Methods for making recombinant protein

Abstract

To provide methods of producing recombinant plasminogen in a mammalian expression system.SOLUTION: A method for producing plasminogen comprises: (i) providing a host cell comprising a first polynucleotide encoding plasminogen and a second polynucleotide encoding plasminogen activator inhibitor-1 (PAI-1), wherein the first and second polynucleotides are provided in a single vector; and (ii) culturing the host cell in a suitable culture medium under conditions to effect expression of plasminogen from the first polynucleotide and expression of PAI-1 from the second polynucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing recombinant plasminogen in a mammalian expression system.

[0002] Related Applications This application claims priority to Australian Provisional Application No. AU2019902468, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] The production of large quantities of relatively pure polypeptides and proteins is important for the manufacture of many pharmaceutical preparations. Recombinant DNA technology has been adopted for the production of many proteins, in part, because large amounts of exogenous proteins can be expressed in host cells.

[0004] Plasmin is the major fibrinolytic enzyme in mammals. This protein is a serine protease belonging to the chymotrypsin-like family that is derived from the inactive zymogen precursor plasminogen that circulates in plasma.

[0005] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is synthesized primarily in the liver and is abundant in most extracellular fluids. The concentration of plasminogen in plasma is approximately 2 μM. Therefore, plasminogen constitutes a large potential source of proteolytic activity in tissues and body fluids.

[0006] Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The native, secreted, uncleaved form is called Glu-plasminogen because it has an amino-terminal (N-terminal) glutamic acid. However, in the presence of plasmin, Glu-plasminogen is cleaved at Lys76-Lys77 to give Lys-plasminogen. Compared to Glu-plasminogen, Lys-plasminogen has a higher affinity for fibrin and is activated at a higher rate by plasminogen activators, but there is no evidence that Lys-plasminogen is found in the circulation.

[0007] Plasminogen can be produced by tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (urokinase-type plasminogen activator). Arg561-Val562 peptide by either lasminogen activator (uPA) Plasmin is activated by cleavage of the Pan-apple bond of native plasminogen (including Glu-plasminogen, whose N-terminus is glutamic acid) to form plasmin. This cleavage results in an α-heavy chain consisting of one pan-apple and five kringle domains, four of which have lysine-binding sites, and a β-light chain containing the catalytic triad, His603, Asp646, and Ser741. Active plasmin is involved in the lysis of fibrin clots in the host. When bound to a fibrin clot, it dissolves the Pan-apple domain of native plasminogen (including Glu-plasminogen, whose N-terminus is glutamic acid). The main protein has been shown to be readily cleaved and converted to N-terminally lysine-modified plasminogen (83 kDa) (Lys-plasminogen).

[0008] Two major glycoforms of plasminogen exist in human plasma: type 1 plasminogen, which contains at least two glycosylated moieties (N-linked to N289 and O-linked to T346), and type 2 plasminogen, which contains at least one O-linked sugar (O-linked to T346). Type 2 plasminogen is preferentially mobilized to cell surfaces, whereas type 1 plasminogen is predominantly mobilized by blood clots.

[0009] Plasminogen can exist in two conformations: closed and open. Native Glu-plasminogen in circulation is in the closed form, and therefore the activation site is not exposed. Upon binding to a target, such as a fibrin clot or a cell surface receptor, via a lysine-binding site on the kringle region, it changes to an open conformation in which its activation site is exposed. The molecular dimensions between these two conformations are significantly different.

[0010] Plasmin is a fundamental component of the fibrinolytic system and is the primary enzyme involved in clot dissolution and clearance of leaked fibrin. Additionally, plasmin cleaves diverse biological targets, including basement membranes, extracellular matrices, cell receptors, cytokines, and complement. Plasminogen is therefore vital in wound healing, cell migration, tissue remodeling, angiogenesis, and embryogenesis. Plasminogen has been shown to be involved in multiple cellular processes during all phases of wound healing, namely inflammation, proliferation, and remodeling. These processes include fibrin degradation, platelet activation, cytokine and growth factor release, apoptotic cell clearance, keratinocyte activation, and epithelial-mesenchymal transition of fibroblasts, cell migration, and extracellular matrix degradation.

[0011] There are numerous technical difficulties associated with obtaining sufficient quantities of sufficiently pure preparations of recombinant plasminogen for use as a therapeutic agent. Due to the complex structure of the full-length plasminogen molecule, bacterial expression systems have not proven useful for recombinant plasminogen production. Plasminogen is produced in the form of insoluble inclusion bodies, which make it impossible to refold. Furthermore, expression of plasminogen in mammalian cells is complicated by the intracellular activation of plasminogen to plasmin and the resulting cytotoxicity. While production of fully active plasminogen using insect cells is feasible, this system is not suitable for large-scale production due to low yields.

[0012] As a result of the difficulty of obtaining recombinant plasminogen of suitable quantity and quality using recombinant systems, most plasminogen produced for use in clinical settings today is obtained from fractions of plasma. Obtaining plasminogen directly from human plasma presents its own problems, such as the need to rely on ample supplies of source material and the risk of pathogen contamination. Summary of the Invention [Problem to be solved by the invention]

[0013] There is a need for new methods and compositions for obtaining plasminogen in sufficient quantities and with sufficient activity for use in treating conditions requiring plasminogen replacement.

[0014] The reference to any prior art in the specification is not an admission or suggestion in any jurisdiction that this prior art forms part of the common general knowledge or that this prior art could reasonably be expected to be understood, regarded as relevant to, and / or combined with other pieces of prior art by a person skilled in the art. [Means for solving the problem]

[0015] In one aspect, the present invention provides a method for producing plasminogen, comprising: (i) providing a host cell containing a first recombinant polynucleotide encoding plasminogen and a second recombinant polynucleotide encoding a plasminogen activator inhibitor; (ii) culturing said host cells in a suitable culture medium under conditions that allow expression of plasminogen from the first polynucleotide and plasminogen activator inhibitor from the second polynucleotide; The present invention provides a method comprising:

[0016] Preferably, the present invention provides a method for producing plasminogen, comprising: (i) a first recombinant polynucleotide encoding plasminogen and a second recombinant polynucleotide encoding plasminogen activator inhibitor-1 (PAI-1) or a variant thereof; providing a host cell containing the nucleotide; (ii) culturing said host cells in a suitable culture medium under conditions that allow expression of plasminogen from the first polynucleotide and PAI-1 or a variant thereof from the second polynucleotide; The present invention provides a method comprising:

[0017] In one aspect, the present invention provides a method for producing recombinant plasminogen, comprising the steps of: (a) providing a first polynucleotide encoding plasminogen; (b) providing a second polynucleotide encoding PAI-1 or a variant thereof, the first and second polynucleotides are operably linked to a promoter to allow expression of the polynucleotides; (c) providing a host cell; (d) transforming or transfecting a host cell with the polynucleotides of a) and b); (e) providing a cell culture medium; (f) culturing the transformed or transfected host cells in a cell culture medium under conditions sufficient for expression of plasminogen and a polynucleotide encoding PAI-1 or a variant thereof; and Optionally, (g) recovering or purifying the plasminogen from the host cells and / or cell culture medium. The present invention provides a method comprising:

[0018] In one embodiment, the first and second polynucleotides are provided in a single polynucleotide molecule. In an alternative embodiment, the first and second polynucleotides are provided in different polynucleotide molecules. For example, a polynucleotide encoding plasminogen and a plasminogen activator inhibitor (preferably PAI-1) or a variant thereof may be provided in a single vector. Alternatively, a polynucleotide encoding plasminogen and a plasminogen activator inhibitor (PAI-1) or a variant thereof may be provided in a single vector. The variants may be provided in separate vector constructs, each vector having a different type of selectable marker than the other vector.

[0019] In another aspect, the present invention also provides a method for producing recombinant plasminogen, comprising the steps of: (a) providing a vector comprising a polynucleotide encoding plasminogen; (b) providing a vector comprising a polynucleotide encoding PAI-1 or a variant thereof; (c) providing a host cell; (d) transforming or transfecting a host cell with the vectors of steps (a) and (b); (e) providing a cell culture medium; (f) culturing the transformed or transfected host cells in a cell culture medium under conditions sufficient for expression of plasminogen and a polynucleotide encoding PAI-1 or a variant thereof; and Optionally, (g) recovering or purifying the plasminogen from the host cells and / or cell culture medium. Also provided is a method comprising:

[0020] In any embodiment of the method of the present invention, the method further comprises the step of mixing PAI-1 or a variant thereof into the culture medium.

[0021] In another aspect, the present invention provides a method for producing plasminogen, comprising: (i) providing a host cell containing a recombinant polynucleotide encoding plasminogen; (ii) culturing the host cells in a suitable culture medium under conditions that allow expression of plasminogen from the polynucleotide; Including, The culture medium comprises PAI-1 or a variant thereof.

[0022] In another aspect, the present invention provides a method for producing recombinant plasminogen, comprising the steps of: (a) providing a polynucleotide encoding plasminogen, the polynucleotide is operably linked to a promoter to allow expression of the polynucleotide; (c) providing a host cell; (d) transforming or transfecting a host cell with the polynucleotide of (a); (e) providing a cell culture medium comprising PAI-1 or a variant thereof; (f) culturing the transformed or transfected host cells in a cell culture medium under conditions sufficient for expression of the polynucleotide encoding plasminogen; and Optionally, (g) recovering or purifying the plasminogen from the host cells and / or cell culture medium. The present invention provides a method comprising:

[0023] In either embodiment of the method of the present invention, the method provides for transient or stable expression of a polynucleotide encoding plasminogen, and transient or stable expression of a polynucleotide encoding PAI-1 or a variant thereof.

[0024] The plasminogen can correspond to any mammalian plasminogen sequence. In any embodiment, the plasminogen is human plasminogen, non-human primate plasminogen, porcine, mouse, rat, sheep, goat, horse, cow, cat, dog, or other mammalian plasminogen. Preferably, the plasminogen is human plasminogen.

[0025] In any embodiment of the present invention, the plasminogen is selected from the group consisting of Glu-Plg, Lys-Plg, Midi-Plg, Mini-Plg and Micro-Plg.

[0026] The plasminogen may comprise a wild-type plasminogen sequence, or a variant or modified sequence thereof. In any embodiment of the present invention, the plasminogen is selected from the group consisting of Glu-Plg, Lys-Plg, midi-Plg, mini-Plg, and micro-Plg. In an alternative embodiment, the plasminogen may comprise a plasminogen sequence containing amino acid substitutions in the protease active site, activation site, and combinations thereof, and / or amino acid substitutions that result in increased protease activity.

[0027] In certain embodiments, the polynucleotide encoding plasminogen is SEQ ID NO: 1, 5, 6, 8, 10, 12 or 14, or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1, 5, 6, 8, 10, 12 or 14.

[0028] In certain embodiments, the plasminogen encoded by the polynucleotide or produced according to any of the methods described herein comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19, or 20, or a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19, or 20. In one embodiment, the plasminogen produced according to any of the methods described herein does not contain a signal sequence, including any of the signal sequences described herein.

[0029] Preferably, the plasminogen activator inhibitor is PAI-1. More preferably, the PAI-1 comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 4. Alternatively, the PAI-1 sequence may comprise, consist of, or consist essentially of the sequence of unmodified (i.e., wild-type) PAI-1, the wild-type sequence consisting of the sequence of SEQ ID NO: 4, with residues at positions 197 and 355 being glutamine and glycine, respectively.

[0030] In certain embodiments, the polynucleotide encoding the plasminogen activator inhibitor comprises the nucleic acid sequence of SEQ ID NO:3, or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO:3.

[0031] In certain embodiments in which the first and second polynucleotides (i.e., polynucleotides encoding plasminogen and plasminogen activator inhibitor) are provided in a single polynucleotide construct, e.g., a single vector construct, the single polynucleotide construct comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:5, or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence set forth in SEQ ID NO:5.

[0032] The host cell is preferably a mammalian host cell, including, but not limited to, cells selected from the group consisting of Expi293, variants of Expi293, CHO (Chinese Hamster Ovary) cells and derivatives thereof, HeLa (human cervical carcinoma) cells, COS and Vero cells.

[0033] In another aspect, the present invention also provides a vector or construct comprising a first polynucleotide sequence encoding plasminogen and a second polynucleotide sequence encoding PAI-1 or a variant thereof. Preferably, the first and second polynucleotides The first polynucleotide sequence is operably linked to a promoter to allow expression of the polynucleotide. In certain embodiments, plasminogen and PAI-1 are encoded in a single polynucleotide construct to allow bicistronic expression. In certain embodiments, the vector or construct contains an internal ribosome entry site (IRES) between the first polynucleotide sequence and the second polynucleotide sequence to allow translation initiation independent of the cap.

[0034] Typically, the first polynucleotide sequence encodes a plasminogen selected from the group consisting of Glu-Plg, Lys-Plg, midiPlg, miniPlg and microPlg.

[0035] In certain embodiments, the first polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 6, 8, 10, 12, or 14, or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1, 6, 8, 10, 12, or 14.

[0036] In certain embodiments, the second polynucleotide comprises, consists of, or consists essentially of the nucleic acid sequence of SEQ ID NO:3, or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence set forth in SEQ ID NO:3.

[0037] In certain embodiments, the plasminogen encoded by the first polynucleotide comprises, consists of, consists essentially of, or has an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19, or 20.

[0038] In one embodiment, the plasminogen activator inhibitor encoded by the second polynucleotide is plasminogen activator inhibitor-1 (PAI-1) or a variant thereof. More preferably, the PAI-1 comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 4. Alternatively, the PAI-1 sequence may comprise, consist of, or consist essentially of the sequence of unmodified (i.e., wild-type) PAI-1, the wild-type sequence consisting of the sequence of SEQ ID NO: 4, with residues at positions 197 and 355 being glutamine and glycine, respectively.

[0039] In one embodiment, the vector or construct comprises the nucleic acid sequence set forth in SEQ ID NO:5, or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the sequence set forth in SEQ ID NO:5.

[0040] In another aspect, the present invention provides a host cell comprising an inventive vector or construct described herein.

[0041] In another aspect, the present invention provides isolated, purified, substantially purified, or recombinant plasminogen produced by the methods of the invention described herein. The plasminogen may be any one of those described herein, for example, may comprise, consist of, or consist essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19, or 20, or a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19, or 20. Preferably, the plasminogen does not contain a signal sequence, including any of the signal sequences described herein.

[0042] In another aspect, the present invention provides a composition comprising plasminogen and a plasminogen activator inhibitor, preferably PAI-1 or a variant thereof, isolated, purified, or substantially purified from culture medium from the inventive methods described herein.

[0043] In a further aspect, the present invention provides isolated, purified, substantially purified, or recombinant plasmin derived from or obtained from plasminogen produced by the inventive methods described herein.

[0044] Still further, the present invention provides the use of isolated, purified, substantially purified, or recombinant plasminogen (or plasmin derived therefrom) in a method of treating a condition in an individual, the condition requiring the administration of exogenous plasminogen (or plasmin).

[0045] As used herein, unless the context otherwise requires, the term "comprise" and variations of this term, such as "including", "comprises", and "comprised" is not intended to exclude additional additives, ingredients, integers or steps.

[0046] Further aspects and further embodiments of the aspects of the invention set out in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 shows a Coomassie-stained 10% SDS-PAGE of protein fractions eluted from the affinity column. The protein band at approximately 100 kDa represents the eluted rPlg. [Figure 2] Ion exchange chromatography of rPlg. (A) Purification of rPlg (indicated by the double arrow) by anion exchange chromatography using a gradient of high-salt buffer B (in this case, rPlg elutes as a single peak). Contaminants (indicated by "c") are separated from affinity-purified rPlg (see Figure 1), which is later eluted in the presence of 100% buffer B. (B) Coomassie-stained 10% SDS-PAGE showing the rPlg-containing fraction indicated by the double arrow in (A). [Figure 3] Figure 1 shows size exclusion chromatography of rPlg. (A) Size exclusion profile of rPlg purified on a Superdex 200 column. (B) Coomassie-stained 12% SDS-PAGE showing purified rPlg. [Figure 4] Coomassie-stained 12% SDS-PAGE showing recombinant plasmin (rPlm) generated by activation of rPlg with tPA. Under reducing conditions, rPlm separates into heavy and light chains (residues Glu1 to Arg561 and Val562 to Asn791, respectively). [Figure 5] Figure 1 shows a progress curve illustrating tPA (500 nM)-mediated activation of Plg and rPlg, the native Plg glycoforms I and II (Plg GI and Plg GII), as measured by hydrolysis of the plasmin fluorogenic substrate H-Ala-Phe-Lys-AMC. [Figure 6] Figure 1 shows Michaelis-Menten analysis of Plg activation by tPA in the presence of 1 μM EACA. Results from activation of native Plg GI and GII, rPlg, show that rPlg is the most readily activatable (as indicated by KM and Vmax). [Figure 7] Figure 1 shows the radius of gyration of native Plg GI, GII, and rPlg. Experiments were performed using small-angle X-ray scattering, which measures the dimensions of macromolecules in solution. Closed and open conformations were recorded in the presence or absence of 20 mM EACA, respectively. In the closed form, the radius of gyration of GI and rPlg is similar. [Figure 8] Figure 1 shows a SAXS titration experiment to study the conformational changes of Plg in response to EACA. Overall, the titration curves are comparable between GI, GII, and rPlg. Kopen is the EACA concentration at which 50% of Plg is in the open conformation. [Figure 9] Figure 1 shows the binding of rPlg and native Plg to α2-AP. Plg binding (at the concentrations indicated) to α2-AP immobilized on a Ni2+-NTA chip was measured in real time. The colored lines represent experimental curves, and the dotted lines represent fitted curves. A two-state reaction model was used to calculate the kinetics and affinity constants for native Plg binding (bottom), and a 1:1 Langmuir binding model was used to calculate the kinetics and affinity constants for rPlg binding (top) using Biacore T200 evaluation software (Biacore AB). [Figure 10]Figure 1 shows the binding of rPlm and native Plm to α2-AP. Binding of Plm (at the concentrations indicated) to α2-AP immobilized on a Ni2+-NTA chip was measured in real time. The colored line represents the experimental curve, and the dotted line represents the fitted curve. Data were fitted with a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB) and used to calculate kinetic and affinity constants. [Figure 11] Binding of rPlg and native Plg to streptokinase (SK). Binding of recombinant and native plasminogen to SK immobilized on a Ni2+-NTA chip measured in real time. The colored line represents the experimental curve, and the dotted line represents the fitted curve. The data were fitted with a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB) and used to calculate the kinetics and affinity constants. [Figure 12] Figure 1 shows the binding of rPlm and native Plm to streptokinase. Binding of recombinant plasmin and native plasmin to streptokinase immobilized on a Ni2+-NTA chip was measured in real time. The colored line represents the experimental curve, and the dotted line represents the fitted curve. The data were fitted with a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB) and used to calculate the kinetics and affinity constants. [Figure 13] Figure 1 shows the binding of rPlg to cellular receptors (HEK293 cells). rPLG binds to the Plg receptor on mammalian cells. [Figure 14]A. rPlg (labeled with Alexa Fluor 790) accumulates at the site of bone and muscle injury and reduces dystrophic mineralization after injury. A. Top panel: rPlg accumulates at the fracture site. Alexa Fluor-labeled fibrin and rPlg were injected IP. The image shows the accumulation of fibrin and rPlg at the fracture site. Bottom panel: rPlg accumulates at the site of muscle injury. Cardiotoxin was injected into the right leg to induce muscle injury, and rPlg was administered IP at 1 mg / day. The image shows the accumulation of rPlg in the injured leg and kidney, but not in the uninjured one. B. Top panel: rPlg accumulation at the site of muscle injury. Cardiotoxin was injected into the leg to induce muscle injury. rPlg labeled with Alex Fluor dye was injected IP at 1 mg / day, and images were recorded 1 to 7 days after injury. Lower panel: rPlg accumulation at the injury site prevents muscle calcification in Plg+ / - animals. Cardiotoxin was injected into the leg to induce muscle injury. rPlg was injected IP at 1 mg / day, and images were recorded 7 days after injury. Muscle calcification is evident in Plg+ / - but not WT animals and can be rescued by inhibition of rPlg or alpha2-antiplasmin (α2AP) expression using α2AP antisense oligonucleotides. [Figure 15] Figure 1 shows the results of a coexpression study using α2-AP. Representative SDS-PAGE gel (A) and Western blot (B) show the results of coexpression of plasminogen with either PAI-1 or α2-AP. Lanes 1-3: Plg / PAI-1, days 1, 3, and 5; Lanes 4-6: Plg / α2-AP, days 1, 3, and 5. [Figure 16]Figure 1 shows the results of coexpression studies using PAI-2 and PAI-3. A. Representative Western blot showing the results of coexpression of plasminogen with PAI-1, PAI-2, or PAI-3. Lanes 1-3: Plg / PAI-1 stable, days 1, 3, and 5. Lanes 4-6: Plg / PAI-1, days 1, 3, and 5; Lanes 7-9: Plg / PAI-2, days 1, 3, and 5; and Lanes 10-12: Plg / PAI-3, days 1, 3, and 5. B and C. Plasminogenic activity (RFU / sec) of recoverable recombinant plasminogen obtained when coexpressed with PAI-1, PAI-2, or PAI-3. Note that in 16B, the lines with almost no RFU are Plg+PAI-2 and Plg+PAI-3, whereas the curves displaying significant RFU are Plg+PAI-1stable and Plg+PAI-1. DETAILED DESCRIPTION OF THE INVENTION

[0048] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

[0049] Specific embodiments of the present invention will be described in detail below. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

[0050] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0051] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0052] For the purposes of interpreting this specification, any term in the singular where used shall include the plural and vice versa.

[0053] The expression of large amounts of protein in recombinant expression systems is a convenient method for obtaining proteins for clinical use. However, there have been significant difficulties in successfully producing intact plasminogen in various expression systems. This has been attributed to the ubiquitous presence of intracellular plasminogen activators in mammalian cells, which degrade plasminogen or cause toxicity in the cell. Expression in non-mammalian systems has also been explored, but this has limited clinical application given the formation of inclusion bodies when expressed in bacterial systems and limitations regarding proper protein folding and glycosylation in bacterial and non-mammalian eukaryotic systems. As a result of the inherent difficulty in obtaining sufficient quantities of plasminogen with the purity and activity required for clinical use, most plasminogen produced for clinical use today is obtained from fractions of human plasma.

[0054] The present inventors have developed a new approach for producing plasminogen in a recombinant system. Surprisingly, the present inventors have been able to utilize a mammalian expression system to produce significant quantities of recombinant plasminogen. Furthermore, the present inventors have demonstrated that the recombinant protein produced is biologically active and, in fact, has superior potency compared to commercially available preparations of plasminogen purified from plasma.

[0055] In addition, the method developed by the present inventors is easy to handle, and the resulting recombinant plasminogen is free of pathogenic contaminants and can be purified in a simple three-step process. Thus, the present inventors have developed a robust and simple method for obtaining functional, pure plasminogen in sufficient quantities for use in clinical settings.

[0056] The present inventors have surprisingly found that co-expression of plasminogen with plasminogen activator inhibitor (PAI-1) allows the production of large amounts of full-length functional plasminogen in recombinant mammalian cells. The yields achieved by the inventors represent a significant improvement over prior art methods for recombinant expression of plasminogen, including co-expression of other components of the plasmin / fibrinolytic pathway.

[0057] Plasminogen Plasminogen is the inactive precursor form of plasmin, the major fibrinolytic enzyme in mammals. Plasmin also plays an important role in cell migration, tissue remodeling, and bacterial invasion. Plasmin is a serine protease that preferentially cleaves Lys-Xaa and Arg-Xaa bonds with higher selectivity than trypsin. Plasminogen activators, such as tissue plasminogen activator (tPA) or Iodokinase converts the human plasminogen molecule to Arg 560 -Val 561The plasminogen cleaves the bond at the endonuclease site to produce active plasmin. The two chains of the resulting plasmin are held together by two interchain disulfide bridges. The light chain (25 kDa) contains the catalytic center (which contains the catalytic triad) and has sequence similarity to trypsin and other serine proteases. The heavy chain (60 kDa) consists of five highly similar triple-loop structures called kringles. Part of the kringles contains a lysine-binding site that mediates the plasminogen / plasmin interaction with fibrin. Plasmin belongs to the peptidase family Si.

[0058] The amino acid sequence of human Glu-Plg is provided in SEQ ID NO: 2 (see also SEQ ID NO: 6). SEQ ID NO: 16 shows the "mature" amino acid sequence, i.e. the amino acid sequence after cleavage of the signal peptide.

[0059] It will be understood that the present invention includes recombinant production of plasminogen from human and non-human sources. Thus, plasminogen produced according to the methods of the present invention may be used in any The plasminogen may comprise or consist of the amino acid sequence of a mammalian plasminogen or plasminogen variant. In any embodiment, the plasminogen is human plasminogen, non-human primate plasminogen, porcine, mouse, rat, hamster, sheep, goat, horse, cow, cat, dog, or other mammalian plasminogen. Preferably, the plasminogen is human plasminogen.

[0060] Additionally, the present invention includes the expression of functional variants of plasminogen, such as, but not limited to, those further described herein. More specifically, the present invention contemplates methods for the recombinant production of Glu-plasminogen (Glu-Plg), Lys-plasminogen (Lys-Plg), and mini-, midi-, and micro-plasminogen.

[0061] Lys-plasminogen is an N-truncated form of Glu-Plg formed from the cleavage of Glu-plasminogen by plasmin. Lys-plasminogen exhibits a higher affinity for fibrin than Glu-Plg and is more easily activated by uPA and tPA.

[0062] The amino acid sequence of human Lys-plasminogen is provided in SEQ ID NO: 9. SEQ ID NO: 17 shows the "mature" amino acid sequence, i.e., the amino acid sequence after cleavage of the signal peptide.

[0063] Midiplasminogen contains kringle regions 4 and 5 and the light chain of plasminogen (serine protease domain). It is formed by cleavage of kringle regions 1-3 from Glu-plasminogen.

[0064] The amino acid sequence of human midiplasminogen is provided in SEQ ID NO:11. SEQ ID NO: 18 shows the "mature" amino acid sequence, ie the amino acid sequence after cleavage of the signal peptide.

[0065] Miniplasminogen (also known as 442Val-Plg or neoplasminogen) is generated by the action of elastase on Glu-plasminogen at residue 442 (located within kringle region 4). Miniplasminogen therefore contains kringle region 4, kringle region 5 and part of the serine protease domain of plasminogen. The amino acid sequence of human miniplasminogen is provided in SEQ ID NO: 13. SEQ ID NO: 19 shows the "mature" amino acid sequence, i.e., the amino acid sequence after cleavage of the signal peptide.

[0066] Microplasminogen consists of the proenzyme domain of plasminogen with a stretch connecting a peptide and several residues of kringle 5 attached to its N-terminus. It is produced by the action of plasmin on plasminogen. Thus, microplasminogen (or microPlg) is a protein derived from plasminogen (a serine protease). It contains a light chain of the ATPase domain (the ATPase domain) and does not contain a kringle region. (See, e.g., Shi et al. (1980) J. Biol. Chem. 263:17071-5.) Like plasminogen, microplasminogen is activated by tPA and urokinase to form a molecule with proteolytic activity. Human microplasmin has a molecular weight of approximately 29 kDa and has a lower affinity for fibrin than plasmin.

[0067] The amino acid sequence of human microplasminogen is provided in SEQ ID NO: 15. SEQ ID NO: 20 shows the "mature" amino acid sequence, i.e., the amino acid sequence after cleavage of the signal peptide.

[0068] Other variants: e.g., variants of plasminogen that contain modifications or mutations at the lysine binding sites found in the kringle region. It will be understood that the methods of the present invention themselves result in the expression of any of a number of plasminogen variants, including but not limited to, recombinant plasminogen having modifications at one or more sites.

[0069] PAI-1 Plasminogen activator inhibitor-1 (PAI-1) also acts as an endothelial plasminogen activator. PAI-1, also known as fibrinolysis inhibitor or serpin E1, is a protein encoded by the SERPINE1 gene in humans. PAI-1 is a serine protease inhibitor (serpin) that functions as a primary inhibitor of the plasminogen activators tissue plasminogen activator (tPA) and urokinase (uPA). Thus, PAI-1 is one or the primary inhibitor of fibrinolysis in vivo.

[0070] Other plasminogen activator inhibitors include plasminogen activator inhibitor-2 (PAI-2), protein C inhibitor (PAI-3), and protease nexin-1 (SERPINE2), which act as inhibitors of tPA and urokinase. However, the inventors have found that the methods of the present invention are particularly useful when PAI-1 or a variant thereof is co-expressed with plasminogen.

[0071] The amino acid sequence of human PAI-1 in which the sequence has been modified at Q197 and G355 to introduce cysteine ​​residues is provided in SEQ ID NO:4.

[0072] Exemplary nucleic acid and amino acid sequences for PAI-2 and PAI-3 are provided in NCBI accession numbers NM_002575.3 and NM_000624.6, respectively.

[0073] It will be understood that the present invention also contemplates the use of "wild-type" PAI-1 (ie, the sequence is unaltered at residues 197 or G355 as shown in SEQ ID NO:4).

[0074] As used herein, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide is used synonymously with "variant." Variants with respect to a given reference sequence may include naturally occurring allelic variants. A "variant" includes any protein or amino acid sequence containing at least one amino acid mutation compared to the wild-type. Mutations can include substitutions, insertions, and deletions. Preferably, a variant retains the ability to inhibit plasminogen activators to a level equivalent to or at least about 99%, 98%, 97%, 96%, 96%, 0.4%, 93%, 92%, 91%, 90%, 85%, or 80% of the wild-type. For example, a PAI-1 variant retains the ability to inhibit plasminogen activators to a level at least about 99%, 98%, 97%, 96%, 96%, 0.4%, 93%, 92%, 91%, 90%, 85%, or 80% of the wild-type PAI-1. The wild-type PAI-1 can be any of those described herein, including SEQ ID NO: 4. In one embodiment, the PAI-1 variant is not PAI-2 or PAI-3. Preferably, the PAI-1 variant has greater potency in inhibiting tPA and / or uPA compared to PAI-2, PAI-3, or PAI-2 and PAI-3.

[0075] nucleic acid An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source. An isolated nucleic acid molecule is present in a form or setting other than that in which it is found in nature. Thus, an isolated nucleic acid molecule is one that is different from the form or setting in which it is found in natural cells. However, isolated nucleic acid molecules include nucleic acid molecules ordinarily contained in cells that express, for example, plasminogen, where, for example, the nucleic acid molecule is in a chromosomal configuration that is different from that of natural cells.

[0076] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or purified form. A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the present invention, such nucleic acid sequences include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0077] The polynucleotides of the present invention can be synthesized according to methods well known in the art, for example as described in Sambrook et al. (1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

[0078] The polynucleotide molecules of the present invention may be provided in the form of an expression cassette, which contains a control sequence operably linked to the inserted sequence, thereby allowing the expression of the polypeptide of the present invention in vivo in the targeted subject. These expression cassettes are then typically provided within a vector (e.g., a plasmid or recombinant viral vector) suitable for use as a reagent for nucleic acid immunization. Such expression cassettes may be administered directly to the host subject. Alternatively, a vector containing the polynucleotide of the present invention may be administered to the host subject. Preferably, the polynucleotide is prepared and / or administered using a genetic vector. A suitable vector may be any vector capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptide of the present invention.

[0079] The present invention therefore includes expression vectors comprising such polynucleotide sequences.

[0080] It will further be understood that the compositions and products of the invention may comprise a mixture of polypeptides and polynucleotides. Thus, the invention provides a composition or product as defined herein in which, in place of any one of the polypeptides, there is present a polynucleotide capable of expressing said polypeptide.

[0081] Expression vectors are constructed routinely in the field of molecular biology and may include, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements that may be necessary and positioned in the correct orientation for expressing the peptide of the invention, such as polyadenylation signals, etc. Other suitable vectors will be apparent to those skilled in the art.

[0082] Thus, the methods of the present invention include the steps of delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, the polynucleotide for use in the present invention in a vector is capable of effecting expression of the coding sequence by the host cell. operably linked to the control sequence, i.e. the vector is an expression vector.

[0083] "Operably linked" refers to the arrangement of elements designed so that the components so described perform their normal functions. Thus, a given regulatory sequence, e.g., a promoter, operably linked to a nucleic acid sequence is capable of directing the expression of that sequence when the appropriate enzymes are present. The promoter need not necessarily be contiguous with the sequence, so long as it functions to direct the expression of the sequence. Thus, for example, intervening untranslated but transcribed sequences can exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0084] Various expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include a transcription promoter sequence and transcription start and stop sequences. The vectors of the present invention may be, for example, plasmid, virus, or phage vectors, which are provided with an origin of replication, an optional promoter for the expression of the polynucleotide, and optionally a regulator of the promoter. A "plasmid" is a vector in the form of an extrachromosomal genetic element. The vector may contain one or more selectable marker genes, such as an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example, for producing DNA or RNA, or to transfect or transform host cells, such as mammalian host cells. Vectors may also be adapted for use in vivo, for example, to allow expression of a polypeptide in vivo.

[0085] A "promoter" is a nucleotide sequence that initiates and controls transcription of a polynucleotide encoding a polypeptide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms "promoter" or "control element" are intended to include full-length promoter regions and functional (e.g., transcriptional or translational) segments of these regions.

[0086] As used herein, the term "promoter" shall be interpreted in its broadest sense and includes the transcriptional regulatory sequences of genomic genes, including the TATA box or initiation element required for accurate transcription initiation, which may or may not have additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers, and silencers) that alter nucleic acid expression, for example, in response to developmental and / or exogenous stimuli or in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe a recombinant, synthetic, or fusion nucleic acid, or derivative, that confers, activates, or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial and / or temporal expression of the nucleic acid.

[0087] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-alpha promoter (EF1), the small nuclear RNase I promoter (SRNase I), and the human ER promoter (ERI). A promoter (U1a and U1b), α-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus promoter These include late promoters, β-actin promoters; hybrid regulatory elements containing the CMV enhancer / β-actin promoter or immunoglobulin promoter, or active fragments thereof. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); the human embryonic kidney line (suspension culture medium); These cells are 293 or 293 cells subcloned for growth in culture; baby hamster kidney cells (BHK, ATCC CCL10); or Chinese hamster ovary cells (CHO).

[0088] A polynucleotide, expression cassette or vector according to the invention may additionally comprise a signal peptide sequence, which is generally inserted in operable linkage with a promoter, thereby facilitating the expression of the signal peptide and secretion of a polypeptide encoded by a coding sequence that is also operably linked with the promoter.

[0089] Typically, a signal peptide sequence encodes a peptide of 10 to 30 amino acids, e.g., 15 to 20 amino acids. Often, the amino acids are predominantly hydrophobic. In a typical situation, the signal peptide targets a growing polypeptide chain to which the signal peptide is attached to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved in the endoplasmic reticulum, allowing secretion of the polypeptide via the Golgi apparatus. Thus, the peptides of the present invention can be delivered to an individual by expression from cells within the individual and secretion from those cells.

[0090] Any suitable expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, NY: 1985)) and corresponding suitable host can be employed for the production of recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species in the genera Escherichia, Bacillus, Pseudomonas, and Salmonella; mammalian; or insect host cell systems, including baculovirus systems (e.g., as described by Luckow et al., Bio / Technology 6: 47 (1988)); and established cell lines, such as COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines. Those skilled in the art will recognize that the choice of expression host has ramifications for the type of polypeptide produced. For example, yeast or mammalian cells (e.g., COS-7 cells) can be used. Glycosylation of polypeptides produced in bacterial cells, such as Escherichia coli, is expected to differ from that of polypeptides produced in bacterial cells, such as Escherichia coli.

[0091] Polypeptides "Isolated," as used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are typically substances that would be expected to interfere with diagnostic or therapeutic uses of the polypeptide, and examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated protein will be free of at least one component of the polypeptide's natural environment, and therefore includes the polypeptide in situ within a recombinant cell. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.

[0092] A "fragment" is a portion of a polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as a polypeptide of the present invention, as can be determined using the assays described herein.

[0093] "Percent (%) amino acid sequence identity" or "percent (%) identical" thereto with respect to a polypeptide sequence, i.e., a polypeptide of the invention as defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not take into account any conservative substitutions as part of the sequence identity.

[0094] Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared (non-limiting examples are described below). When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or containing a particular percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) can be calculated as follows: percent amino acid sequence identity = X / Y100, where X is the number of amino acid residues scored as identical matches by the alignment of A and B by a sequence alignment program or algorithm, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A.

[0095] In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. MoI. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped Alignment can be used. BLAST (in BLAST 2.0) was performed according to Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment can also be performed manually by visual inspection. Another non-limiting example of a mathematical algorithm used to compare sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns entire amino acid or DNA sequences, thereby providing data on sequence conservation across the entire amino acid sequence. The ClustalW algorithm is used in a number of commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen, Carlsbad, CA). After alignment of amino acid sequences using ClustalW, percent amino acid identity can be assessed. Non-limiting examples of software programs useful for analyzing ClustalW alignments are GENEDOC™ or JalView (http: / / www.jalview.org / ). GENEDOC™ is a method for identifying amino acid (or DNA) similarities between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such algorithms are available from GCG Wisconsin Group. The ALIGN program (version 10) is part of the genomics software package (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0096] The polypeptide desirably comprises an amino terminus and a carboxyl terminus. The polypeptide may comprise D-amino acids, L-amino acids, or a mixture of D- and L-amino acids. However, the D-form of amino acids is particularly preferred, as polypeptides composed of D-amino acids are predicted to retain their biological activity to a greater extent in vivo.

[0097] Polypeptides can be prepared by any of a number of conventional techniques. Polypeptides can be isolated or purified from naturally occurring sources or from recombinant sources. Recombinant production is preferred. For example, in the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be subcloned into an appropriate vector using well-known molecular genetic techniques (e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd ed. (see Cold Spring Harbor Laboratory, 1989) (See references). In vitro, the fragment can be transcribed and then translated into a polypeptide. Commercially available kits can also be employed (e.g., those manufactured by Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, NJ; InVitrogen, Carlsbad, Calif., etc.). Polymerase chain reaction is optionally and can be employed in the manipulation of nucleic acids.

[0098] The term "conservative substitution," as used herein, refers to the replacement of an amino acid present in the native sequence of a peptide with a naturally occurring or non-naturally occurring amino acid or a peptidomimetic with similar steric properties. If the side chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be made with a naturally occurring amino acid, a non-naturally occurring amino acid, or a peptidomimetic moiety that is also polar or hydrophobic (in addition to having the same steric properties as the side chain of the replaced amino acid).

[0099] Conservative amino acid substitution tables showing functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that can be considered conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0100] Naturally occurring amino acids are typically grouped according to their properties, and conservative substitutions with naturally occurring amino acids can be determined by keeping in mind that the replacement of a charged amino acid with a spatially similar uncharged amino acid is considered a conservative substitution. Amino acid analogs (synthetic amino acids) well known in the art can also be used to generate conservative substitutions with non-naturally occurring amino acids. Peptide mimetics of naturally occurring amino acids are well documented in the literature known to those skilled in the art, and non-natural or unnatural amino acids are further described below. When affecting a conservative substitution, the substituting amino acid should have the same or similar functional group in the side chain as the original amino acid. It is.

[0101] The phrase "non-conservative substitution" or "non-conservative residue," as used herein, refers to the replacement of an amino acid present in a parent sequence with another naturally occurring or non-naturally occurring amino acid that has different electrochemical and / or steric properties. Thus, the side chain of the substituting amino acid may be significantly larger (or significantly smaller) than the side chain of the naturally occurring amino acid being substituted and / or may have a functional group with significantly different electronic properties than the amino acid being substituted. Examples of this type of non-conservative substitution include phenylalanine or cyclohexylmethyl guanine in place of alanine. Examples include substitutions of isoleucine for lysine, glycine with -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Non-conservative substitutions include any mutation that is not considered conservative.

[0102] Non-conservative amino acid substitutions can be due to changes in (a) the structure of the amino acid backbone in the area of ​​substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of the amino acid side chain. In general, substitutions predicted to produce the greatest changes in protein properties are those in which (a) a hydrophilic residue is substituted for a hydrophobic residue (or vice versa); (b) proline is substituted for any other residue (or vice versa); (c) a residue with a bulky side chain, such as phenylalanine, is substituted for a residue without a side chain, such as glycine (or vice versa); or (d) a residue with a positively charged side chain, such as lysyl, arginyl, or histidyl, is substituted for a negatively charged residue, such as glutamyl or aspartyl (or vice versa).

[0103] Alterations to the native amino acid sequence, e.g., by insertion, deletion, and / or substitution, to generate mutant polypeptides can be accomplished by various means known to those skilled in the art. For example, site-specific mutations can be introduced by ligating synthetic oligonucleotides containing modified sites into an expression vector. Alternatively, Alternatively, oligonucleotide-directed site-directed mutagenesis procedures, such as those disclosed in Walder et al., Gene 42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Patent Nos. 4,518,584 and 4,737,462, may be used. A preferred means for introducing mutations is the QuikChange site-directed mutagenesis kit (Stratagene, LaJolla, Calif.).

[0104] The terms "N-terminal" and "C-terminal" are used herein to designate the relative position of any amino acid sequence or polypeptide domain or structure to which they are applied. The relative positioning will be clear from the context; that is, an "N-terminal" feature will be located at least closer to the N-terminus of a polypeptide molecule than another feature discussed in the same context (the other feature can be referred to as "C-terminal" to the first feature). Similarly, the terms "5'-" and "3'-" can be used herein to designate the relative position of polynucleotide features.

[0105] Recombinant polypeptides produced according to the methods of the invention may also be modified, conjugated, or fused to other moieties to facilitate purification of the polypeptide or for use in immunoassays using methods known in the art. For example, polypeptides of the invention may be modified by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, etc.

[0106] Modifications contemplated herein include, but are not limited to, modifications to the side chains, These include incorporating unnatural amino acids and / or their derivatives during polypeptide synthesis, and using cross-linking agents and other methods to impose conformational constraints on the polypeptides of the present invention.Any modification is contemplated herein, including post-translational modifications that reduce the ability of molecules to form dimers.Examples include modifications incorporated by click chemistry, as known in the art.Exemplary modifications include glycosylation.

[0107] Examples of side chain modifications contemplated by the present invention include modification of amino groups, such as reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and modification of amino groups by pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

[0108] The guanidine groups of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0109] The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide.

[0110] Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation of performic acid to cysteic acid; formation of hybrid disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides; formation of mercury derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonate, phenylmercuric chloride, 2-chloromercuri-4-nitrophenol and other mercurials; and carbamoylation with cyanate at alkaline pH.

[0111] Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides, while tyrosine residues may be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0112] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

[0113] Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or the use of D-isomers of amino acids. Table 1 provides a list of unnatural amino acids contemplated herein.

[0114] [Table 1-1]

[0115] [Table 1-2]

[0116] The crosslinker may be, for example, a homobifunctional crosslinker, such as (CH2) n Bifunctional imidoesters with spacer groups (where n=1 to n=6), glutaraldehyde, N-hydroxysuccinimide esters, and heterobifunctional reagents that typically contain an amino-reactive moiety, e.g., N-hydroxysuccinimide, and another group-specific reactive moiety, can be used to stabilize the 3D conformation.

[0117] As used herein, the N-terminal domain "homologous to the kringle region of native human plasminogen" Polypeptides referred to herein as having an N-terminal domain "homologous to kringle 1" exhibit structural and functional characteristics similar to the native kringle region of plasminogen. Furthermore, polypeptides referred to herein as having an N-terminal domain "homologous to kringle 1" exhibit characteristics similar to native kringle 1, at least to the extent that the polypeptide may have a higher affinity for o-aminocarboxylic acids (and functional homologs, e.g., trans-4-aminomethylcyclohexane-1-carboxylic acid, a cyclic acid) than for kringle 5. Conditions and protocols for comparing the binding of isolated kringle region polypeptides to aminopentanoic acid (5-APnA); 6-aminohexanoic acid (6-AHxA), also known as epsilon-aminocaproic acid (EACA); 7-aminoheptanoic acid (7-AHpA); and trans-4 aminomethylcyclohexane-1-carboxylic acid (t-AMCHA) are described in detail in the literature. See, e.g., Chang, Y. et al., Biochemistry 37:3258-3271 (1998), incorporated herein by reference. References to kringle regions "homologous to kringle 4" are similarly defined as above with respect to the phrase "homologous to kringle 1." That is, they exhibit functional characteristics similar to kringle 4 of native human plasminogen, as discussed above. These polypeptides also bind to immobilized lysines, as described above.

[0118] The polypeptides produced according to the methods of the present invention bind to immobilized ricin. As used herein, the phrase "binds to immobilized ricin" means that when subjected to column chromatography using ricin-Sepharose as a chromatographic medium, the polypeptides so characterized proceed more slowly than proteins that do not bind to ricin. Typically, the polypeptides of the present invention can be eluted from such chromatographic medium (ricin affinity resin) using a solution containing a specific ligand, such as EACA, as an eluent.

[0119] cell culture Those skilled in the art will be familiar with standard methods for transfecting host cells, such as mammalian cells, with nucleic acid vectors and culturing the host cells under conditions suitable for expressing the genes encoded by the vector. Representative methods for transfecting and culturing mammalian cells to produce recombinant proteins are described, for example, in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).

[0120] Means for introducing isolated nucleic acids, vectors, or expression constructs containing same into cells for expression are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into cells include, among others, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection using, for example, lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and transfection using, for example, DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA). ) to bombard microparticles.

[0121] The host cells used in accordance with the present invention can be cultured in a variety of media depending on the cell type used. Commercially available media, such as Ham's F10 (Sigma), minimum Essential medium (MEM, Sigma), RPM1-1640 (Sigma), and Dulbecco's modified Eagle's medium The medium (DMEM, Sigma) is suitable for culturing mammalian cells. Media for culturing other cell types are known in the art.

[0122] Additionally, one of skill in the art would be familiar with methods for purifying expressed recombinant proteins from cell culture medium, such as using size exclusion and affinity chromatography methods, and combinations thereof.

[0123] If the protein is secreted into the culture medium, the supernatant from such an expression system is first filtered through a commercially available protein concentration filter, e.g., Amicon or Millipore. The supernatant may be concentrated using a Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants. Alternatively, or in addition, the supernatant may be filtered and / or separated from the protein-expressing cells, for example, using continuous centrifugation.

[0124] Proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., lysine affinity columns), or any combination of the foregoing. These methods are known in the art and are described, for example, in WO 99 / 57134 or in Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0125] Those skilled in the art will also recognize that proteins can be modified to include tags to facilitate purification or detection, such as a polyhistidine tag, e.g., a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein may then be purified using methods known in the art, such as affinity purification. For example, a protein containing a hexa-His tag may be purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-His tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively, or in addition, a ligand or antibody that binds to the tag may be used in the affinity purification method.

[0126] Assay of recombinant plasminogen activity Recombinant plasminogen (or recombinant plasmin derived therefrom) produced according to the present invention can be evaluated for biological activity using standard methods known in the art and as described later in the Examples herein.

[0127] For example, recombinant plasminogen can be converted to plasmin via cleavage with tPA or uPA using standard techniques. Cleavage of recombinant plasminogen with tPA occurs at residues Glu1 to Arg, respectively. 561 and Val 562 ~Asn 791 (For examples of methods for converting plasminogen to plasmin, see Chapter 20 of Mutch and Booth, Hemostasis and Thrombosis: Basic Principles and Clinical Practice, by Victor J. Marder, William C. Aird, Joel S. Bennett, Sam Schulman, and II Gilbert C. White, incorporated herein by reference.)

[0128] Furthermore, the ability of recombinant plasminogen to bind to physiological binding targets or ligands , can be assessed using conventional techniques. For example, binding to recombinant plasminogen (or plasmin derived therefrom) can be assessed with respect to binding to alpha2-antiplasmin (α2-AP) and streptokinase. Methods for assessing binding to α2-AP and streptokinase are described, for example, in Horvath et al. (2011) Methods in Enzymology, 501:223-235 and Zhang et al. (2012) Journal of Biological Chemistry, 287:42093-42103, respectively, the contents of which are incorporated herein by reference.

[0129] The binding of recombinant proteins produced according to the invention to cell surface receptors, such as mammalian plasminogen receptors, can be determined, for example, as described in Example 4.

[0130] Finally, the therapeutic efficacy of recombinant proteins produced according to the present invention can be assessed according to standard techniques, including those utilized for the assessment of the quality of plasminogen and plasmin isolated from human and non-human plasma.

[0131] composition Recombinant plasminogen (or plasmin derived therefrom) can be provided in the form of a pharmaceutically acceptable composition for administration to an individual in need thereof. For example, recombinant proteins produced according to the present invention find use in treating wounds (e.g., skin wounds such as abrasions and burns, bone wounds such as fractures, muscle injuries), providing plasminogen replacement in traumatic injury situations, plasminogen replacement therapy, in the presence of congenital defects, and in the treatment of heterotopic ossification and dystrophic mineralization.

[0132] In some instances, the recombinant plasminogen (or plasmin derived therefrom) described herein can be administered parenterally, topically, intraventricularly, via a reservoir implanted in a dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier, or by any other convenient dosage form. The term "parenteral," as used herein, includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0133] Methods for preparing recombinant plasminogen in a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the United States Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0134] The pharmaceutical compositions of this disclosure are particularly useful for parenteral administration, e.g., intravenous administration or administration into a body cavity, organ, or joint cavity. Compositions for administration will generally comprise a solution of plasminogen dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. The compositions may contain pharmaceutically acceptable adjuvants, such as pH adjusters and buffers, toxicity control agents, and the like, as needed to approximate physiological conditions, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the plasminogen of the present disclosure in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as mixed oils and ethyl oleate, can also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity or chemical stability, e.g., buffers and and preservatives.

[0135] Upon formulation, recombinant plasminogen produced in accordance with the present disclosure will be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically / prophylactically effective. [Example]

[0136] Example 1: Transient expression of recombinant plasminogen material Expi293 Expression Medium (Cat. No. A1435101), Erlenmeyer flask, phosphate-buffered saline (1x), glucose (300 mg / mL), polyethyleneimine (PEI) (1 mg / mL), Lupin (125 g / L), Glutamax (100x), pcDNA3.1Plg (0.5 μg per mL of cell culture), pcDNA3.1PAI-1 (0.5 μg per mL of cell culture).

[0137] method Day 1: Dilute cells before transfection.

[0138] Day 2: Add DNA diluted in PBS and PEI to the cells. Incubate the cells at 37°C, 5% CO, and 110-140 rpm.

[0139] Days 3–7: Adjust lupin and glucose levels in the culture medium.

[0140] Day 8: Harvest the medium by centrifuging the culture at 2000 xg for 15 minutes at 4°C.

[0141] Example 2: Stable expression of recombinant plasminogen material Expi293 expression medium (catalog no. A1435101) supplemented with 4 g / L lupin and 125 mg / L geneticin (G418), Erlenmeyer flasks, glucose (300 mg / mL), and glutamax (100x).

[0142] method ·Expi293 cells were transfected with pcDNA3.1Plg IRES PAI-1. The cells were then passaged in the presence of the selection antibiotic geneticin. Geneticin-resistant clones were isolated in 96-well plates (one colony per well). ·Streptokinase was used to test for the presence of secreted Plg.

[0143] Typical yields from transient expression are 30-50 mg per L of cell culture. Typical yields from stably transfected Expi293 cells (suitable for large-scale expression) are approximately 80-100 mg per L of cell culture.

[0144] Example 3: Purification of recombinant plasminogen For every 100 mL of clarified supernatant, add 20 mL of 0.5 M NaH2PO4, 5 g of glycerol, and one Roche protease inhibitor tablet (aprotinin, bestatin, calpain inhibitors I and II, chymostatin, E-64, leupeptin, pefabloc SC / PMSF, pepstatin, TLCK-HCl, trypsin inhibitor, antipain dihydrochloride, phosphoramidite). Add the phosphoramidon (containing phosphoramidon) and mix.

[0145] 1. Lysine affinity column Buffer A: 100 mM NaHPO, pH 8.0, 5% glycerol, 0.02% azide Buffer B: 100 mM NaHPO, pH 8.0, 25 mM EACA (epsilon aminocaproic acid), 5% glycerol CV = column volume. a) Use 20 mL of Lysine Hyper D resin per 100 mL of culture supernatant. b) In a gravity flow column, wash the resin with 2 CV of MQ H2O and equilibrate with 2 CV of Buffer A. c) Add the equilibrated lysine resin to the clarified medium (from step 1) and allow to batch bind for 1 hour at 4°C. d) Allow the medium to pass through and collect the flow-through. e) Wash the resin with 2 CV of buffer A. f) Elute with Buffer B, typically half the resin volume at a time (e.g., 10 mL fractions for 20 mL of resin). Determine the elution endpoint using Bradford reagent. g) Fractionate on 10% SDS-PAGE. h) Pooling fractions for the next purification step.

[0146] Figure 1 shows an image of a representative Coomassie-stained 10% SDS-PAGE gel of fractions eluted from the lysine affinity column. The approximately 100 kDa band represents eluted rPlg after transient expression.

[0147] 2. HiTrap Q FF Buffer A: 50 mM Tris, pH 9.0, 5 mM EACA, 10% glycerol, 30 mM NaCl, 0.02% azide Buffer B: 50 mM Tris, pH 9.0, 5 mM EACA, 10% glycerol, 1 M NaCl, 0.02% azide. a) Concentrate the sample to 5 ml (50K MWCO), keep the pre-column sample and dilute to 50 ml using Buffer A. b) Pre-equilibrate the HiTrap Q with 5 CV MQ H2O and 5 CV Buffer A. c) Load the sample at 1 ml / min and collect the effluent. d) Wash with 5 CV of buffer A. e) Elution gradient: - 0 to 25% B in 20CV (100ml) - 20% to 100% B at 0CV - 100% for 2CV.

[0148] Plg typically elutes as a predominant peak at approximately 10% B. Purity is determined by running a gel and fractions are pooled.

[0149] The pooled fractions are dialyzed overnight at 4° C. in 25 mM Tris, pH 7.4, 150 mM NaCl, 5% glycerol. Dialysis is repeated for an additional 2 hours to ensure removal of EACA.

[0150] Figure 2 shows representative results of anion exchange chromatography of rPlg.

[0151] 3. Gel filtration S200 16 / 60 Buffer: 25 mM Tris, pH 7.4, 150 mM NaCl, 5% glycerol, 1 mM sodium EDTA, 0.02% azide, 1x Roche protease inhibitor cocktail. (Tris can be substituted with Hepes or Na2HPO4). a) Concentrate to at least 5 ml and gel filter on Superdex 200 16 / 60. b) The elution volume is approximately 73 ml. c) Run the gel and pool relevant fractions.

[0152] Plasminogen can be concentrated to 15 mg / ml in a 50K MWCO concentrator in the presence of 5% glycerol.

[0153] Plasminogen can be stored frozen after snap freezing in liquid N2.

[0154] Figure 3 shows representative results of size exclusion chromatography of rPlg.

[0155] Mini-plg and micro-plg were also successfully expressed and purified using the methods described herein and shown to be active (data not shown).

[0156] Example 4: Qualitative evaluation of recombinant plasminogen 1. rPlg can be activated to plasmin (rPlm) Figure 4 shows a Coomassie-stained 12% SDS-PAGE showing tPA-cleaved recombinant plasmin (rPlm) resulting from cleavage of rPlg by tPA. Under reducing conditions, rPlm is cleaved at residues Glu1 to Arg, respectively. 561 and Val 562 ~Asn 791 It is separated into heavy and light chains composed of

[0157] FIG. 5 shows a progress curve demonstrating tPA activation of 500 nM rPlg as measured by hydrolysis of the plasmin fluorogenic substrate H-Ala-Phe-Lys-AMC.

[0158] 2. rPlg is more similar to natural Plg GI Figure 6 shows tPA activation of native Plg glycoform 1 (G1I), Plg glycoform II (G2I), and rPlg. rPlg was obtained according to the methods described herein. Native Plg glycoforms I and II were obtained in-house and purified from human plasma using standard techniques.

[0159] The results showed that rPlg was the most readily activatable of the three forms tested (lowest K m and the highest V max indicates that the

[0160] [Table 2]

[0161] Figure 7 shows that the open conformation of Plg is induced by the presence of 20 mM EACA. In the closed form, glycoform I and rPlg have a larger radius of gyration (R) similar to that of glycoform II. g(The radius of gyration of a body about an axis of rotation is defined as the radial distance from the axis of rotation of the point at which, if the entire mass of the body were assumed to be concentrated, its moment of inertia about that given axis would be expected to be the same as for its actual mass distribution. X-ray scattering also reveals the excess dimensions of macromolecules rotating in solution. It can be used to measure the R. Plg can assume both closed and open conformations. The dimensions of the open form are very similar. In the closed form, there is a small R. g indicates small, closely packed molecules. This measurement is used as an indicator of how stable / well packed the closed conformation is.

[0162] [Table 3]

[0163] SAXS titration studies (Figure 8) measured the conformational changes of Plg in response to EACA. The titration curves are similar for glycoforms I and II and rPlg. rPlg most readily adopts the open form. K open is the EACA concentration at which 50% of Plg is in the open conformation.

[0164] [Table 4]

[0165] 3. rPlm binds to alpha2-antiplasmin (α2-AP), a Plm-specific inhibitor. Recombinant α2-AP was immobilized on a Ni2+-NTA chip, and the binding of native or recombinant Plg and Plm was monitored in real time. Figures 9 and 10 show sensorgrams demonstrating the binding of rPlg and rPlm, as well as native Plg and Plm, to alpha2-antiplasmin (α2-AP).

[0166] rPlg and rPlm were obtained by the methods described herein: Native Plg was purchased from Merck (purified from human plasma) and native Plm was purchased from Haematologic Technologies.

[0167] 4. rPlg and rPlm are strains derived from Streptococcus pyrogenes. Binds to streptokinase Recombinant SK was immobilized on a Ni2+-NTA chip, and the binding of native or recombinant Plg and Plm was monitored in real time. rPlg and rPlm were obtained by the methods described herein. Native Plg was purchased from Merck (purified from human plasma), and native Plm was purchased from Haematologic Technologies. The results are shown in Figures 11 and 12.

[0168] All experiments were fitted using a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB) (except for the α2-AP / native Plg case, where a two-state reaction model was used to describe the conformational change of Plg glycoform II upon binding). The reaction rates (k a and k d ) and affinity constant (K D ) are summarized.

[0169] [Table 5]

[0170] The above results demonstrate that recombinant plasminogen (or plasmin derived therefrom) produced according to the methods of the present invention binds to physiologically relevant binding partners with affinity similar to or greater than that of commercially available preparations of native plasminogen / plasmin purified from plasma.

[0171] 5. rPlg binds to mammalian Plg receptors Briefly, HEK293 cells were resuspended in PBS-EDTA + 2% FCS and then 5 × 10 cells were added per sample. 5 The cells were incubated with 5 μg / mL of nPlg or rPlg and 10 μg / mL of Alexa-488-labeled Plg antibody for 30 minutes. The median fluorescence intensity of at least 10,000 events was measured using a FACSCalibur (BD Biosciences) flow cytometer in the FL1 channel (488 nm laser excitation source). Figure 13 shows the binding of rPlg to the plasminogen receptor in HEK293 cells.

[0172] 6. rPlg accumulates at the site of bone and muscle injury and reduces dystrophic mineralization after injury Figure 14 shows the results after rPlg injection following bone and muscle injury. A. The top panel shows that rPlg accumulates at the fracture site in bone. Alexa fluor-labeled fibrin and rPlg were injected IP. The image shows the accumulation of fibrin and rPlg at the fracture site. B. The bottom panel shows that rPlg accumulates at the site of muscle injury. Cardiotoxin was injected into the right leg to induce muscle injury, and rPlg was administered IP at 1 mg / day. The image shows the accumulation of rPlg in the injured leg and kidney, but not in the uninjured leg / kidney.

[0173] B. The upper panel also shows rPlg accumulation at the site of muscle injury. Cardiotoxin was injected into the leg to induce muscle injury. rPlg labeled with Alex Fluor dye was injected IP at 1 mg / day, and images were recorded 1 to 7 days after injury. Lower panel: rPlg accumulation at the site of injury prevents muscle calcification in Plg+ / - animals. Cardiotoxin was injected into the leg to induce muscle injury. rPlg was injected IP at 1 mg / day, and images were recorded 7 days after injury. Muscle calcification is evident in Plg+ / - but not WT animals and can be rescued by inhibition of rPlg or alpha2-antiplasmin (α2AP) expression using α2AP antisense oligonucleotides. .

[0174] Example 5: Co-expression of recombinant plasminogen with other inhibitors Expi293 cells were transfected with constructs encoding: · recombinant Plg / α2-AP; Recombinant Plg / PAI-1 stable ; · Recombinant Plg / PAI-1; · Recombinant Plg / PAI-2; Recombinant Plg / PAI-3.

[0175] All constructs were premixed in a 1:1 (w / w) ratio before transfection.

[0176] The expression levels of Plg at 1, 3, and 5 days after transfection were determined by Western blot using anti-Plg antibody.

[0177] The results shown in Figures 15 and 16 demonstrate that the yield of recombinant Plg was significantly higher when expressed with stably transfected or transiently transfected PAI-1 compared with when expressed with either α2-AP, PAI-2, or PAI-3.

[0178] Conclusion: PAI-1 is important for the expression of recombinant Plg. Results from experiments comparing the expression of Plg when co-expressed with α-AP indicate that inhibition of Plm activation is more important than inhibition of Plm activity to maximize protein yield.

[0179] Sequence information Exemplary nucleic acid sequence of human plasminogen (SEQ ID NO: 1) ACCAGGAAGTGAACCTGGAACCTCACGTGCAGGAAATCGAGGTGTCCAGACTGTTCTGGAACCCACCCGGAAGGATATCGCCCTGCTGAAGCTGAGCAGCCCTGCCGTGATCACCGACAAAGTGATT CCCGCCTGCCTGCCCAGCCCCAACTATGTGGTGGCCGACAGAACCGAGTGCTTCATCACCGGCTGGGGCGAGACACAGGGCACATTTGGAGCCGGCCTGCTGAAAGAGGCCCAGCTGCCTGTGATCGAG AACAAAGTGTGCAACCGCTACGAGTTCCTGAACGGCAGAGTGCAGAGCACCGAGCTGTGTGCCGGACATCTGGCTGGCGGCACAGATAGCTGTCAGGGCGATTCTGGCGGCCCTCTCGTGTGCTTCGAG AAGGACAAGTACATCCTGCAGGGCGTGACCAGCTGGGGCCTGGGATGTGCCAGACCTAACAAGCCCGGCGTGTACGTGCGCGTGTCCAGATTTGTGACCTGGATCGAGGGCGTGATGCGGAACAACTGA

[0180] An exemplary amino acid sequence of human plasminogen (SEQ ID NO: 2) with the signal peptide underlined. MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHA HGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPE NYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0181] Exemplary nucleic acid sequence of recombinant PAI-1 (SEQ ID NO:3). Mutations to improve serpin stability include: Q197C and G355C (underlined in the sequence below) (according to Chorostowska-Wynimko J et al. (2003) Molecular Cancer Therapeutics. 2003;2(1):19-28. doi: 10.1186 / 1476-4598-2-19). ATGCAGATGTCTCCCGCCCTGACCTGCCTGGTGCTGGGCCTGGCCCTGGTGTTCGGAGAGGGCTCTGCCGTGCACCACCCACCTAGCTACGTGGCACACCTGGCCTCCGACTTCGGCGTGAGGGTGTTTCAGCAGGTGGCCCAGGCC AGCAAGGATCGCAACGTGGTGTTCAGCCCTTATGGCGTGGCCTCCGTGCTGGCCATGCTCCAGCTGACCACAGGAGGAGAGACCCAGCAGCAGATCCAGGCAGCTATGGGCTTCAAGATCGACGATAAGGGAATGGCACCCGCCCTG AGGCACCTGTACAAGGAGCTGATGGGCCCTTGGAATAAGGACGAGATCAGCACCACAGATGCCATCTTTGTGCAGCGCGACCTGAAGCTGGTGCAGGGCTTCATGCCACACTTCTTTCGGCTGTTCCGGAGCACCGTGAAGCAGGTG GACTTCAGCGAGGTGGAGAGGGCCCGCTTTATCATCAACGATTGGGTGAAGACCCACACAAAGGGCATGATCAGCAATCTGCTGGGCAAGGGAGCAGTGGATCAGCTGACCAGGCTGGTGCTGGTGAACGCCCTGTACTTCAATGGC TGCTGGAAGACCCCATTTCCCGACAGCTCCACACACCGGAGACTGTTCCACAAGTCCGATGGCTCTACAGTGAGCGTGCCTATGATGGCCCAGACCAACAAGTTCAATTATACAGAGTTTACCACACCTGACGGCCACTACTATGACATCCTGGAGCTGCCATACCACGGCGACACCCTGAGCATGTTTATCGCCGCCCCTTATGAGAAGGAGGTGCCACTGTCCGCCCTGACAAACATCCTGTCCGCCCAGCTGATCTCTCACTGGAAGGGCAATATGACCAGGCTGCCAAGGCTGCTGGTGCTGCCTAAGTTCTCCCTGGAGACAGAGGTGGACCTGCGGAAGCCTCTGGAGAACCTGGGCATGACCGATATGTTCAGACAGTTTCAGGCCGACTTTACATCTCTGAGCGATCAGGAGCCACTGCACGTGGCACAGGCCCTCCAGAAGGTGAAGATCGAGGTGAACGAGTCC TGT ACCGTGGCCTCTAGCTCCACAGCCGTGATCGTGTCTGCCAGGATGGCCCCAGAGGAGATCATCATGGATCGGCCCTTCCTGTTTGTGGTGAGACACAATCCAACCGGCACAGTGCTGTTCATGGGCCAGGTCATGGAGCCCTGA

[0182] Amino acid sequence of recombinant PAI-1, Q197C, G355C (SEQ ID NO: 4) MQMSPALTCLVLGLALVFGEGSA VHHPPSYVAHLASDFGVRVFQQVAQASKDRNVVFSPYGVASVLAMLQLTTGGETQQQIQAAMGFKIDDKGMAPALRHLYKELMGPWNKDEISTTDAIFVQRDLKLVQGFMPHFFRLFRSTVKQVDFSEVERARFIINDWVKTHTKGMISNLLGKGAVDQLTRLVLVNALYFNG CWKTPFPDSTHRRLFHKSDGSTVSVPMMAQTNKFNYTEFTTPDGHYYDILELPYHGDTLSMFIAAPYEKEVPLSALTNILSAQLISHWKGNMTRLPRLLVLPKFSLETEVDLRKPLENLGMTDMFRQFQADFTSLSDQEPLHVAQALQKVKIEVNES C TVASSSTAVIVSARMAPEEIIIMDRPFLFVVRHNPTGTVLFMGQVMEP

[0183] Nucleic acid sequence of hPlg-IRES2-PAI-1 expression cassette (construct for stable plasminogen and PAI-1 expression) (SEQ ID NO: 5) TACTATGACATCCTGGAGCTGCCATACCACGGCGACACCCTGAGCATGTTTATCGCCGCCCCTTATGAGAAGGAGGTGCCACTGTCCGCCCTGACAAACATCCTGTCCGCCCAGCTGATCTCTCACTGGAAGGGCAATATGACCAGGCTGCCAAGGCTGCTGGTGCTGCCTAAGTTCTCCCTGGGAGACAGAGGTGGACCTGCGGAAGCCTCTGGAGAACCTGGGCATGACCGATATGTTCA GACAGTTTCAGGCCGACTTTACATCTCTGAGCGATCAGGAGCCACTGCACGTGGCACAGGCCCTCCAGAAGGTGAAGATCGAGGTGAACGAGTCCTGTACCGTGGCCTCTAGCTCCACAGCCGTGATCGTGTCTGCCAGGATGGCCCCAGAGGAGATCATCATGGATCGGCCTTCCTGTTTGTGGTGAGCACAATCCAACCGGCACAGTGCTGTTCATGGGCCAGGTCATGGAGCCCT.

[0184] Exemplary nucleic acid sequence of ヤglu-Plg

[0185] Exemplary amino acid sequence of human glu-Plg with signal peptide underlined (SEQ ID NO: 7) MEHKEVVLLLLLFLKSGQG EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCR NPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVA VTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTM NYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVP QCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPA CLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0186] Exemplary nucleic acid sequence of human Lys-Plg (SEQ ID NO: 8)

[0187] Exemplary amino acid sequence of human Lys-Plg with signal peptide underlined (SEQ ID NO: 9) MEHKEVVLLLLLFLKSGQG KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQ CLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKK CSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0188] Exemplary nucleic acid sequence of human midiPlg (SEQ ID NO: 10) ATGGAACACAAAGAAGTGGTGTTGCTCCTGCTGCTGTTCCTGAAGTCCGGCCAGGCGATTGCTACCACGGCGACGGCCA

[0189] Exemplary amino acid sequence of human midiPlg with signal peptide underlined (SEQ ID NO: 11) MEHKEVVLLLLLFLKSGQG DCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMF GNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSW PWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFI TGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0190] Exemplary nucleic acid sequence of human mini-Plg (SEQ ID NO: 12)

[0191] Exemplary amino acid sequence of human mini-Plg with signal peptide underlined (SEQ ID NO: 13) MEHKEVVLLLLLFLKSGQG EDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVN LEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0192] Exemplary nucleic acid sequence of human microPlg (SEQ ID NO: 14) atggaacacaaagaagtggtgttgctcctgctgctgttcctgaagtccggccagggcgcccctagcttcgattgtggcaagccccaggtggaacccaagaaatgccccggcagagtcgtgggcggatgtgtggcccatcctcactcttggccttggcaggtgtccctgcggaccagattcggcatgcacttttgcggcggcaccctgatcagccccgagtgggtgctgacagccgcccactgtctggaaaagtcccccagacccagcagctacaaagtgatcctgggagcccaccaggaagtgaacctggaacctcacgtgcaggaaatcgaggtgtccagactgttcctggaacccacccggaaggatatcgccctgctgaagctgagcagccctgccgtgatcaccgacaaagtgattcccgcctgcctgcccagccccaactatgtggtggccgacagaaccgagtgcttcatcaccggctggggcgagacacagggcacatttggagccggcctgctgaaagaggcccagctgcctgtgatcgagaacaaagtgtgcaaccgctacgagttcctgaacggcagagtgcagagcaccgagctgtgtgccggacatctggctggcggcacagatagctgtcagggcgattctggcggccctctcgtgtgcttcgagaaggacaagtacatcctgcagggcgtgaccagctggggcctgggatgtgccagacctaacaagcccggcgtgtacgtgcgcgtgtccagatttgtgacctggatcgagggcgtgatgcggaacaactga

[0193] Exemplary amino acid sequence of human microPlg (SEQ ID NO: 15) with the signal peptide underlined MEHKEVVLLLLLFLKSGQGAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPAC LPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0194] Exemplary amino acid sequence of human Glu-Plg with signal peptide removed (SEQ ID NO: 16) EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHA HGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPE NYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0195] Exemplary amino acid sequence of human Lys-Plg with signal peptide removed (SEQ ID NO: 17) KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQ CLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKK CSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPS SYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0196] Exemplary amino acid sequence of human midiPlg (SEQ ID NO: 18) DCYHGDGQSYRGTSSTTTTGKCCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRA TTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLIS PEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0197] Exemplary amino acid sequence of human mini-Plg (SEQ ID NO: 19) EDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVN LEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

[0198] Exemplary amino acid sequence of human microPlg (SEQ ID NO: 20) APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Claims

1. 1. A method for producing plasminogen, comprising: (i) a host cell comprising a first recombinant polynucleotide encoding plasminogen and a second recombinant polynucleotide encoding plasminogen activator inhibitor-1 (PAI-1); providing host cells; (ii) culturing said host cells in a suitable culture medium under conditions that allow expression of plasminogen from the first polynucleotide and PAI-1 from the second polynucleotide; A method comprising:

2. 1. A method for producing recombinant plasminogen, comprising: (a) providing a first polynucleotide encoding plasminogen; (b) providing a second polynucleotide encoding PAI-1, the first and second polynucleotides are operably linked to a promoter to allow expression of the polynucleotides; (c) providing a host cell; (d) transforming or transfecting a host cell with the polynucleotides of (a) and (b); (e) providing a cell culture medium; (f) culturing the transformed or transfected host cells in a cell culture medium under conditions sufficient for expression of plasminogen and PAI-1; and Optionally, (g) recovering or purifying the plasminogen from the host cells and / or cell culture medium. A method comprising:

3. The method of claim 1 or 2, wherein the first and second polynucleotides are provided in a single vector.

4. The method of claim 1 or 2, wherein the first and second polynucleotides are provided in separate vectors.

5. 5. The method of claim 4, wherein each vector has a selectable marker of a different type than the other vector.

6. The method of any one of claims 1 to 4, further comprising the step of mixing PAI-1 into the culture medium.

7. 1. A method for producing plasminogen, comprising: (i) providing a host cell containing a recombinant polynucleotide encoding plasminogen; (ii) culturing said host cells in a suitable culture medium under conditions that allow expression of plasminogen from the polynucleotide; Including, The culture medium comprises PAI-1.

8. 1. A method for producing recombinant plasminogen, comprising: (a) providing a polynucleotide encoding plasminogen, the polynucleotide is operably linked to a promoter to allow expression of the polynucleotide; (c) providing a host cell; (d) transforming or transfecting a host cell with the polynucleotide of (a); (e) providing a cell culture medium comprising PAI-1; (f) culturing the transformed or transfected host cells in a cell culture medium under conditions sufficient for expression of plasminogen; and Optionally, (g) recovering or purifying the plasminogen from the host cells and / or cell culture medium. A method comprising:

9. 9. The method of any one of claims 1 to 8, wherein the plasminogen is selected from the group consisting of Glu-Plg, Lys-Plg, midi-Plg, mini-Plg and micro-Plg as described herein.

10. 10. The method of any one of claims 1 to 9, wherein the polynucleotide encoding plasminogen comprises, consists of, or essentially consists of a nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 5, 6, 8, 10, 12, or 14.

11. 10. The method of any one of claims 1 to 9, wherein the polynucleotide encoding plasminogen comprises, consists of, or essentially consists of a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1, 5, 6, 8, 10, 12 or 14.

12. 10. The method of any one of claims 1 to 9, wherein the plasminogen comprises, consists of, or essentially consists of an amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19 or 20, or has a sequence that is at least 75% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 9, 13, 15, 16, 17, 18, 19 or 20.

13. The method of any one of claims 1 to 12, wherein the polynucleotide encoding PAI-1 comprises the nucleic acid sequence of SEQ ID NO:

3.

14. 13. The method of any one of claims 1 to 12, wherein the polynucleotide encoding PAI-1 comprises, consists of, or consists essentially of a nucleic acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO:

3.

15. 13. The method of any one of claims 1 to 12, wherein the PAI-1 comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:

4.

16. 4. The method of any one of claims 1 to 3, wherein the first and second polynucleotides (i.e., the polynucleotides encoding plasminogen and PAI-1) are provided in a single polynucleotide construct, wherein the single polynucleotide construct comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO:

5.

17. 17. The method of claim 16, wherein the single polynucleotide construct comprises a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:

5.

18. 18. The method of any one of claims 1 to 17, wherein the host cell is a mammalian cell.

19. The mammalian cells are selected from the group consisting of Expi293, variants of Expi293, CHO (Chinese Hamster Ovary), HeLa, COS or Vero cells.

19. The method of claim 18.

20. A vector or nucleic acid construct comprising a first polynucleotide sequence encoding plasminogen and a second polynucleotide sequence encoding PAI-1.

21. 21. The vector or nucleic acid construct of claim 20, wherein the first and second polynucleotides are operably linked to a promoter to allow expression of the polynucleotides.

22. 22. The vector or nucleic acid construct of claim 20 or 21, wherein the first polynucleotide sequence encodes a plasminogen selected from the group consisting of Glu-Plg, Lys-Plg, midi-Plg, mini-Plg and micro-Plg.

23. 23. A vector or nucleic acid construct according to any one of claims 20 to 22, wherein the polynucleotide encoding plasminogen comprises, consists of, or essentially consists of a nucleic acid sequence set forth in any one of SEQ ID NOs: 1, 5, 6, 8, 10, 12 or 14.

24. 24. A vector or nucleic acid construct according to any one of claims 20 to 23, wherein the polynucleotide encoding plasminogen comprises, consists of, or essentially consists of a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 1, 5, 6, 8, 10, 12 or 14.

25. 24. A vector or nucleic acid construct according to any one of claims 20 to 23, wherein the plasminogen comprises, consists essentially of, or has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 9, 11, 13, 15, 16, 17, 18, 19 or 20.

26. 26. The vector or nucleic acid construct of any one of claims 20 to 25, wherein the polynucleotide encoding PAI-1 comprises, consists of, or consists essentially of the nucleic acid sequence of SEQ ID NO:

3.

27. 26. A vector or nucleic acid construct according to any one of claims 20 to 25, wherein the polynucleotide encoding PAI-1 comprises, consists of, or consists essentially of a nucleic acid sequence having at least 75% identity to the sequence set forth in SEQ ID NO:

3.

28. 28. The vector or nucleic acid construct of claim 27, wherein the vector encodes a PAI-1 comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:

4.

29. 22. A vector or nucleic acid construct according to claim 20 or 21, wherein the vector comprises the nucleic acid sequence set forth in SEQ ID NO:

5.

30. 22. A vector or nucleic acid construct according to claim 20 or 21, wherein the vector comprises a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:

5.

31. 31. A host cell comprising the vector or nucleic acid construct of any one of claims 20 to 30.

32. 20. Isolated, purified, substantially purified, or recombinant plasminogen produced by the method of any one of claims 1 to 19.

33. 20. Isolated, purified, substantially purified, or recombinant plasmin obtained from recombinant plasminogen produced by the method of any one of claims 1 to 19.

34. 20. A composition comprising isolated, purified, or substantially purified plasminogen and plasminogen activator inhibitor from the culture medium from the method of any one of claims 1 to 19.

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