 2 T7tag-Clost(51-526) before activation in 2 M urea.
  • Lanes 3, 4 T7tag-Clost(51-526) after activation in activation buffer containing 2 M urea at room temperature for 1 hr. The gel was stained with
  • Figure 7 shows a comparison of in vitro processing of clostripain proenzyme, core protein, and core protein mutant containing linker mutations. Inclusion bodies were extracted from 0.15 OD 60 o of IPTG-induced cells. Lane M: molecular weight marker. Lanes 1-3: clostripain proenzyme from pET23 a-proClost(28-526)HMS 174DE3. Lanes 4-6 : clostripain core protein from pET23a-Clost(51-526)/HMS174(DE3).
  • proteins were activated in activation buffer containing 2 M urea for 0 min (lanes 1, 4), 20 min (lanes 2, 5), or 60 min (lanes 3, 6). Proteins were then loaded on to a 4-20 % Tris-Glycine SDS gel for electrophoresis. The protein bands were detected by Coomassie Brilliant Blue staining. Banding positions of clostripain subunits are indicated by arrows: ( *- ) heavy chain, ( ⁇ - ) light chain from Clost(28- 526), ( ⁇ - ) light chain from Clost(51-526). Protein molecular weight markers are indicated in kDa (lane M).
  • Clostripain was recombinantly expressed in E. coli host cells and recovered from such cells in the form of inclusion bodies (LBs) in accordance with the preceding Examples.
  • the host cells were harvested by centrifugation and mechanically lysed in 50 mM Tris, 2.5 mM EDTA, pH 7.8, at 12, 000 psi.
  • the LB's were washed with water and collected by centrifugation at 15,000- 17,000 x g to form an LB paste.
  • This paste was solubilized in 8 M urea, containing 25 mM HEPES at pH 7.6, at a ratio of about 50 mL of urea solution/g LB followed by centrifugation at 6-8 °C at 15,000-17,000 x g to pelletize cell debris.
  • the centrifugation supernatant contained solubilized inactive clostripain.
  • the solubilized 8 M urea LB mixture was adjusted with 8 M urea, 25 mM HEPES at pH 7.6 to a concentration of 4 mg/mL of LB, and diluted to various concentrations of urea (1, 2, 3 and 4 M) in an activation buffer comprised of HEPES and CaCl 2 (e.g., 25 mM HEPES, 2 - 5 mM CaCl 2 , pH 7.6), to yield a final clostripain concentration of about 0.5 mg/mL, as reflected in Table 1.
  • an activation buffer comprised of HEPES and CaCl 2 (e.g., 25 mM HEPES, 2 - 5 mM CaCl 2 , pH 7.6), to yield a final clostripain concentration of about 0.5 mg/mL, as reflected in Table 1.
  • DTT dithiothreitol
  • Table 1 reflects the specific activities noted for each of the urea concentration levels of the dilution reaction mixtures. Kinetic activity was measured by Method 2, Example 11. Enzyme purity was assessed by HPLC assay Method 3, Example 11.
  • the maximum activity at the optimum urea concentration of 2 M obtained in the presence of 10 mM DTT was found to be 35 % higher than that obtained in the absence of DTT.
  • the areas of the clostripain light and heavy chain peaks in HPLC chromatograms were on average 50 % greater for samples prepared in the presence of 10 mM DTT, suggesting greater homogeneity of the activated clostripain.
  • Solubilized clostripain prepared by homogenizing clostripain inclusion bodies isolated as in Example 8, was activated by dilution into an activation buffer comprised of 25 mM HEPES and 5 mM CaCl 2 at pH 7.6, in a way that resulted in a urea concentration of 1 to 3 M.
  • Sodium chloride was added to the activated clostripain solution to a concentration of 0.1 M prior to loading on a DEAE column. A substantial portion of clostripain was recovered in the fractionation breakthrough (unbound fraction).
  • Figures 9, 10 and 11 illustrate the typical HPLC profiles for the starting material, retentate and permeates after diafiltration using a 10 K Regenerated Cellulose membrane in accordance with the process of the instant invention.
  • the starting purity for freshly activated clostripain frequently ranges from approximately 20-40 %.
  • Analysis of the permeate shows that the smaller hydrophilic compounds (peptides and degradation products of clostripain and cellular proteins) have passed through the membrane (Figure 11), while that of the retentate shows the enrichment in clostripain as its light and heavy chains, the peaks at 11 and 20 minutes respectively, in Figure 10.
  • Enzyme degradation products although mostly small enough to pass through the membrane, can still be large enough to be retained by the 10 K membrane.
  • the purity of clostripain does not directly indicate the kinetic activity of the enzyme.
  • the HPLC method gives an indication of the impurities present, the activity is determined by the BAEE kinetic assay.
  • HPLC Method 1 A Hewlett-Packard HP 1100 system was used, a Nydac C4 column (5 ⁇ particle size, 4.6 mm X 150 mm) and the following gradient program using solvent A: 5 % acetonitrile: 95 % water: 0.1 % TFA; solvent B: 95 % acetonitrile: 5 % water: 0.1 % TFA: The gradient was 15-50 % B (10 min), 50-100 % B (0.5 min) 100 % B (1 min), 100-15 % B (0.1 min), and 15 % B (3.4 min). Flow rate was 1.5 mL/min with detection at 280 nm. This HPLC assay was used to monitor the purity of clostripain as defined by the sum of the peak areas of its heavy and light chains relative to the other species present.
  • BAEE kinetic assay the substrate ⁇ -Benzoyl-L-Arginine ethyl ester (BAEE) was used for the kinetic assay at a temperature of 25°C.
  • the assay buffer was 25 mM HEPES, pH 7.6, 2.5 mM DTT, 0.25 mM BAEE, 5 mM CaCl 2 .
  • the assay was started by the addition of enzyme. Enzyme was diluted as needed to ensure a linear rate of 0.02-0.04 absorbance units per minute. This assay served as a marker of enzyme activity. Enzyme that would hydrolyze BAEE would also amidate peptides in a transpeptidation reaction. Clostripain BAEE-activity units are defined as that amount of enzyme which will hydrolyze one micromole of BAEE per minute under the conditions specified here.
  • HPLC assay a Beckman system Gold System was use with a Nydac C4 column (10 ⁇ m particle size, 4.6 x 250 mm). Elution was at a flow rate of 1.5 mL/min, at a temperature of 45 °C, and with detection at 280 nm. The gradient was 10-30 % B (3 min), 30-45 % B (20 min), 45-100 % B(3min), 100 % B (0.5 min), 100-10 % B (1 min). A was 5 % acetonitrile, 0.1 % TFA, B was 95 % acetonitrile, 0.1 % TFA. EXAMPLE 12
  • T7tag-Clost(51-526) and MRI inclusion bodies were solubilized in 8 M urea and then activated at a concentration of 2 and 4 M urea for either 1 hour at room temperature or 18 hours at room temperature (Figure 12).
  • Figures 12 and 13 indicate lanes for the solubilized constructs and molecular weight markers.
  • EXAMPLE 14 Effect of pH on the activation of clostripain
  • Washed inclusion bodies of clostripain were prepared as in Example 8, as a solution of 2 mg/mL in 8 M urea. Aliquots of 2 mL were diluted with 6 mL of buffers at different pH values to a final protein concentration of 0.5 mg/mL. The clostripain activity of each dilution was followed for about 3 hours by Method 2, Example 11.
  • the buffers were: from pH 6 to 7.7 : 0.1 M NaCl, 0.1 M HEPES, 0.1 M MES, 5 mM CaCl 2 ; from pH 7.7 to 9.4: 0.1 M NaCl, 0.1 M HEPES, 0.1 M ammonia, 5 mM CaCl 2 .
  • the diluent solutions were adjusted to the following pH values prior to adding the 8 M urea solution of clostripain inclusion bodies: 6.09, 6.32, 7.05, 7.35, 7.65, 7.70, 8.06, 8.32, 8.98, 9.38.
  • Maximum activities, attained within 2 to 3 hours, were normalized to the values at pH 7.7 (100%) and are reported in Figure 1. It was thereby established that maximum activation was obtained in a pH range around 7 to 8.5.

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Abstract

L'invention concerne un procédé permettant d'activer la clostripaïne, consistant à dissoudre dans une solution d'urée des corps d'inclusion de clostripaïne prélevés dans un mélange de fermentation comprenant des cellules hôtes recombinées ayant exprimé de la clostripaïne, puis à diluer ladite solution d'urée dans un tampon d'activation calcique jusqu'à obtenir une concentration finale en urée inférieure à environ 3 M. Cette activation par dilution à des concentrations en urée modérément élevées entraîne une élévation surprenante de l'activation de la clostripaïne et de la pureté de la solution résultante.
PCT/US2003/016646 2002-07-29 2003-05-23 Procede d'activation de la clostripaine Ceased WO2004011482A2 (fr)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AGRAZ ET AL: 'Renaturation, purification, and characterization of recombinant D-2-hydroxyisocaproate dehydrogenase from Escherichia coli' ENZYME AND MICROBIAL TECHNOLOGY vol. 17, 1995, pages 558 - 563, XP002978658 *
TAN ET AL: 'Cloning, Overexpression, Refolding, and Purification of the Nonspecific Phospholipase C from Bacillus cereus' PROTEIN EXPRESSION AND PURIFICATION vol. 10, 1997, pages 365 - 372, XP004451772 *
WITTE ET AL: 'Heterologous expression of the clostripain gene from Clostridium histolyticum in Escherichia coli and Bacillus subtilis: Maturation of the clostripain precursor is coupled with self-activation' MICROBIOLOGY vol. 140, 1994, pages 1175 - 1182, XP002978932 *

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