CZ2021400A3 - A strain producing clostripain-like protein for use in an admixture with recombinant collagenase ColG and ColH to isolate pancreatic islets of Langerhans - Google Patents

Abstract

Genetically modified E. coli bacterial strain deposited in the Czech Collection of Microorganisms under the number CCM 9176 with the introduced plasmid pET28 containing the gene for clostripain peptidase having the amino acid sequence SEQ ID NO:1, producing the peptidase enzyme, and its use.

Description

The invention relates to the preparation of recombinant proteins in the bacterium E. coli, their subsequent purification and the use of a mixture of purified proteins for the isolation of islets of Langerhans (LO) from pancreatic tissue. Specifically, these are two types of collagenases (ColG and ColH) and mainly a new type of clostripain, which acts synergistically with the two mentioned collagenases in the isolation of LO.

Current state of the art

Isolation of the islets of Langerhans (LO) has its specific characteristics compared to the separation of other cell types. It must not result in a complete division of the same cell types. Within this separation of tissue cells, a balanced procedure is still sought, in which tissue disintegration is necessary, but at the same time the opposite process, keeping the same cell cluster (LO) together. Considerable progress is being made in this field, and to date LO isolation processes have been set up and a number of enzymes have been identified and tested, thanks to which LO can be isolated in relatively good quality. There are still plenty of areas where improvement is needed. One of the main ones is the production of standardized batches of collagenase enzymes and auxiliary enzymes such as clostripain and neutral proteases and mixing their mixtures. So far, no specific enzyme mixture has been developed that is routinely used for LO isolation. The reason is, on the one hand, still insufficient detailed information on the composition of pancreatic tissue and, on the other hand, insufficient knowledge of the synergy of individual collagenase enzymes, clostripain and other proteases, during pancreatic tissue disintegration. Enzymes are often described in detail biochemically and individually in their own right, but there is a lack of data on how they may act in vivo in the LO isolation process.

Collagenases are collagen-degrading proteases that are widely used in cosmetics, food, industry, and medicine. For therapeutic purposes, such as the isolation of LO, microbial collagenases are used, which, unlike eukaryotic collagenases, can cleave collagen in multiple places. For high-quality optimization and balancing of LO isolation, it is necessary to find a suitable ratio of the enzymes used, which have a synergistic effect.

A mixture of enzymes isolated from the lysate of the bacterium Clostridium histolyticum was originally used to isolate LO, when it was a mixture of up to 12 different enzymes involved in tissue degradation. However, depending on the quality of purification, this mixture also contained various ballast proteins such as pigments or endotoxins. These proteins could be undesirable for the LO isolation process and may even have a pro-inflammatory effect (Berney et al., 2001; Vargas et al., 1998). Although these mixtures were and are always produced in the same way, their nature leads to ambiguous and uninterpretable results. This makes their quality use in medicine difficult. Such enzyme mixtures, which are partially purified, are still used for isolation today, but there are efforts to use highly purified enzymes directly from production bacteria, or to use the production of recombinant proteins in well-described systems such as E. coli bacteria (Ducka et al., 2009 ; Volpe et al., 2016), Bacillus (Jung et al., 1996), etc. However, the production of recombinant proteins also entails pitfalls, as they do not occur in their natural organism, they have different tags. For that reason, for example, the codons for the production of clostridial collagenase in E. coli were optimized (Alipour et al., 2014).

Already in the 1990s, it was shown that collagenase class I and II (ColH and ColG) are sufficient to successfully isolate LO from pancreatic tissue (Vos-Scheperkeuter et al., 1997; Gerrit H.J. Wolters et al., 1995). Adding more purified enzymes would increase the cost of the entire LO isolation process due to the complexity of the production process. Several biotechnological companies focused on the purification of bacterial collagenases and thereby contributed to the expansion of the LO isolation and transplantation method

- 1 CZ 2021 - 400 A3 to patients suffering from diabetes. In the first place, the production of these purified enzymes on an industrial scale contributed to the fact that the method could become a routine medical practice. However, there are still many aspects that can be improved, one of them is precisely better specificity and purity, to which the synergistic effect of additional enzymes such as clostripain can contribute.

For complete isolation and separation of LO from the surrounding tissue, collagenase ColG and ColH need to act together in a specific ratio and require the synergistic effect of additional enzymes such as neural proteases or thermolysin (O'Gorman et al., 2005; G.H.J. Wolters et al., 1992). Although ColH and ColG collagenases are capable of cleaving pancreatic tissue independently, or alone with the addition of neutral proteases, there are studies on the effective ratios of these two forms of C. histolyticum collagenases. The ideal ratio of ColH to ColG is reported to be 0.64:1.0 (Brandhorst et al., 2008), suggesting that ColG is essential for cleavage in this combination. Although ColH alone with admixture of neutral proteases is capable of cleaving pancreatic tissue. The ratio of the two mentioned collagenases also determines the amount of neutral proteases required to isolate LO. These ratios also determine the quality of tissue disintegration, which leads to the quality and higher viability of the islets, which is required for the success of the transplant itself.

In addition to using specific ColG and ColH ratios, specially mixed mixtures containing supporting lytic proteins are ideal for LO isolation. In addition to neutral proteases, thermolysin and dispase (used in pig pancreas experiments), another important enzyme is clostripain, which is responsible for tryptic activity in LO isolation mixtures. Experiments with LO isolations from porcine pancreas indicated that purified collagenases that showed high tryptic activity and low neutral protease activity had a higher yield of LO. Further experiments in rats have shown that the use of clostripain leads to a reduction in the time of tissue digestion and does not cause a violation of the LO as with the use of other mixtures (Klock et al., 1996). The basis for the isolation of LO is therefore the use of the correct proportion of various specific enzymes, which can also differ in the way they are produced. There are several studies that confirm that the presence of clostripain increases the yield and quality of LO when isolated (Brandhorst et al., 2009). Unlike the synergistic effect of clostripain and neutral proteases, its use with thermolysin has no positive effect. However, the use of different enzymes may also differ depending on the material for isolation (pig pancreas, rat or human material). In the isolation of LO from the pancreas of human origin, better isolation occurred when clostripain was used (Stáhle et al., 2015). The mechanism of action of clostripain has not yet been fully described, according to hypotheses, its activity helps the degradation of elastin as part of the extracellular matrix.

In addition to the use of collagenases and proteolytic enzymes, originating directly from the production organisms, well-defined systems for recombinant proteins can be used for production. The preparation of such strains may not be easy, as the protein may not be well produced in the target organism. Already in 1996, collagenase ColH from C. histolyticum was prepared, produced in the bacterium Bacillus subtilis, as it is more closely related to C. histolyticum than the commonly used E. coli (Jung et al., 1996). However, functional recombinant collagenase was also prepared in E. coli (Ducka et al., 2009), which can be used for LO isolation.

The enzymes thus prepared were successfully used for LO isolation in experiments (Brandhorst et al., 2003; Loganathan et al., 2018) and no significant significant differences were observed between the purified collagenase and the recombinant version of the protein. Some properties of LO were better when using clostridial collagenase and others with that produced in E. coli. However, the collagenase ratio used is still the subject of research, and the possibility of ratio variants and combinations with other enzymes (clostripain, neutral proteases) increase the number of all possible combinations for LO isolation.

Similar to recombinant collagenase, recombinant clostripain was also prepared, but it is different from the enzyme produced according to this invention. It is a protein that, like antibodies, has a light and heavy amino acid chain and can be found in protein databases under the accession number WP_138210406.1. This protein was also prepared as a recombinant, but due to its nature, a more complex preparation had to be chosen than for common bacterial

- 2 CZ 2021 - 400 A3 proteins. (Kim et al., 2007; Witte et al., 1994). There are several studies in which clostridial purified or recombinant purified clostripain was used, which increased the efficiency of LO isolation (Brandhorst et al., 2019; Dendo et al., 2015; Stáhle et al., 2015), but it was always the protein mentioned above (WP_138210406.1).

The essence of the invention

The essence of the invention is the strain Escherichia coli CCM 9176, producing in the pET28 plasmid a recombinant protein identified as clostripain. Furthermore, the method of production, purification and use of this protein (clostripain Clos) in a mixture with recombinant collagenase G and H (ColG and ColH) for the isolation of islets of Langerhans. When using the addition of clostripain produced by strain CCM 9176, a higher number of LOs is always obtained in the isolation than when using crude collagenase alone or a mixture of recombinant collagenases produced by strains CCM 9174 and CCM 9175. Examples also include the production of collagenase G and collagenase H produced by strains E. coli CCM 9174 and CCM 9175 created in the laboratory with which this synergistic effect can be guaranteed.

Using bioinformatic tools, genes for collagenase type I and type II (ColG and ColH) and also genes for subunits of clostripain, commonly used in mixtures for LO isolation, were identified in the genome of the bacterium C. histolyticum (=Hathewaya histolytica) (WP_138210406.1). Furthermore, a gene was identified that was annotated automatically by some databases as clostripain, but sequenced differently from the commonly used clostripains used in studies with LO isolates. The sequence of this clostripain-like protein designated as clostripain CCM 9176 is shown in Figure 1 (SEQ ID NO: 1). This gene was cloned into an expression plasmid using restriction cloning (NcoI and BamHI) and a histidine anchor was placed at the C-terminus of the protein sequence. The tag cleavage site was not integrated, as the protein is functional even without removal. The theoretical isoelectric point of the protein including the his-tag is 8.31 and the molecular weight (mw) is 45.7 kDa. After analysis of the amino acid sequence, it appears that the protein contains a cysteine-peptidase domain belonging to the peptidase family C11 (Figure 4), which is referred to as the clostripain family of enzymes and contains chymotrypsin-like specific sequences (Barrett & Rawlings, 2014).

Clostripain has a positive effect on the use of a mixture of collagenases for LO isolation and was tested with recombinant collagenases produced in E. coli strain CCM 9174 and CCM 9175. These are collagenases ColG and ColH (SEQ ID NO: 2 and 3, Figure 2 and 3 ) originating from C. histolyticum and produced from plasmid pET28 using E. coli expression cells. In the case of ColH, it is a protein with a histidine anchor added to its C-terminus, which does not have a site for cleavage of the amino acid sequence. The protein produced has a predicted molecular weight of 114 kDa and an isoelectric point of 5.68. In the case of ColG-tagged collagenase, the his-tag is at the N-terminus of the protein, followed by a cleavage site for thrombin to cleave the anchor. Both proteins show biochemical activity even without cleavage of the histidine anchor. ColG has a molecular weight of 123 kDa and a theoretical isoelectric point of 5.58. All procedures are optimized to use identical conditions for protein production and purification. Although highly purified enzymes are not produced and purified, the processes are easily performed for all mentioned proteins.

The essence of the invention

The whole process, expression and purification and incorporation is designed to be the same for all proteins, collagenase G, collagenase H and especially the newly described clostripain, which has a major positive effect on LO isolation. This means that the procedure described below can be used for all bacterial cultures (E coli CCM 9174, CCM 9175 and CCM 9176) and all produced and purified proteins (ColG, ColH and Clostripain).

Preparation of canned bacterial culture: Work with bacterial culture is carried out sterilely, tips with a filter are used when pipetting cultures. Up to 50 ml of LB medium (tryptone 5 g/l, yeast extract 10 g/l, NaCl

- 3 CZ 2021 - 400 A3 g/l, pH 7) 50 μΐ of kanamycin stock solution (final concentration 0.05 mg/ml) and 20 μΐ of culture containing plasmid pET28 for production of Clostripain or ColH or ColG are added. It is cultivated in a 200 ml Erlenmayer flask, in an incubator on a shaker, at 37 °C overnight (14 to 18 hours). The next day, the culture is centrifuged (3500 x g, 20 min, 4 °C), the supernatant is removed. The pellet is dissolved in 1 ml of cooled 10% glycerol and divided into sterile tubes of 200 μl each, which will serve as storage cans. Prepared preserves can be frozen down to -80 °C and can be stored this way for a long time. They are used in the preparation of all proteins (ColG, ColH and Clos).

Production of collagenases and clostripain: When preparing the production culture, add 600 μl of kanamycin stock solution to 600 ml of LB medium and inoculate 50 μl of the stock culture in glycerol, culture in a 2 l Erlenmayer flask. Cultivation takes place in a thermobox at 37 °C with intensive shaking. The culture is allowed to grow to OD 600 = 0.5 for approximately 3 hours. After reaching the targeted optical density, expression is induced by adding IPTG to a final concentration of 0.2 mM. After the addition of the inducer, the culture is then cultured at 22°C for 18 hours and still intensively aerated. The next morning, the grown culture is harvested by centrifugation. Centrifuge at 3500 rpm, 20 min, 4 °C. The supernatant is pooled and 10 to 100 ml of cooled sonication buffer (10 ml in each cuvette) is added to the pellets, cooled with ice. Centrifuge cuvettes are rinsed with an additional 10 ml of sonication buffer and the rest of the cells are transferred to the main fraction of the cell pellet in a suitable container.

Culture processing after protein expression: To release proteins from the cells, sonicate 3x10 min on ice, or disintegrate the cells in another suitable way, after every 10 min a 5 min break is made so that the lysate does not heat up too much. Sonicate at 70% power, at higher values the lysate heats up very quickly. After sonication, the lysate is transferred to ultracentrifuge tubes (Beckman coulter). Centrifuge at 1400 x g, 30 min, 4°C. The obtained supernatant is filtered through a 0.22 μm filter with a PES membrane so that during the purification, larger parts of the cell residues do not clog the chromatographic column. The pellet is stored in the refrigerator for later SDSPAGE analysis.

Protein purification: Purification of a protein with a histidine anchor is performed using metallochelation chromatography. For example, a HisTrap HP 5ml column (filled and charged with Ni Sepharose High Performance) is used. The methodology of column equilibration, sample loading and sample elution is the same for all systems. The used buffers and water must be deaerated in an ultrasonic bath for 10 to 15 minutes before purification. The abbreviation CV used below stands for column volume. The column is equilibrated with at least 5 CV buffer to set suitable conditions for purification (50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.5). After equilibration of the column, a lysate containing the required soluble protein (ColG, or ColH, or Clostripain) is applied to it. After applying the entire volume to the column, the protein is captured on the column using a histidine anchor and the ballast proteins are removed. The column is washed again with equilibration buffer (3 CV) for additional removal of untrapped impurities. An 8% elution buffer (50 mM Tris, 300 mM NaCl, 500 mM imidazole, pH 7.5) is applied to the column to remove weakly binding proteins. Clostripain remains trapped on the column, although some may be washed away, but the product is purified. In the second step, a 50% elution buffer solution (50mM Tris, 300mM NaCl, 500mM imidazole, pH 7.5) is applied to the column, which causes the release of clostripain from the column. This fraction (Figure 5) is used in the next step.

The step following purification is the removal of imidazole and transfer to the final buffer (50mM Tris, 300mM NaCl, pH 7.5), for this a HiTrap Desalting chromatographic column of 5 ml is used (the filling is based on dextran - Sephadex G-25 Superfine). The method works on the principle of SEC (size exclusion chromatography). If a precipitate forms in the purified sample, it must be filtered through a 0.45 μm filter before changing the buffer. Again, a syringe or peristaltic pump can be used to work with the HiTrap Desalting column. The syringe is filled with 25 mL exchange buffer (50 mM Tris, 300 mM NaCl, pH 7.5) and a Luer Lock attachment is attached. The column is connected to the extension in such a way that no air enters the column ("drop to drop"). The column is equilibrated with 25 ml of buffer at a flow rate of 5 ml/min (the maximum recommended flow rate for the column is 10 ml/min, and a pressure of 0.25 bar = 25 kPa). If air gets into the column, it is purged

- 4 CZ 2021 - 400 A3 until the bubble comes out (turning the column during flushing removes air more effectively).

Figure 5 shows a photo of the gel after SDS PAGE of individual purified clostripain fractions. Clostripain is present in all fractions, but the A10 fraction is the purest. The elution buffer concentration used for fraction A10 was used as the final elution concentration.

Removal of imidazole from the sample: The sample is applied to the column with a 2 to 5 ml syringe. The syringe is filled with exchange buffer and reattached to the column without air penetration. The sample from the column is eluted with a volume of buffer depending on the volume of the applied sample. The desired protein is then present in the eluate in a new imidazole-free buffer. Classical dialysis against the same buffer can also be used with a suitable dialysis hose with corresponding porosity. It is dialyzed against a 10x volume of buffer to remove unwanted imidazole.

Lyophilization: After removal of imidazole and transfer to a suitable buffer, the sample is filtered through a 0.22 μm PES filter. 50 to 100 μ are taken into the new eppendorf tube! sample for further analyzes characterizing the final product (measurement of protein concentration - Bradford, determination of product purity - SDS-PAGE, enzyme activity). The filtered sample is sterilely filled into weighed vials according to the required amount of units needed in one vial. Filled vials can be frozen at -80°C for at least 1 hour. After freezing the lyophilizer and freezing the sample, the vials can be lyophilized. If the sample volume is up to 10 ml, the sample is lyophilized within 24 h. After lyophilization, the vial with the lyophilisate is weighed and the weight of the product is determined.

Clarification of drawings

Figure 1 shows the amino acid sequence of the clostripain protein according to the invention produced by the strain E. coli CCM 9176. The genetically modified strain retains the sequence determined by the sequencing of the strain Clostridium histolyticum CCM 8656, described in the Czech invention application PV 2017-537, for which patent CZ 308157 was granted.

Figure 2 shows the amino acid sequence of collagenase ColG produced by strain E. coli CCM 9174. The genetically modified strain retains the sequence determined by sequencing of strain Clostridium histolyticum CCM 8656, described in the Czech invention application PV 2017-537, for which patent CZ 308157 was granted.

Figure 3 shows the amino acid sequence of ColH produced by the strain E. coli CCM 9175. The genetically modified strain retains the sequence determined by the sequencing of the strain Clostridium histolyticum CCM 8656, described in the Czech invention application PV 2017-537, for which patent CZ 308157 was granted.

Figure 4 presents a schematic representation of the peptidase domain predicted in the clostripain protein of the invention.

Figure 5 is a photograph of a gel after SDS PAGE of individual purified clostripain fractions. Clostripain is present in all fractions, but the A10 fraction is the purest. The elution buffer concentration used for fraction A10 was used as the final elution concentration.

Examples of implementation of the invention

1. Example of cultivation/production in a fermenter:

When preparing the production culture, 10 ml of the kanamycin stock solution is added to 10 liters of LB medium and 10 ml of the stock culture is inoculated in glycerol, cultivated in a volume of 10 l in a fermenter at

- 5 CZ 2021 - 400 A3 at a temperature of 37 °C with intensive mixing and aeration. The culture is allowed to grow to OD600 = 0.5. After reaching the targeted optical density, expression is induced by adding IPTG to a final concentration of 0.2 mM. After the addition of the inducer, the culture is then cultured at 22°C for 18 hours and still intensively aerated. The next morning, the grown culture is centrifuged in 0.5 L cuvettes at 3500 x g, 20 min, 4 °C. The supernatant is pooled and 10 to 100 mL of chilled sonication buffer (10 mL per cuvette) is added to the pellets in an ice-cooled falcon.

2. Example of purification by centrifugation:

Protino Ni-NTA Agarose can be used for purification in ordinary centrifuges without the need to use FPLC, etc. During equilibration, Protino Ni-NTA Agarose is resuspended by thorough shaking. The necessary amount of suspension is immediately taken into a suitable test tube (the volume is selected at least 10 times larger than the volume of the agarose beads - gel. For example, 12 ml test tubes are used for 1 ml of gel). The gel is sedimented by centrifugation at 500 xg, 5 minutes. Add 10 volumes of equilibration buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole) to calibrate the gel. Repeated inversion of the test tube mixes its contents. The gel is sedimented at 500 xg, 5 minutes. Carefully remove the supernatant into the waste container. Filtered bacterial lysate containing the required proteins is applied to bind the proteins to the gel. The entire suspension is gently mixed for 30 to 60 min (bonding between His-tag proteins and Ni ²⁺ ions). The gel is sedimented by centrifugation at 500 xg, 5 minutes. Carefully remove the supernatant into the waste container. Add 10 volumes of wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole) to wash the gel. Repeated inversion of the test tube mixes its contents. The gel is sedimented at 500 xg, 5 minutes. The supernatant is carefully collected in a waste container and the rinsing step is repeated. For protein elution, 1 volume of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole) is added to the sedimented gel. The suspension is gently stirred for 2 min at room temperature. The bound Histag proteins (Clostripain, or ColG or ColH) will be released from the gel. The gel is sedimented at 500 xg, 5 minutes. Carefully remove the supernatant with the desired proteins into a new tube. Eluted proteins are stored on ice. The elution steps are repeated a total of 5 times. The eluted fractions are always collected in a new tube. Imidazole is removed from the sample by dialysis, ultrafiltration or using a desalting column (HiTrap desalting).

3. Isolation of LO from the pancreas of experimental animals (wistar rats):

Rat pancreases were treated with control collagenase (Sigma Aldrich) and collagenase produced by strains E. coli CCM 9174 and CCM 9175 with the addition of clostripain produced by strain E. coli CCM 9176. A volume of 15 ml of the mixture solution with a collagenase activity of 2 U/ml was used. The enzyme ratio was ColG:ColH:Clos (5:5:1). Digestion takes place at a temperature of 37 °C for 10 to 40 minutes, depending on the state of the pancreas, and individual cellular tissues are subsequently separated using discontinuous gradient centrifugation. Isolated islets were cultured in CMRL-1066 medium supplemented with 10% FBS, 5% HEPES, 1% Glutamax, and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. The quality of isolated LOs was determined using vital staining and stimulated insulin secretion.

4. Isolation of LO from the pancreas of experimental animals in other ratios A:

Rat pancreases were treated with control collagenase (Sigma Aldrich) and collagenase ColH according to the invention with the addition of clostripain Clos according to the invention. A volume of 15 ml of the mixture solution with a collagenase activity of 2 U/ml was used. The collagenase ratio was ColH:Clos (5:2). Digestion takes place according to example No. 3.

5. Isolation of LO from the pancreas of experimental animals in other ratios B:

Rat pancreases were treated with control collagenase (Sigma Aldrich) and collagenase CCM 9174 and CCM 9175 supplemented with clostripain CCM 9176. A volume of 15 mL of the mixture solution was used

- 6 CZ 2021 - 400 A3 with collagenase activity 2 U/ml. The ratio of collagenases was ColG:ColH:Clos (7:4:2). Digestion takes place according to example No. 3.

Industrial applicability

A mixture of recombinant collagenase G and H containing a new type of clostripain can be used to make LO isolation more efficient. The new type of recombinant clostripain produced by E. coli CCM 9176 can also be added to crude collagenase mixtures or used in other recombinant collagenase mixtures than the aforementioned CCM 9174 and CCM 9175 mixture.

List of sequences:

SEQ ID NO: 1

Clostripain according to the invention Clos

MENKKFKKWTILIYTDGNNEMEDIMFKSMNDCKSVEVKEDINLVMQIGLLGESKLYNK NKFSGVRRYYLNAKEPMLLENLGKANMGDPNILYNFIRWGIKEFPAHHYMVIISGHGTS FVGALTDTSLDKNYIMGIPEMIKAISLGCKELKSIIDILVLDMCFMNSIEILYELSQYESIDR MITYTDTAPYGGLGYKKLVEAIEKNCNLKDIDLFTKNLITNLNFNLTAYDLDKFKLELIK KKFSYLAFNYLNNKERSKNPLDIINSIHPSNPYYSFNNTYYDYFNEINGTIKSTIIHDKSKF NGINSSIKIENKDINNLIAFYRKLEFSRENSWTDLLYNNSNTQKRDINKVKVRTPTSSLTTT HYILQFNPKDPNSSSVDKLAAALEHHHHHH

SEQ ID NO: 2

Sequence produced by collagenase ColG

MGSSHHHHHHSSGLVPRGSHMEPIENTNDTSIKNVEKLRNAPNEENSKKVEDSKNDKVE HVKNIEEAKVEQVAPEVKSKSTLRSASIANTNSEKYDFEYLNGLSYTELTNLIKNIKWNQI NGLFNYSTGSQKFFGDKNRVQAIINALQESGRTYTANDMKGIETFTEVLRAGFYLGYYN DGLSYLNDRNFQDKCIPAMIAIQKNPNFKLGTAVQDEVITSLGKLIGNASANAEVVNNC VPVLKQFRENLNQYAPDYVKGTAVNELIKGIEFDFSGAAYEKDVKTMPWYGKIDPFINE LKALGLYGNITSATEWASDVGIYYLSKFGLYSTNRNDIVQSLEKAVDMYKYGKIAFVAM ERITWDYDGIGSNGKKVDHDKFLDDAEKHYLPKTYTFDNGTFIIRAGDKVSEEKIKRLY WASREVKSQFHRVVGNDKALEVGNADDVLTMKIFNSPEEYKFNTNINGVSTDNGGLYIE PRGTFYTYERTPQQSIFSLEELFRHEYTHYLQARYLVDGLWGQGPFYEKNRLTWFDEGT AEFFAGSTRTSGVLPRKSILGYLAKDKVDHRYSLKKTLNSGYDDSDWMFYNYGFAVAH YLYEKDMPTFIKMNKAILNTDVKSYDEIIKKLSDDANKNTEYQNHIQELADKYQGAGIPL VSDDYLKDHGYKKASEVYSEISKAASLTNTSVTAEKSQYFNTFTLRGTYTGETSKGEFK DWDEMSKKLDGTLESLAKNSWSGYKTLTAYFTNYRVTSDNKVQYDVVFHGVLTDNAD ISNNKAPIAKVTGPSTGAVGRNIEFSGKDSKDEDGKIVSYDWDFGDGATSRGKNSVHAY KKAGTYNVTLKVTDDKGATATESFTIEIKNEDTTTPITKEMEPNDDIKEANGPIVEGVTV KGDLNGSDDADTFYFDVKEDGDVTIELPYSGSSNFTWLVYKEGDDQNHIASGIDKNNSK VGTFKSTKGRHYVFIYKHDSASNISYSLNIKGLGNEKLKEKENNDSSDKATVIPNFNTTM QGSLLGDDSRDYYSFEVKEEGEVNIELDKKDEFGVTWTLHPESNINDRITYGQVDGNKV SNKVKLRPGKYYLLVYKYSGSGNYELRVNK

SEQ ID NO: 3

Sequence produced by collagenase ColH

MVQNESKRYTVSYLKTLNYDLVDLLVKTEIENLPDLFQYSSDAKEFYGNKTRMSFIMD EIGRRAPQYTEIDHKGIPTLVEVVRAGFYLGFHNKELNEINKRSFKERVIPSILAIQKNPNF

- 7 CZ 2021 - 400 A3

KLGTEVQDKIVSATGLLAGNETAPPEVVNNFTPILQDCIKNIDRYALDDLKSKALFNVLA APTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYGKNDNNSWIIDNGIYHIAPLG KLHSNNKIGIETLTEVMKVYPYLSMQHLQSADQIKRHYDSKDAEGNKIPLDKFKKEGKE KYCPKTYTFDDGKVIIKAGARVEEEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDI LTMVIYNSPEEYKLNSVLYGYDTNNGGMYIEPEGTFFTYEREAQESTYTLEELFRHEYTH YLQGRYAVPGQWGRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVSNIHNTTRN NRYKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLNDLAKNNDVDGYDNY IRDLSSNYALNDKYQDHMQERIDNYENLTVPFVADDYLVRHAYKNPNEIYSEISEVAKL KDAKSEVKKSQYFSTFTLRGSYTGGASKGKLEDQKAMNKFIDDSLKKLDTYSWSGYKT LTAYFTNYKVDSSNRVTYDVVFHGYLPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEG SFDPDGKIVSYEWDFGDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIK DLSENKLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDFSSEQNPS HVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEPNNSKETASGPIVPGIP VSGTIENTSDQDYFYFDVITPGEVKIDINKLGYGGATWVVYDENNNAVSYATDDGQNLS GKFKADKPGRYYIHLYMFNGSYMPYRINIEGSVGRDPNSSSVDKLAAALEHHHHHH

Literature review

Alipour, H., Raz, A., Dinparast Djadid, N., Rami, A., & Mahdian, S. M. A. (2014). Codon optimization of Col H gene encoding Clostridium histolyticum collagenase to express in Escherichia coli. Codon Optimization of Col H Gene Encoding Clostridium Histolyticum Collagenase to Express in Escherichia Coli. https://doi.org/10.7287/peeij.preprints.754

Barrett, A.J., & Rawlings, N.D. (2014). Evolutionary Lines of Cysteine Peptidases. Biological Chemistry. https://doi.org/ 10.1515/bchm.2001.382.5.727

Berney, T., Molano, R.D., Cattan, P., Pileggi, A., Vizzardelli, C., Oliver, R., Ricordi, C., & Inverardi, L. (2001). Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis. Transplantation. https://doi.org/10.1097/00007890-200101150-00020

Brandhorst, H., Brandhorst, D., Hesse, F., Ambrosius, D., Brendel, M., Kawakami, Y., & Bretzel, R.G. (2003). Successful human islet isolation utilizing recombinant collagenase. Diabetes. https://doi.org/10.2337/diabetes.52.5.1143

Brandhorst, H., Friberg, A., Andersson, H.H., Felldin, M., Foss, A., Salmela, K., Lundgren, T., Tibell, A., Tufveson, G., Korsgren, O., & Brandhorst, D. (2009). The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation. Transplantation. https://doi.org/10.1097/TP.0b013e31819499f0

Brandhorst, H., Johnson, P.R., Monch, J., Kurfurst, M., Korsgren, O., & Brandhorst, D. (2019). Comparison of Clostripain and Neutral Protease as Supplementary Enzymes for Human Islet Isolation. Cell Transplantation. https://doi.org/10.1177/0963689718811614

Brandhorst, H., Raemsch-Guenther, N., Raemsch, C., Friedrich, O., Huettler, S., Kurfuerst, M., Korsgren, O., & Brandhorst, D. (2008). The ratio between collagenase class I and class II influences the efficient islet release from the rat pancreas. Transplantation. https://doi.org/10.1097/TP.0b013e31816050c8

Dendo, M., Maeda, H., Yamagata, Y., Murayama, K., Watanabe, K., Imura, T., Inagaki, A., Igarashi, Y., Katoh, Y., Ebina, M., Fujimori, K., Igarashi, K., Ohuchi, N., Satomi, S., & Goto, M. (2015). Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation. Transplantation. https://doi.org/10.1097/TP.0000000000000662

Ducka, P., Eckhard, U., Schonauer, E., Kofler, S., Gottschalk, G., Brandstetter, H., & Nuss, D.

- 8 CZ 2021 - 400 A3 (2009) . A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253009-1953-4

Jung, C.M., Matsushita, O., Katayama, S., Minami, J., Ohhira, I., & Okabe, A. (1996). Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification. Microbiology and Immunology. https://doi.org/10.1111/j.13480421.1996.tb01161.x

Kim, C.K., Lee, S.Y., Kwon, O.J., Lee, S.M., Nah, S.Y., & Jeong, S.M. (2007). Secretory expression of active clostripain in Escherichia coli. Journal of Biotechnology. https://doi.org/10.1016/j.jbiotec.2007.07.936

Klock, G., Kowalski, M.B., Hering, B.J., Eiden, M.E., Weidemann, A., Langer, S., Zimmermann, U., Federlin, K., & Bretzel, R.G. (1996). Fractions from commercial collagenase preparations: Use in enzymic isolation of the islets of Langerhans from porcine pancreas. Cell Transplantation. https://doi.org/10.1016/0963-6897(96)00023-1

Loganathan, G., Subhashree, V., Breite, A.G., Tucker, W.W., Narayanan, S., Dhanasekaran, M., Mokshagundam, S.P., Green, M.L., Hughes, M.G., Williams, S.K., Dwulet, F.E., McCarthy, M. R.C., & Balamurugan, A.N. (2018). Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield. American Journal of Transplantation. https://doi.org/10.1111/ajt.14542

O’Gorman, D., Kin, T., McGhee-Wilson, D., Shapiro, A.M.J., & Lakey, J.R.T. (2005). Multilot analysis of custom collagenase enzyme blend in human islet isolations. Transplantation Proceedings. https://doi.org/10.1016/j.transproceed.2005.09.139

Stahle, M., Foss, A., Gustafsson, B., Lempinen, M., Lundgren, T., Rafael, E., TufVeson, G., Korsgren, O., & Friberg, A. (2015). Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation. Transplant Direct.

https://doi.org/10.1097/txd.0000000000000528

Vargas, F., Vives-Pi, M., Somoza, N., Armengol, P., Alcalde, L., Marti, M., Costa, M., Serradell, L., Dominguez, O., Fernández-Llamazares , J. , Julian , J.F. , Sanmarti , A. , & Pujol-Borrell , R. (1998). Endotoxin contamination may be responsible for the unexplained failure of human pancreatic islet transplantation. Transplantation. https://doi.org/10.1097/00007890-19980315000020

Volpe, L., Salamone, M., Giardina, A., & Ghersi, G. (2016). Optimization of a Biotechnological Process for Production and Purification of Two Recombinant Proteins: Col G and Col H. Chemical Engineering Transactions. https://doi.org/10.3303/CET1649011

Vos-Scheperkeuter, G.H., Van Suylichem, P.T.R., Vonk, M.W.A., Wolters, G.H.J., & Van Schilfgaarde, R. (1997). Histochemical Analysis of the Role of Class I and Class II

Clostridium Histolyticum Collagenase in the Degradation of Rat Pancreatic Extracellular Matrix for Islet Isolation. Cell Transplantation. https://doi.org/10.1177/096368979700600407

Witte, V., Wolf, N., Diefenthal, T., Reipen, G., & Dargatz, H. (1994). 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. https://doi.org/10.1099/13500872-140-5-1175

Wolters, G.H.J., Vos-Scheperkeuter, G.H., van Deijnen, J.H.M., & van Schilfgaarde, R. (1992).

- 9 CZ 2021 - 400 A3

An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Diabetology. https://doi.org/10.1007/BF00429093

Wolters, Gerrit H.J., Vos-Scheperkeuter, G.H., Lin, H.C., & Van Schilfgaarde, R. (1995).

Different roles of class I and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation. Diabetes. https://doi.org/10.2337/diab.44.2.227

Claims (4)

1. Genetically modified E. coli bacterial strain stored in the Czech Collection of Microorganisms under number CCM 9176 with inserted plasmid pET28 containing the gene for clostripain peptidase 5 having the amino acid sequence SEQ ID NO:1, producing the peptidase enzyme. 2. Use of the purified strain according to claim 1 for the production of clostripain peptidase with the sequence SEQ ID NO: 1. 3. Use of the E. coli strain deposited in the Czech Collection of Microorganisms under the number CCM 9176 for the production of clostripain peptidase with the sequence SEQ ID NO: 1 suitable in combination with collagenase G 10 and/or collagenase H for the isolation of pancreatic islets of Langerhans. 4. Use according to claim 3, where collagenase G with the sequence SEQ ID NO: 2 produced by the strain E. coli CCM 9174 and/or collagenase H with the sequence SEQ ID NO: 3 produced by the strain E. coli CCM 9175 is used.