EP1472346A2 - Method for producing recombinant proteins in micro-organisms - Google Patents

Abstract

The invention relates to a method for producing a recombinant functional plasminogen in micro-organisms, and to a method for identifying plasminogen activators. The nucleic acid sequence coding for the functional part of the plasminogen is fused with a nucleic acid molecule coding for at least one signal peptide. The nucleic acid molecule coding for the plasminogen and the nucleic acid molecule coding for the signal peptide are combined with codons for interfaces of proteases which ensure the separation of the signal peptide. The recombinant plasminogen or the corresponding plasmine is suitable for treating wounds which are slow to heal or not healing, by application of the enzyme in an appropriate formulation.

Description

 Process for the production of recombinant proteins in microorganisms

description

The human fibrinolytic system contains the protease plasmin (Pm) as a central element. On the one hand, plasmin is able to break down fibrin and, on the other hand, it can activate matrix metalloproteinases (MMP) and growth factors, which in turn are responsible for the breakdown of the extracellular matrix and wound healing.

Plasmin arises from its Norlauf molecule, the plasminogen. So far, two physiological activators of plasminogen (also known as plasminogen activators, PA) are known. These are the tissue-type plasminogen activator (tissue-type PA; t-PA) and the urokinase-type plasminogen activator (urokinase-type PA; u-PA). The system is additionally characterized by a number of protease inhibitors, e.g. B. α2-antiplasmin, regulated. The two most important biological properties of the plasminogen and the plasmin are linked directly to the two different activators.

The so-called t-PA-mediated pathway is responsible for fibrin homeostasis, while the u-PA-mediated pathway in cell migration and tissue remodeling should be emphasized. In particular, it was shown that chronic, non-healing wounds develop in u-PA deficient mice. The same applies to mice whose genes for plasminogen and t-PA or u-PA have been switched off. In addition, the lifespan of the animals was shortened significantly, which is due among other things to thrombosis and organ failure. An overview of the plasminogen / plasmin system was published by Desire Collen (Thrombosis and Haemostasis, 82, 1999 (i.

The therapeutic use of plasmin is suitable for the treatment of heart attack or stroke patients in whom a rapid dissolution of the fibrin clot is crucial for survival, and is therefore an alternative treatment to that with plasminogen activators that only hydrolyses the fibrin clot reach indirectly.

As the mouse models mentioned above show, plasmin is also a potential therapeutic that can be used to treat wounds that do not heal or only heal slowly.

Plasminogen is usually activated by t-PA only in the presence of fibrin, i.e. after the blood coagulation cascade has ended. In the absence of a substrate, plasmin is inhibited almost immediately by α ₂ -antiplasmin. The plasmin binds to fibrin However, the interaction slows down significantly, which enables the fibrin clot to break down.

Since the resolution of blood clots in a heart attack or stroke is often unavoidable for the survival of the patient, various strategies of plasminogen activation are used for therapy. For example, the infusion of streptokinase leads to a rapid recanalization of the vascular lumens. The activation of plasminogen with streptokinase, a bacterial protein, is not based on proteolytic activation but on complexation. This complex can then activate other plasminogen molecules to form plasmin.

Urokinase is also used therapeutically, but like streptokinase at the molecular level, it cannot distinguish fibrin-bound plasminogen from free plasminogen. Therefore, recombinant human t-PA was developed, which was shown to be superior to streptokinase in the clinical studies. However, these findings could not be confirmed in other studies.

The recombinantly produced plasminogen activators such as rt-PA (plus various derivatives), recombinant single chain urokinase-PA and recombinant staphylokinase emphasize the importance of the production systems generated with molecular genetic methods for the production of recombinant proteins for use in modern therapy.

Plasminogen is the norwegian molecule of the fibrinolytic enzyme plasmin. The cDΝA (Malinowski et al., Biochemistry, 23, 1984 (12); Forsgren et al., FEBS Lett. 213, 1987 (2)) and the gene including the non-coding introns (Petersen et al., J. Biol. Chem., 265, 1990 (3)) for human plasminogen have already been published in the scientific literature.

Human plasminogen (hPg), the proenzyme of the serine protease plasmin, is a glycoprotein consisting of a polypeptide chain of 791 amino acids with a molecular weight of 92,000 and a theoretical isoelectric point of 7.1. The carbohydrate content is 2% (Collen, 1999, (1)). Plasminogen is formed in the liver, the plasma concentration is around 200 mg / 1 (1.5-2 μM).

The molecule is divided into 7 structural domains; these include the Ν-terminal preactivation peptide (Glu-1 - Lys-77), five partially homologous Kringle domains and the catalytically active proteinase domain (Nal-562 - Asn-791; Collen, 1999 (1)). The structural motif of the catalytic triad common to all serine proteases is made up of the Amino acids His-603, Asp-646 and Ser-741 together. The Kringle domain 1 serves as a recognition sequence for binding the plasminogen to fibrin (Petersen et al., 1990 (3)) and various cell surface receptors.

Of particular note among the post-translational modifications are the two glycosylation sites Asn-289 and Thr-346, which are essential for the function of plasminogen (can be activated by various proteinases or streptokinase, receptor binding properties), both of which are located in the Kringle domain 3. These modifications distinguish two main forms of the plasminogen:

- Plasminogen I has the glycosylation pattern described above

- Plasminogen II lacks the modification to Asn-289

Another glycosylation site is the amino acid Ser-248. The amino acid Ser-578 can be phosphorylated.

Activation takes place in the organism through proteolytic cleavage between the amino acids Arg-561 and Nal-562. This is followed by a further proteolytic activation between Lys-77 and Lys-78 to Lys-78-hPg. Alternatively, this bond can first be hydrolyzed directly in the Glu-Pg. The active plasmin Lys-78-hPm is always linked via disulfide bridges. The heavy chain of the hPm (1 / 78-561) is for the interaction with the substrates, e.g. B. fibrinogen and fibrin. The light chain (562-791) resulting from the C-terminus represents the catalytically active subunit.

A method is already known from the literature with which the fibrin binding domain of the plasminogen in Pichia pastoris was recombinantly produced in a yield of 17 mg / l (Duman et al., Biotechnol Appl Biochem. 28; 39-45, 1998 (4)) , The authors were able to demonstrate the glycosylation of this domain (Kringle 1-4). Another reference describes the production of the two domains Kringle 4 and 5 of human plasminogen (Guan et al, Sheng Wu Gong Cheng Xue Bao, 17, 2001 (5)). The aim was to identify the domain that can inhibit the growth of endothelial cells.

However, the plasminogen domains produced by the two working groups recombinantly in Pichia pastoris do not have the catalytic domain that is decisive for the physiological functionality.

Gonzalez-Gronow et al. (Biochimica et Biophysica Acta, 1039, 1990 (6)) compared the expression of recombinant human plasminogen in Escherichia coli and COS cells, a monkey kidney cell line. Microbial production in E. coli failed what the authors attribute the lack of glycosylation. The production of the peptide chain was successful, but in a form that could not be activated, ie treatment with activators (urokinase and t-PA) did not lead to active plasmin.

The lack of glycosylation thus results in a protein which lacks the important physiological functions both in terms of activatability (no enzyme activity detectable) and in terms of endothelial cell recognition (Gonzalez-Gronow et al., Biochimica et Biophysica Acta, 1039, 1990 (6) ). The post-translational modification with the carbohydrates also significantly influences the half-life in the blood of mammals.

In contrast, the authors were able to produce functional plasminogen in COS cells. Other authors describe the functional expression in insect cells (Whitefleet-Smith et al., Arch. Biochem. Biophys., 271, 1989 (7)). However, the complex and cost-intensive cultivation conditions when using mammalian and insect cells and the low protein amounts that can be achieved are disadvantageous. Furthermore, due to the intracellular expression and the proteases in the cytoplasm, mammalian cells are unsuitable for producing larger amounts of a proenzyme (Nilsen and Castellino, Protein Expression and Purification, 16, 1999 (8) and Busby et al, J. Biol. Chem., 266, 1991 (9)). The expression yields of the baculovirus / lepidopteran (insect cell) system are typically only 3-10 mg / ml.

WO0250290 has disclosed the recombinant production of functional mini and microplasminogen in yeast. For this purpose, the authors expressed the genes for the catalytic domain of human plasminogen with (miniplasminogen) or without a Kringle domain (microplasminogen) in the host organism Pichia pastoris. The mini or microplasminogen produced in such a recombinant manner was then purified, processed to mini or microplasmin and its activity demonstrated in animal experiments. The claimed yield of the recombinant proteins is 100 mg / 1 for miniplasminogen and 3 mg / 1 for microplasminogen. However, the larger a protein is, the more difficult it is to produce it recombinantly, which is confirmed by the significant decrease in the yield by two orders of magnitude from micro to mini plasminogen in the disclosure of WO0250290. An embodiment for the expression of longer plasminogen variants such. B. Lys or Glu plasminogen was not shown.

The recombinant production of functional plasminogen in microorganisms has not yet been disclosed, so that a person skilled in the art can carry it out. The object of the present invention is therefore to produce functional human plasminogen in an inexpensive process and to process it to catalytically active plasmin.

This object is achieved by a recombinant production process for the production of plasminogen with a microorganism according to claim 1. Further solutions are mentioned in the independent claims. The dependent claims reflect preferred embodiments.

Surprisingly, it was found that recombinant microbial production of functional glu and lys plasminogen is possible in microorganisms. Furthermore, it was found that the recombinant production of micro, mini, Lys and Glu plaminogen is possible in unexpectedly high amounts.

The invention relates to the cloning of the plasminogen gene, preferably the human micro and mini plasminogen gene and more preferably the glu or lys plasminogen gene or a functional variant thereof in each case, into expression vectors and the recombinant production of functional plasminogen, preferably functional human plasminogen, using molecular genetic methods. The invention further describes the identification of proteases which catalyze the activation of plasminogen to plasmin. The plasminogen or plasmin, which is produced by this invention, is free of contaminants such as. B. animal proteins or viruses that occur naturally when isolated from humans, cattle and other mammals and which can lead to side effects in the patient.

The invention is characterized by a recombinant production method, comprising at least the following step: a.) Fusion of the nucleic acid sequence coding at least for the functional part of the plasminogen peptide with a nucleic acid sequence coding for at least one signal peptide, the one for the functional plasminogen peptide coding nucleic acid sequence and the nucleic acid sequence coding for at least the signal peptide are linked to codons for interfaces of proteases which ensure the cleavage of the signal peptide. The production of therapeutic proteins is increasingly being carried out using recombinant production systems. Because of cost factors, the aim is to carry out recombinant production in microbial, in particular bacterial, organisms. These systems have the advantage that, in addition to comparatively inexpensive production, protein yields in the g / 1 range can be achieved and the recombinant proteins are not infected with viruses or proteins such as, for. B. Prions are contaminated, which can be harmful to the patient. Since bacterial production systems are often unable to produce correctly folded protein, In addition to the in-fold refolding of the misfolded proteins, production is often carried out in eukaryotic systems such as yeast, insect cells or mammalian cells. The eukaryotic production strains and cell lines offer the advantage that they can be used to produce glycosylated proteins. For insect cells or mammalian cells in particular, recombinant protein production is very cost-intensive and the yields are often very low. They also have the disadvantage that they can also be contaminated with viruses and proteins which are harmful to humans. This is not the case when using eukaryotic microorganisms. The equipment for the cultivation of eukaryotic microorganisms is comparable to that of bacterial organisms, contamination with mammalian viruses and proteins is not present and protein yields in the g / 1 range are also possible. The eukaryotic host organism used particularly preferably belongs to the department of fungi, preferably to the Ascomycota. It is further preferred that it belongs to the Saccharomycotina, in particular to the class of the Saccharomycetes, here in particular to the order of the Saccharomycetales. According to particularly preferred embodiments, the host organism also belongs to the family Saccharomycetaceae, here in particular to the genus Pichia. Eukaryotic microorganisms which are preferably used according to the invention are, for example, the baker's yeast Saccharomyces cerevisiae, further examples are Candida, the methanothrophic yeasts Pichia pastoris, Pichia methanolica and Hansenula polymorpha or filamentous fungi of the genus Aspergillus, such as, for. B. Aspergϊllus niger, Aspergillus oryzae and Aspergillus nidulans. Pichia pastoris is particularly preferred.

The recombinant production process is further characterized in that a nucleic acid molecule coding for at least the functional part of plasminogen is inserted into an expression vector for this microorganism, the nucleic acid molecule preferably coding for human plasminogen with that for at least one signal peptide, preferably a prepropeptide, preferably for the transport into the endoplasmic reticulum, coding nucleic acid molecule is fused, codons for protease interfaces are inserted between the two nucleic acid molecules, which enable the cleavage of the signal sequence or the prepropeptide in the host organism. A nucleic acid molecule coding for human plasminogen is preferably used. In addition to a nucleic acid molecule coding for human plasminogen, nucleic acid molecules can be used which code for plasminogen from other mammals. This leads to the production of plasminogen in the respective mammals. Furthermore, the recombinant human plasminogen is formed by overexpression in accordance with the present method and, if desired, can be secreted into the culture medium from which it can be separated from the host cells by centrifugation, filtration or sedimentation and thus can be used for protein purification without complex cell disruption processes can, which can be carried out using methods known to those skilled in the art. The Activation of plasminogen to plasmin is solved by proteases that are able to process plasminogen to catalytically active plasmin.

Terms used in the context of the present invention are defined below:

“Recombinant production method” means that a peptide or a protein from a nucleic acid sequence, preferably a DNA sequence, is expressed by a suitable host organism, the nucleic acid sequence resulting from the cloning and fusion of individual nucleic acid segments.

“Cloning” is intended to encompass all cloning methods known according to the prior art, but not all of them are described in detail because they are a matter of course for the skilled worker.

“Expression in a suitable expression system” is intended here to encompass all expression methods known according to the prior art, in particular those which are addressed in the claims.

The “functional plasminogen peptide part” is to be understood as the part of the plasminogen or plasminogen peptide which can perform the biologically relevant functions of the plasminogen. These biologically relevant functions are at least the ability to activate plasmin by plasminogen activators such as twe plasminogen activator, urokinase , vampire-bat plasminogen activator, streptokinase, stapyhlokinase, Pia protein from Yersinia pestis etc., and the proteolytic activity, which is characterized by the hydrolysis of fibrin. Under the term "plasminogen activator (s)" used in the description and the examples both proteolytic and non-proteolytic plasminogen activators are understood.

In the case of glu-plasminogen, the processability to lys-plasminogen is also to be understood by the plasmin-catalyzed cleavage of the preactivation peptide.

The biological functions also include the fact that plasminogen can be activated up to a factor of 1000 after binding to fibrin, laminin, fibronectin, vitronectin, heparan sulfate proteoglucan, type 4 collagen and other substrates.

Among the biologically relevant functions of plasmin, which must be ensured after processing the plasminogen, are the degradation of laminin, the degradation of fibronectin, of vitronectin, of heparan sulfate proteoglucan, the activation of ProCoUagenases, the activation of Promatrix metalloproteases, the activation of To understand latent macrophages elastase, prohormones and growth factors such as TGFß-1 (latent transforming growth factor), VEGF (vascular endothelial growth factor) or bFGF (basic fϊbroblast growth factor).

Another biological function is the ability to be inhibited by plasmin inhibitors such as α2-antiplasmin and α2-macroglobulin.

In addition, the biologically relevant functions include binding to fibrin, laminin, fibronectin, vitronectin, heparan sulfate proteoglucan and type 4 collagen, binding to receptors such as, for example, α-enolase, Annexin II or amphoterin.

Plasminogen is initially formed as an inactive glu-plasminogen. This glu-plasminogen can be converted from plasmin into Lys-plasminogen by splitting off the so-called preactivation peptide. Both are converted by tissue plasminogen activators (in this case only by the above-mentioned proteolytic activators) by proteolytic cleavage into plasmin, which consists of subunits connected via sulfide bridges. The smaller subunit contains the proteolytic domain and the phosphorylation site, the larger subunit carries the three glycosylations and is responsible for binding to fibrin. Furthermore, the glycosylations are important for stability in the plasma. By forming a 1: 1 complex with streptokinase or staphylokinase, plasminogen can additionally be converted into a proteolytically active enzyme which is able to process plasminogen into plasmin.

Functional plasminogen is therefore plasminogen, which can be processed into proteolytically active plasmin by plasminogen activators. Furthermore, functional plasminogen preferably contains the fibrin-binding domain and can preferably contain at least one of the three glycosylations.

The smallest forms of functional plasminogen are micro and mini plasminogen, a larger form lys plasminogen. Glu-plasminogen, which still contains the preactivation peptide, is also functional plasminogen. However, it is conceivable that regions, in particular within the larger chain, can be omitted without significantly impairing the above-mentioned functionality (including proteolysis, fibrin binding).

It is obvious to a person skilled in the art to produce different forms of the plasminogen (hereinafter referred to as plasminogen derivatives) which contain a functional catalytic domain. Functionally, as already described, means that the plasminogen variant has proteolytic activity after activation with plasminogen activators such as streptokinase or urokinase. • The catalytic domain can contain deletions and amino acid exchanges or can be fused with other amino acids or peptides or proteins.

• The large domain can contain all intermediates from Glu20 to Arg580 (based on the sequence of the pre-plasminogen) which can be activated with plasminogen activators to form active plasmin.

As a concrete example, three forms of Lys plasminogen are mentioned:

Variant 1: N-terminal amino acid: Met88 Variant 2: N-terminal amino acid: Lys97 Variant 3: N-terminal amino acid: Val98

The plasminogen derivatives are preferably a number of 1 to 50 amino acids shorter or longer than the corresponding micro, mini, Lys or Glu plasminogen or preferably have an exchange of 1 to 10 amino acids, these derivatives furthermore having the property to be activated by plasminogen activators. Between the respective micro, mini, Lys or Glu plasminogen and the associated plasminogen derivative there is a sequence homology (sequence agreement) of over 80%, preferably over 85%, more preferably over 90%, further preferably over 95% , particularly preferably of over 98% and furthermore particularly preferably of over 99%.

The plasminogen derivatives preferably have the following characteristics:

• The catalytic domain can contain at least one deletion and / or at least one amino acid exchange and / or be fused with at least one further amino acid or at least one further peptide or at least one further protein.

• The large domain can contain all intermediates from Glu20 to Arg580 (based on the sequence of the pre-plasminogen), which can be activated with plasminogen activators to active plasmin.

A plasminogen derivative has an amino acid sequence homology (match) of preferably over 80%, further preferably over 85%, further preferably over 90%, particularly preferably over 95% and further particularly preferably over 99%.

"Microorganism" encompasses all those life forms which have only small dimensions. Both eukaryotic and prokaryotic microorganisms are to be included. Bacteria, yeasts, fungi and viruses should be mentioned in particular. “Nucleic acid” is intended to encompass both DNA and RNA, both in all conceivable configurations, for example in the form of double-stranded nucleic acid, in the form of single-stranded nucleic acid, combinations thereof, and linear or circular nucleic acids.

“Signal sequence” is understood to mean a peptide sequence which is able to ensure the transport of a further peptide sequence into or across a membrane, for example into the endoplasmic reticulum. This can be, for example, a prepropeptide, a prepeptide or a propeptide.

“Interface” denotes those sites in a peptide sequence which make it possible to split off a signal sequence, a prepropeptide or propeptide from the further peptide sequence or generally to split a peptide sequence into two parts in a host organism.

A “nucleic acid coding for at least one signal peptide or a prepropeptide” is a nucleic acid sequence which codes for a peptide or a protein structure which enables the further polypeptide to be introduced into membranes, for example into the endoplasmic reticulum.

A primer oligonucleotide is referred to as "primer". By this we mean short-chain, single-stranded oligoribo or deoxyribo nucleotides which are complementary to a region on a single-stranded nucleic acid molecule and can hybridize with it to form a double strand. The free 3'- The hydroxyl end in this double strand serves as a substrate for DNA polymerases and as a starting point for the polymerization reaction of the entire single strand to form the double strand.The primers are used in particular for PCR, ie the polymerase chain reaction known to the person skilled in the art.

The term "plasmid" denotes the nucleic acid molecules which are present in many prokaryotic and some eukaryotic microorganisms and are not integrated into the chromosome and have a length of approximately 2 kb to more than 200 kb.

"Ligation" is the term for the connection of the ends of two nucleic acid molecules with the aid of a ligase or in the context of a self-ligation, ie by an intramolecular ring closure reaction, in which the two single-stranded ends of a linear DNA molecule join together, provided that their ends are together Can form base pairs.

"Restriction endonuclease" is the name for a class of bacterial enzymes that cleave phosphodiester bonds in both strands of a DNA molecule within specific base sequences. "Electroporation" is a method for introducing nucleic acids into cells. The cell membranes of the exponentially growing recipient cells in suspension are made permeable to high molecular weight molecules by short electrical pulses of high field strength while being exposed to the nucleic acid solution.

“Overexpression” is understood to mean an increased production of functional plasminogen by a cell in comparison to a production by the wild type of this cell. In general, one speaks of an overexpression when the expressed foreign gene in the case of intracellular production accounts for about 1-40% of the total cellular protein of the host cell.

“Expression vector” is to be understood as those vectors which, after introduction into a suitable host cell, allow the transcription of the foreign gene cloned into the vector and the subsequent translation of the mRNA (messenger RNA) formed. Expression vectors generally contain those for the expression of genes control signals required in cells of prokaryotes or eukaryotes.

To control gene expression in yeasts such. B. Pichia pastoris are preferred in the present invention by methanol inducible promoters such. B. the AOX1 promoter or particularly preferably constitutive promoters such. B. the YPTl promoter or the GAP promoter used. The constitutive GAP promoter is particularly preferred.

"AOX1" is a gene of alcohol oxidase 1 from P. pastoris;

"GAP" is a gene of glyceraldehyde-3-phosphate dehydrogenase from P. pastoris and

"YPT1" is a gene of a GTP binding protein from P. pastoris.

The signal peptides of the proteins encoded by the genes PHO-1, SUC-2, PHA-E or alpha-MF are often used for secretory production in yeasts.

"PHO1" is a gene of the acidic phosphatase from P. pastoris;

"SUC-2" is a gene of the secretory invertase from S. cerevisiae;

"PHA-E" is a gene of the acid phosphatase from Phaseolus vulgaris Agglutinis; and

"Alpha-MF" is a gene of the alpha-mating factor from S. cerevisiaea.

The codons for the interfaces of proteases and codons for the interfaces for splitting off the propeptide for the protease Kex 2 or the protease Ste 13 are particularly preferred. The link in step a) above is particularly preferably carried out with codons which are for a Kex 2 interface and additionally code two Ste 13 interfaces. In a preferred embodiment of the present invention, this is for the signal peptide or Prepropeptide-encoding nucleic acid molecule from yeast, in particular the yeast Saccharomyces cerevisiae. A still more preferred embodiment is directed to a nucleic acid molecule coding for the signal peptide or the prepropeptide, which encodes the signal peptide or prepropeptide of the α-factor of the yeast Saccharomyces cerevisiae. The resulting fusion product described in step a) above is preferably amplified by PCR and then further preferably purified.

WO02 / 50290 discloses the recombinant production of mini and microplasminogen with the expression vector pPICZαAD D which contains the inducible AOX1 promoter and the prepropeptide of the yeast alpha factor and is suitable for yeast. These smaller variants of plasminogen either have none at all (like microplasminogen) or only a Kringle domain (like mini plasminogen). The expression vector pPICZαAD contains the interfaces for the proteases Kex2 and Stel3. When cloning the corresponding

Expression vectors of mini and microplasminogen, however, deleted the Stel3 interfaces.

A number of promoters are known for inducible expression systems in yeast. These include the AOXl promoter, AOX2, CUP1 (Koller A, Valesco J, Subramani S., Yeast 2000: 16 (7), 651-6), PHO1 (EP0495208), HIS4 (US 4885242), FLD1 (Shen et al., Gene 1998: 216 (1), 93-10) and the XYLl promoter (Den Haan and Van Zyl, Appl. Microbiol. Biotechnol. 2001: 57 (4), 521-7).

With the help of the methanol-inducible AOXl promoter, heterologous protein production can be controlled and a homogeneous biomass can be achieved. Before the expression of the foreign protein is induced, the host organisms can achieve a high growth density without any selection disadvantages that would arise from the expression of a foreign protein.

In contrast to their smaller variants, which are expressed in WO02 / 50290 under the control of the AOXl promoter, the glu and Lys plasminogen recombinantly produced in the present invention contain all five Kringle domains, making their recombinant production difficult for the following reasons makes:

- Possible loss of the expression cassette due to growth disadvantages for the host organisms when expressing the foreign proteins;

- Proteolytic degradation of the expressed proteins and

- Low yield.

Due to the disadvantages shown, the production of glu or Lys plasminogen has not been disclosed in WO02 / 50290. These difficulties were solved in the present invention in that the recombinant protein contains a signal peptide, a Kex2 and at least one Stel3, preferably two Stel3, protease cleavage sites. Furthermore, in a preferred embodiment, a glycerol feed between 0.1 and 10 ml / h, preferably between 0.5 and 5 ml / h, more preferably between 0.8 and 1.5 ml / h, was carried out as a further C source and the culture medium buffered to neutral pH of 7.0. Care was taken to ensure sufficient oxygen input.

In a preferred embodiment, care was taken to ensure that the recombinant nucleic acid was not connected to the 5 'region of the AOXl gene, but rather to the 5' region of the P. pastorisza glyceraldeydphosphate dehydrogenase gene. integrate. A constitutive promoter was used, not an inducible one. Constitutive promoters that are active and can be used in yeast are the GAP promoter, the YPT1 promoter (Sears et al., Yeast 1998: 14 (8), 783-90), the TKL promoter (Den Haan and Van Zyl, Appl. Microbiol. Biotechnol. 2001: 57 (4), 521-7), the ACT promoter (Kang et al., Appl. Microbiol. Biotechnol. 2001: 55 (6), 734-41) and the PMAl Promoter (Yeast 2000: 16 (13), 1191-203). Preferred promoters are the GAP promoter and the YPTI promoter. A particularly preferred promoter is the GAP promoter.

In contrast to an inducible promoter, a constitutive promoter has the disadvantage that the foreign protein to be expressed is produced constructively, that is to say during the entire wax phase. This creates disadvantages for the host cell, which u. a. expresses in a slower growth. Because of the prevailing selection pressure, host cells that have lost the recombinant expression cassette have an advantage and can overgrow the recombinant host cells. This can result in a heterogeneous mixed population that should be avoided. Surprisingly, however, it was found that the constitutive GAP promoter according to a preferred embodiment of the present invention enables a higher yield.

While using the AOXl promoter yields of lys-plasminogen after 120 hours induction of at least 17 U / 1 (≡ 1.5 mg / 1) more preferably 120 U / 1 (≡ 11 mg / 1), more preferably 180 U / 1 (= 16 mg / 1), further preferably 200 U / 1 (≡ 18 mg / 1), further preferably 220 U / 1 (≡ 20 mg / 1), further preferably 240 U / 1 (≡ 22 mg / 1 1), particularly preferably 260 U / 1 (≡24 mg / 1) and further particularly preferably 280 U / 1 (≡ 25.5 mg / 1), the yields were when using a consumer promoter, in particular the GAP promoter , significantly higher. In a preferred embodiment, a constitutive promoter, e.g. B. the GAP promoter is operatively linked to a nucleic acid which codes for at least the functional part of the plasminogen sequence, and which is fused to a nucleic acid sequence which codes for at least one signal peptide, the nucleic acid sequence coding for the functional plasminogen and for at least the nucleic acid sequence encoding the signal peptide is linked to codons for interfaces of proteases which ensure the cleavage of the signal peptide.

In a particularly preferred embodiment, a constitutive promoter, e.g. B. the GAP promoter with the nucleic acid sequence of the micro, mini, Lys or Glu plasminogen operatively linked, which is fused with the nucleic acid sequence of a signal peptide from the yeast.

Surprisingly, it was found in this regard that the constitutive GAP promoter according to a preferred embodiment of the present invention enables an approximately 10-fold higher yield (see Example 7c, production of Lys-Plasminogen, 1375 U / 1, which corresponds to 125 mg / 1 results). In a further preferred embodiment, a glycerol feed between 0.1 and 10 ml / h, preferably between 0.5 and 5 ml / h, more preferably between 0.8 and 1.5 ml / h, is carried out as a further C source and the culture medium buffered to neutral pH of 7.0. The growth rate μ [1 / h] reaches values between 0.002 and 0.10, preferably between 0.004 and 0.020, more preferably between 0.008 and 0.010.

When using the GAP promoter, yields of lys plasminogen of at least 660 U / 1 (60 mg / 1), preferably 1000 U / 1 (≡ 91 mg / 1), preferably 1500 U / 1 (≡ 136 mg / 1) , more preferably 2000 U / 1 (≡ 182 mg / ml), preferably 2500 U / 1 (≡ 227 mg / 1), particularly preferably and further particularly preferably 2750 U / 1 (≡ 250 mg / 1) after a fermentation time of 250 Received hours.

In the recombinant production of mini and microplasminogen, correspondingly higher yields were obtained. The yields of miniplasminogen are between 100 mg to 2 g per liter, preferably from 300 mg / 1 to 1.5 g / 1, more preferably from 400 mg / 1 to 1 g / 1 and more preferably from 500 mg / 1 to 800 mg / 1 and particularly preferably from 600 to 700 mg / 1. The yields of microplasminogen are also at least 10% higher than that of miniplasminogen. The recombinant production of glu-plasminogen yielded slightly poorer yields in comparison to lys-plasminogen.

The method according to the invention is suitable for the production of mini, micro, Lys and glu plasminogen. Preferred embodiments are therefore directed to the recombinant Production of mini, micro, Lys and Glu plasminogen, which are each coupled to a signal or prepro sequence, in an expression vector which contains a consumer promoter, e.g. B. contains the GAP promoter. In a further preferred embodiment, the signal sequence consists of the signal peptide or prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae. In a particularly preferred embodiment, a constitutive promoter, e.g. B. the GAP promoter, operatively with a nucleic acid of the sequences Seq. ID. No. 7 or 9 or one of the sequences Seq. ID. No. 13 or 15 or one of the sequences Seq. ID. Nos. 50 to 59 linked and expressed in a suitable expression vector.

In a further preferred embodiment, a constitutive promoter, e.g. B. the GAP promoter is operatively linked to a nucleic acid which codes for at least the functional part of the plasminogen sequence. In a particularly preferred embodiment, a constitutive promoter, e.g. B. the GAP promoter, operatively with a nucleic acid of the sequences Seq. ID. Nos. 13, 15, 7 and 9 or one of the sequences Seq. ID. No. 50 to 59 or the sequence Seq. ID. No. 11 linked and expressed in a suitable expression vector

Glu-Plasminogen (data calculated with the program EditSeq ™ (DNASTAR)) Molecular weight: 88431.67 Dalton 791 amino acids

7,121 isoelectric point at pH 7.0: 1,351 glycosylation sites: O-268, N-308, O-365 (the numbering refers to the 810 AS-long pre-plasminogen)

Lys plasminogen (data calculated using the EditSeq ™ (DNASTAR) program) Molecular weight: 79655.71 Daltons 714 amino acids

7,492 isoelectric point at pH 7.0: 5,287 glycosylation sites: O-268, N-308, O-365 (the numbering refers to the 810 AS long pre-plasminogen)

Mini-plasminogen (data calculated with the program EditSeq ™ (DNASTAR)) molecular weight 38169.63 Daltons 348 amino acids

7.203 Isoelectric point at pH 7.0: 0.893 No more glycosylation sites Micro-plasminogen (data calculated with the program EditSeq ™ (DNASTAR)) molecular weight 27230.41 Daltons 249 amino acids

7,934 Isoelectric point at pH 7.0: 3,733 No more glycosylation sites.

The method according to the invention is described in detail below.

The fusion product formed in step a) of the present invention can also be inserted into an expression vector suitable for microorganisms. This expression vector is preferably selected from the group comprising pPICZαA, B and C and pPICZ A, B and C and pGAPZαA, B and C and pGAPZA, B and C and pPIC6αA, B and C and pPIC6A, B and C and pAO815, pPIC3 , 5K and pPIC9K. The introduction into the expression vector is again preferably carried out by ligation. The PCR product and the expression vector are preferably cut with the restriction endonucleases Kspl and Xhόi before they are ligated with a T4 DNA ligase. The ligated nucleic acid can be transformed into a microorganism, preferably E. coli, by electroporation and the DNA can be isolated from the transformed strains thus obtained and separated by endonucleolytic cleavage, preferably with Xhol or Sful and Kspl. The nucleic acid obtained in this way can be a plasmid, preferably selected from the group pMHS476.1, pSM54.2, pSM49.8, pSM82.1, pSM58.1, pAC37.1, pJW9.1, pPLGl.l, pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1, pPLGlO.l, pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2, pPLG15. 1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 and pPLG20.1. Two oligonucleotide primers selected from the group comprising N034 (Sequence ID number 1), N036 (Sequence ID number 2), N036a (Sequence ID number 19), N036b (sequence) are preferably used as primers for the above-mentioned amplification -ID- number 20), N036c (sequence ID number 21), N036d (sequence ID number 22), N036e (sequence ID number 23), N036f (sequence ID number 24), N036g (sequence -ID number 25), N036h (Sequence ID number 26), N036i (Sequence ID number 27), N036j (Sequence ID number 28), N057 (Sequence ID number 3), N037 (Sequence ID -Number 4), N035 (Sequence ID number 5) and N056 (Sequence ID number 6) are used. The following embodiments are particularly preferred according to the present invention:

- Codons which code for the interface of the protease Kex2 and the plasminogen fusion gene which has the nucleic acid sequence shown in Sequence ID number 7 or 13.

- Codons which code for the interface of the protease Kex2 and the plasminogen fusion protein which has the amino acid sequence shown in Sequence ID number 8 or 14. - Codons which code for the interface of the protease Kex2 and the protease Stel3 and the plasminogen fusion gene which has the nucleic acid sequence shown in Sequence ID number 9 or 15.

Codons which code for the protease Kex2 and the protease Stel3 and the plasminogen fusion protein which has the amino acid sequence shown in Sequence ID number 10 or 16.

Preferably, the above-mentioned plasmid, which is preferably selected from the above-mentioned group, is transformed into a microbial host. The transformation can be carried out, for example, by electroporation. The microorganism used is preferably a eukaryotic microorganism which belongs to the department of fungi. Preferred microorganisms belong to the Ascomycota, preferably Saccharomycotina and thereof preferably the class of the Saccharomycetes, more preferably the order of the Saccharomycetales, more preferably the family of the Saccharomycetaceae and particularly preferably the genera Pichia, Saccharomyces, Hansenula and Aspergillus.

According to a particularly preferred embodiment of the present invention, the nucleic acid sequence coding for at least the functional part of the plasminogen is overexpressed from a microbial host organism transformed with the fusion product formed in step a) described above, and at least the functional part of plasminogen is secreted, preferably in the culture medium is secreted. According to a further preferred embodiment, the functional part of the nucleic acid sequence of plasminogen is one of the sequences ID number 60, 61, 62, 63, 64, 65 or 66. According to a further preferred embodiment, the functional part of the nucleic acid sequence of plasminogen corresponds to the complete plasminogen sequence. A human functional plasminogen is preferably produced using the recombinant production method according to the present invention.

This plasminogen, which is obtainable by the recombinant production process according to the present invention, or the plasmin resulting therefrom by the action of proteases, can be used to produce a medicament for the treatment of wounds, in particular for the treatment of slow or poorly healing wounds, for the treatment of thrombotic events , or for the prevention of thrombotic events.

In addition, it was recognized that the plasminogen produced according to the invention and the plasmin obtained therefrom have anti-coagulative properties. These advantageous properties also make it possible to use the plasminogen and / or plasmin as anti-thrombotic and anti-coagulant active ingredients for the prophylaxis and / or treatment of Heart attack, thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammation of the blood vessels and for treatment after a heart attack, after bypass surgery, after angioplasty and after balloon dilatation. The plasminogen can also be used for thrombolytic therapy in acute myocardial infarction, for recanalizing arteriovenous shunts and for reopening (reperfusion) of closed coronary arteries in acute myocardial infarction. Further uses of the plasminogen produced according to the invention include the prophylaxis and treatment of acute pulmonary embolism, of fresh or older clots in the case of venous thrombosis, acute and subacute arterial thrombosis, venous thrombosis, acute arterial occlusion of the extremities, chronic occlusive arteriopathy, thrombosis of arteriovenous thrombosis and the extremities, early thrombosis in the area of the obliterated vessels, acute central vascular occlusion on the eye, conjunctivitis with plasminogen type I deficiency, burns and frostbite, burns and disseminated intravascular coagulation in shock.

Plasminogen and / or plasmin are preferably used for these indications together with an anticoagulant. Heparin, heparin derivatives or acetylsalicylic acid are suitable as anticoagulants.

The present invention is therefore also directed to pharmaceutical compositions comprising a plasminogen which has been prepared according to the recombinant production process of the present invention, or the resulting plasmin, optionally in combination with a pharmaceutically acceptable carrier, additive and / or solvent. In addition, the pharmaceutical compositions can preferably contain an anticoagulant active ingredient, in particular heparin, heparin derivatives or acetylsalicylic acid.

The plasminogen produced according to the invention and / or the plasmin obtained therefrom are preferably used in the external wound treatment in pharmaceutical compositions which are suitable for topical use. Plasminogen and / or plasmin are used in a concentration of 0.01-500 U per gram of pharmaceutical composition, preferably 0.1-500 U, more preferably 0.1-250 U, further preferably 0.5-250 U per gram pharmaceutical composition and particularly preferably used in a concentration of 1 - 150 U plasminogen and / or plasmin per gram of pharmaceutical composition. Will be instead of semi-solid

Preparations in the form of, for example, ointments, pastes, gels, etc. plasters or other dressing materials, the concentration ranges given above per 2 cm plaster surface or surface of the dressing material apply. The pharmaceutical compositions according to the invention are produced in a known manner with the customary solid or liquid carriers or diluents and the commonly used pharmaceutical adjuvants according to the desired type of application in a suitable dosage. The preferred pharmaceutical formulations or preparations are in a dosage form which is suitable for topical external application. Such dosage forms are, for example, ointments, pastes, gels, films, dispersions, emulsions, suspensions or special formulations, such as, for example, nanodisperse systems in the form of liposomes, nanoemulsions or lipid nanoparticles, and also surfactant-free formulations, polymer-stabilized or solids-stabilized emulsions.

Methods for producing various formulations and the various application methods are known to the person skilled in the art and are described in detail, for example, in "Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA".

When using the pharmaceutical compositions for the prophylaxis and / or treatment of heart attack, thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammation of the blood vessels, acute myocardial infarction and for treatment after a heart attack, after a bypass operation, after an angioplasty and after The preparations prepared for parenteral administration are suitable for balloon dilation.

In addition, the pharmaceutical compositions are suitable for various systemic applications, including use for acute pulmonary embolism, thrombolytic therapy for acute myocardial infarction, fish or older clots for venous thrombosis, acute and subacute arterial thrombosis, recanalization of arteriovenous shunts, venous thrombosis, reopening (reperfusion arteries) of closed coronary artery acute heart attack, acute arterial occlusion of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep vein thrombosis of the pelvis and extremities, early thrombosis in the area of desobliterated vessels, acute central vascular occlusion on the eye, conjunctivitis with plasminogen and type I deficiency, burns or disseminated intravascular coagulation in shock.

Another application is for plasminogen deficiency, such as. B. acquired or congenital plasminogen deficiency (homozygous type I plasminogen deficiency), the z. B. can lead to conjunctivitis lignosa or thrombophilia. There is the possibility, for example, of intravenous administration of the recombinant plasminogen, including the forms of glu, lys, mini and microplasminogen, as well as of these derived variants to treat the disease. (Heinz et al., Klin. Monthly Bulletin Ophthalmology 2002, 219 (3): 156-8).

In a further possible application, a decrease in the pseudomembranes and the normalization of the respiratory tract as well as the improved wound healing can be achieved when plasminogen is administered. This application has been described for a newborn child (The New England Journal of Medicine 1998, 339, 23, 1679-1686).

Thus, the recombinantly produced plasminogen may be used together with the plasmin obtained therefrom or also only plasmin in pharmaceutical compositions which are used for the prophylaxis and / or treatment of acute pulmonary embolism, thrombolytic therapy for acute myocardial infarction, fresh or older clots for venous thrombosis, acute and subacute arterial Thrombosis, recanalization of arteriovenous shunts, venous thrombosis, reopening (reperfusion) of closed coronary arteries in acute myocardial infarction, acute arterial occlusion of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep vein thrombosis of the pelvic area and central vein occlusion, amniotic and occlusive areas Eye, conjunctivitis with plasminogen type I deficiency, burns, burns and frostbite, disseminated intravascular coagulation in shock are suitable.

The plasminogen produced recombinantly according to the invention is preferably used in pharmaceutical compositions which are suitable for the topical treatment of burns, frostbite, burns, injuries and / or wounds, in particular poorly healing wounds. The recombinant plasminogen is preferably used together with at least one activator (plasminogen activators such as urokinase or streptokinase). Another preferred possibility consists in converting the plasminogen produced according to the invention in whole or in part by means of an activator into plasmin and using it as plasmin or plasmin with plasminogen in the indications and formulations described herein.

Intravenous, intravascular, intraperitoneal, subcutaneous and intramuscular administration are considered as parenteral applications. In the case of parenteral formulations, in particular in the form of solutions for injection or infusion, the protein is used in a concentration of 0.1-100 million units, preferably 10 to 100 million units per 10 ml solution, further preferably 1 to 10 million units per 10 ml solution and particularly preferably 3 to 5 million units per 10 ml of solution. In formulations suitable for oral administration, the protein is in a concentration of 0.1 to 100,000 units per gram of formulation, preferably 100 to 80,000 units per gram of formulation and particularly preferably 1,000 to 50,000 units per gram of formulation.

Further advantageous formulations are, for example, protease-containing plasters, bandages or other dressing materials. These formulations are particularly suitable for topical use in wound treatment or for the treatment of burns, frostbite, burns and / or injuries. The plasminogen produced recombinantly according to the invention is preferably used in the pharmaceutical compositions, in particular the wound healing agents, plasters and bandaging materials, together with at least one activator (plasminogen activators such as urokinase or streptokinase) or previously converted into plasmin using the activators described above and possibly together with as plasmin Plasminogen and possibly used with at least one activator in and / or on the pharmaceutical compositions and formulations. Particularly preferred is the use of plasminogen, preferably plasminogen with an activator, or plasmin or plasmin together with plasminogen and an activator in and / or on plasters and dressing materials which are used for wound treatment, in particular for treating poorly healing wounds, and for treating burns, Frostbite, burns or other injuries are suitable.

The dressing materials, wound dressings or plasters contain the plasminogen produced according to the invention and / or plasmin obtained therefrom in a concentration of 0.01

500 units of plasminogen and / or plasmin per cm ^{2 of} the pharmaceutical formulation, preferably 0.1 to 500 units of plasminogen and / or plasmin per cm ^{2 of} dressing material or plaster. The pasminogen and / or plasmin is further preferred in a concentration of 0.1-250 units, further preferably 0.5-250 units and particularly preferably 1

- 150 units of plasminogen and / or a resulting plasmin per cm ^{2 of} pharmaceutical formulation in the plaster or dressing material.

Between 100 μg and 1 ng urokinase are used for the activation of 1 mg plasminogen, preferably between 10 μg and 10ng urokinase. Between 1 mg and 1 μg streptokinase are used for the activation of 1 mg plasminogen, preferably between 300 μg and 3 μg streptokinase are used. Between 100 μg and 10 ng protease from S. griseus are used for the activation of 1 mg plasminogen, preferably between 10 μg and 100 ng protease from S. griseus are used. Between 100 μg and 10 ng protease VIII are used for the activation of 1 mg plasminogen, preferably between 10 μg and 100 ng protease VIII. The nucleic acid sequence coding for the functional part of the plasminogen is preferably a DNA sequence.

The present invention also relates to the following plasmids:

Plasmid pPLGl.l

Plasmid pPLG2.1

Plasmid pPLG3.2

Plasmid pPLG4.2

Plasmid ρPLG5.3

Plasmid pPLG6.1

Plasmid ρPLG7.1

Plasmid pPLG8.3

Plasmid ρPLG9.1

Plasmid pPLGl 0.1

Plasmid pPLGl 1.2

Plasmid pPLGl 2.1

Plasmid pPLGl 3.1

Plasmid pPLGl 4.2

Plasmid pPLGl 5.1

Plasmid pPLGl 6.3

Plasmid pPLGl 7.2

Plasmid pPLGl 8.1

Plasmid pPLGl 9.2

Plasmid pPLG20.1

Plasmid pMHS476.1 (accession number: DSM 14678)

Plasmid pSM54.2 (accession number: DSM 14682)

Plasmid pSM49.8 (accession number: DSM 14681)

Plasmid ρSM82.1 (deposit number: DSM 14679)

Plasmid ρSM58.1 (deposit number: DSM 14680)

Plasmid ρAC37.1 (deposit number: DSM 15369)

Plasmid pJW9.1 (accession number: DSM 15368).

(The deposit numbers refer to the deposit with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig)

Furthermore, the present invention relates to a DNA sequence suitable for expression, which comprises at least the nucleic acid sequence coding for the functional part of plasminogen, obtainable by the recombinant production method according to the present invention. It also affects the microbial host organism, which Fusion product obtained in step a) described above and a nucleic acid sequence derived therefrom. The present invention further relates to a vector, a DNA molecule or an RNA molecule which comprises the fusion product obtained in step a) described above or a nucleic acid sequence derived therefrom.

Finally, the present invention also relates to a screening method for the identification of plasminogen activators, in particular plasminogen-activating proteases, the functional plasminogen, produced according to the recombinant production method described above, being used. For this purpose, the resulting piasmin activity is preferably measured after preincubation of the proteases with the functional plasminogen, as produced according to the present invention. The resulting piasmin activity can be measured with a synthetic peptide substrate. The resulting piasmin activity is particularly preferably measured with N-tosyl-Gly-Pro-Lys-pNA.

The invention is explained in more detail by the drawings which show the following:

Fig. 1: Physical map of the plasmid pMHS476.1 (5682 bp). The gene of the prepropeptide of the alpha factor is linked to the human Lys plasminogen gene by the codons for a Kex2 interface and is under the control of the AOX1 promoter.

Fig. 2: Physical map of the plasmid pSM54.2 (5694 bp). The gene of the prepropeptide of the alpha factor is linked to the human Lys plasminogen gene by the codons for a Kex2 interface and two Stel3 interfaces and is under the control of the AOX1 promoter.

Fig. 3: Physical map of the plasmid pSM49.8 (5715 bp). The human preplasminogen gene is under the control of the AOXl promoter.

Fig. 4: Physical map of plasmid pSM82.1 (5913 bp).

The gene of the prepropeptide of the alpha factor is linked to the human Lys plasminogen gene by the codons for a Kex2 interface and is under the control of the AOX1 promoter.

Fig. 5: Physical map of plasmid pSM58.1 (5925 bp). The gene of the prepropeptide of the alpha factor is linked to the human glu-plasminogen gene by the codons for a Kex2 site and two Stel3 sites and is under the control of the AOX1 promoter. Fig. 6: Physical map of plasmid pAC37.1 (11400 bp). The gene of the prepropeptide of the alpha factor is linked to the human Lys plasminogen gene by the codons for a Kex2 interface and two Stel3 interfaces and is under the control of the AOX1 promoter.

Fig. 7: Physical map of plasmid pJW9.1 (5925 bp). The gene of the prepropeptide of the alpha factor is linked to the human Lys plasminogen gene by the codons for a Kex2 site and two Stel3 sites and is under the control of the GAP promoter.

Fig. 8: Physical map of the plasmid pPLGl.l. The gene of the prepropeptide of the alpha factor is linked to the human mini-plasminogen gene by the codons for a Kex2 interface and two Stel3 interfaces and is under the control of the AOX1 promoter.

Fig. 9: Physical map of the plasmid pPLGl 1.2. The prepropeptide of the alpha factor is linked to the human mini-plasminogen gene by the codons for a Kex2 site and two Stel3 sites and is under the control of the GAP promoter.

Fig. 10: Detection of fibrinolysis activity in the clarification yard assay.

According to the invention, all microorganisms which are capable of carrying out the glycosylation and, if desired, the secretion of proteins are suitable as host organisms. Examples include: S. cerevisiae, P. pastoris, P. methanolica and H. polymorpha or the filamentous fungus Aspergillus sp.

In particular, a use of the functional plasminogen or plasmin produced according to the present production method in a pharmaceutical formulation is possible. In such a formulation, the functional plasminogen can be mixed with a pharmaceutically acceptable carrier or auxiliary as well as other suitable hoofs or additives in a manner known to those skilled in the art.

The Kex2 interface ensures that the propeptide is split off by the Kex2 protease located in the Golgi apparatus. This protease, also called protease YscF or kexin, is a proprotein-processing serine protease that cuts C-terminally from basic amino acid pairs (for example: Lys-Arg). The Stel3 interface ensures that the propeptide is split off by the protease Stel3 located in the Golgi apparatus. Stel3 (also called protease YscVI or Dipeptidylaminopeptidase A) is located in the late Golgi and gradually removes N-terminal Xaa-Ala dipeptides, eg from the immature α factor of the yeast S. cerevisiae.

In addition to the interfaces for the proteases Kex2 and Ste 13, further interfaces can be inserted which are recognized as a substrate by proteases located in the endoplasmic reticulum or in the Golgi device.

It is also possible to use only one for transport into the endoplasmic

To fuse the reticulum responsible signal sequence (prepeptide) with the plasminogen gene, d. H. the propeptide e.g. B. the mating factor of yeast S. cerevisiae is not absolutely necessary.

The microbiological, molecular biological and protein chemical methods mentioned in the examples are well known to the person skilled in the art. The following reference works should be mentioned as references: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor press, 1989 (10); Gassen & Schrimpf, Genetic Engineering Methods, Spektrum Akademischer Verlag, Heidelberg, 1999 (11); EasySelect ™ Pichia Expression Kit Instruction Manual, Invitrogen, Groningen, The Netherlands, Catalog No. Kl 740-01. The Pichia pastoris strains and expression systems are also from Invitrogen and are listed above. Instruction Manual described.

In short, pPICZA, B and C are 3.3 kb Pichia pastoris expression vectors. The vectors carry a Zeocin resistance gene for the direct selection of PtcÄt transformants. The vectors also carry a C-terminal tag sequence, which enables rapid purification and the detection of fusion proteins. PPICZalpha A, B and C are 3.6 kb Pichia pastoris expression vectors which also carry the Zeocin resistance gene and the above-mentioned C-terminal tag sequence. They also contain the alpha-factor secretion signal from Saccharomyces cerevisiae for an efficient transport of proteins into the medium.

The plasminogen can also be activated. For this purpose, the plasminogen can be incubated, for example, with a protease that was identified using the screening method according to the invention.

For this purpose, the plasminogen is preferably incubated with protease from S. griseus, with protease VIII or protease XVIII, with ficin, metalloendopeptidase, clostripain, with endoproteinase Glu-C, protease XIII, proteinase A, trypsin, endoproteinase Asp-N or elastase. It is also conceivable to activate plasminogen by incubating plasminogen with one of the proteases t-PA, u-PA, or vb-PA (vampire bat PA).

In a further preferred embodiment, the plasminogen is activated by incubating it with staphylokinase or with streptokinase. Streptokinase or staphylokinase form a 1: 1 complex with plasminogen. As a result of this complex formation, the plasminogen bound in the complex undergoes a conformational change, so that it becomes proteolytically active and is able to activate plasminogen to plasmin.

The functional plasminogen or activated functional plasmin produced according to the present recombinant manufacturing process is capable of hydrolyzing fibrin. It is also able to activate pro-matrix metalloproteases and growth factors.

The invention is explained in more detail below by examples.

Example la: Amplification of the Lys plasminogen gene with incorporation of the codons for a Kex2 interface at the 5 'end

The plasmid pPLGKG (Forsgren et al, FEBS Lett. 1987 Mar 23; 213 (2): 254-60 (2)), which contains the gene for pre-glu-plasminogen, was derived from the strain E. coli HB101 (pPLGKG) isolated using the QIAGEN Plasmid Midi-Kit (QIAGEN, Hilden). 150 ng of pPLGKG-DNA were linearized with 10 U of the restriction endonuclease EcoRI (Röche, Mannheim) and then purified with the QIAquick PCR-Purification Kit (QIAGEN, Hilden). The oligonucleotide-primer pair N034 (Seq. ID No. 1) and N036 (Seq. ID No. 2) were used to amplify the plasminogen gene. In addition to the bases complementary to the plasminogen gene, the oligonucleotide primer N036 bears the codons for the Kex2 interface. For the PCR, 0.5 UJ DNA polymerase (Hybaid, Heidelberg), 400 nM each of the oligonucleotide primers, 200 μM dNTP, 3 ng linearized pPLGKG-DNA and the associated reaction buffer were used in a final volume of 50 μl. The primer binding temperature was 58 ° C.

The resulting PCR product was examined for the expected size by agarose gel electrophoresis and purified using the QIAquick PCR Purification Kit.

Example lb: Cloning of the plasminogen gene in the vector pPICZαA

400 ng of the PCR product were cut with 10 U each of the restriction endonucleases Kspl and Xhό (Röche, Mannheim). 300 ng DNA of the plasmid pPICZαAA (Invitrogen, Groningen, Netherlands), which contains the prepropeptide sequence of the α factor of S. cerevisiae, were also cut with 10 U of the restriction endonucleases Kspl and Xhό. The DNA treated in this way was electrophoretically separated in a 0.9% agarose gel and the fragments obtained were extracted from the gel using the QIAquick Gel Extraction Kit (QIAGEN, Hilden). The vector DNA was combined with the insert DNA and ligated with 1 U T4 DNA ligase (Röche, Mannheim) at 4 ° C. overnight.

The DNA of the ligating ring approach was then purified using the QIAquick PCR purification kit and used to transform E. coli JM109 by electroporation.

The electroporated E. coli JM109 cells were incubated for 1 h at 37 ° C. in 1 ml SOC medium, then plated on LB agar solid medium with 20 μg / μl zeocin (Invitrogen, Groningen, Netherlands) and overnight at 37 ° C incubated.

The DNA was isolated from one of the E. co / z strains thus obtained using the QIAGEN Plasmid Mini-Kit (QIAGEN, Hilden) and separated 300 ng after endonucleolytic cleavage with the enzymes Xhol and Kspl by agarose gel electrophoresis. The isolated plasmid contained a fragment of the expected size and was named pMHS476.1 (Fig. 1). The correct sequence of the fusion gene from the prepropeptide gene of the alpha factor of the yeast Saccharomyces cerevisiae and the Lys plasminogen gene and the codons for the interface sequence of the protease Kex2 was confirmed by sequence analysis (Seq. ID No. 7).

Example lc: Transformation of Pichia pastoris with the plasmid pMHS476.1

With the QIAGEN Plasmid Midi-Kit, plasmid DNA of the plasminogen expression vector pMHS476.1 was isolated from the strain E. coli JM109 (pMHS476.1). 10 μg of pMHS476.1 DNA were linearized with 100 U Pmel (New England Biolabs, Frankfurt) and according to the protocol for electroporation of Pichia pastoris KM71H his 4 :: HIS 4 arg 4 aoxl :: reproduced in the EasySelect ™ Pichia Expression Kit Instruction Manual. ARG 4 genotype from Pichia pastoris Y-11430 (Northern Regional Research Laboratories, Peoria, USA) was used. The colonies grown on YPDS solid medium (EasySelect ™ Pichia Expression Kit Instruction Manual) with 100 μg / ml Zeocin after three to four days were streaked on YPD solid medium with 100 μg / ml Zeocin and used to inoculate liquid cultures. The colonies were designated Pichia pastoris KM71H / pMHS476.1-l / a, where "a" stands for the consecutive numbering of the colonies starting with 1. Example ld: Cultivation of Pichia pastoris KM71H pMHS476.1-l / l to -1/3 and induction of the plasminogen gene expression

To prepare the precultures, 100 ml of BMGY medium (EasySelect ™ Pichia Expression Kit Instruction Manual) were incubated in 1 1 chicane flasks at 28 ° C. and 250 rpm up to an OD ₆₀₀ = 20-30. The precultures were then centrifuged at 4645 g and 4 ° C. for 10 min. The cells harvested in this way were resuspended in BMMY medium (0.5% methanol) in order to obtain a biofum concentration of 80 g / l. 60 ml of these main cultures were incubated in 300 ml baffle flasks at 28 ° C and 250 rpm for 118 h. After 24 and 72 hours, 2% methanol was added. The baffle flask and the high speed of 250 rpm were used to ensure sufficient oxygen entry, which is necessary when using the AOX promoter.

Example le: Measurement of the plasminogen activity in the supernatant of the main cultures after activation with streptokinase

The main culture samples were centrifuged at 16,000 g for 10 min. 300 μl of the supernatant were incubated for 20 min at 37 ° C. with 1 μl streptokinase (S8026) (Sigma, Deisenhofen). To 750 μl 100 mM sodium phosphate buffer pH 8, 0.36 mM CaCl ₂ , 0.9% NaCl was added 100 μl N-tosyl-Gly-Pro-Lys-pNA solution (9.5 mg dissolved in 75 mg glycine / 10 ml, 2% Tween® 20) pipetted and incubated at 37 ° C for 10 min. At the start of the reaction, 250 μl of the supernatant pretreated with streptokinase were added and incubated further at 37 ° C. The increase in absorbance was measured photometrically at 405 nm. Supernatants from a P. pastoris KM71H culture grown in parallel and supernatants without streptokinase activation were used to determine control values. The following activity values were determined for the samples drawn after 72 h induction (1 U / 1 = 1 μmol N-tosyl-Gly-Pro-Lys-pNA conversion per minute per liter of culture supernatant): KM71H / pMHS476.1-1 / 1: 2 U / 1; KM71H / pMHS476.1-l / 2: 2 U / 1; KM71H / pMHS476.1-1 / 3: 1 U / 1. After 118 hours of induction, the following activity values could be determined: KM71H / pMHS476.1-1 / 1: 7 U / 1; KM71H / pMHS476.1-l / 2: 9 U / 1; KM71H / pMHS476.1-l / 3: 8 U / 1.

Example 2a: Amplification of the Lys plasminogen gene with incorporation of the codons of a Kex2 interface and two Stel3 interfaces at the 5 'end

The amplification of the Lys plasminogen gene for cloning into the vector pPICZαA with incorporation of the codons for a Kex2 interface and two Stel3 interfaces was carried out with the two oligonucleotide primers N034 and N057 (Seq. ID No. 3) using the in Example la conditions described performed. The oligonucleotide primer N057 carries in addition to the bases complementary to the plasminogen gene, the codons for the Kex2 interface and the Ste 13 interfaces.

Example 2b: Cloning of the Lys plasminogen gene amplified as described in Example 2a into the vector pPICZαA

The cloning of the Lys plasminogen gene into the vector pPICZαA for the production of a fusion gene from the gene of the prepropeptide of the alpha factor of the yeast S. cerevisiae and the human plasminogen gene with incorporation of the codons for the interfaces of the proteases Kex2 and Stel3 was analogous to the cloning described in Example 1b. The plasmid obtained was designated pSM54.2 (Fig.2). The correct sequence (Seq. ID No. 9) was confirmed by sequence analysis.

Example 2c: Transformation of Pichia pastoris with the plasmid pSM54.2

As described in Example lc for pMHS476.1, Pichia pastoris KM71H was transformed with the plasmid pSM54.2. The colonies obtained were referred to as Pichia pastoris KM71H / pSM54.2-l / a, where “a” in turn stands for the consecutive numbering of the colonies starting with 1.

Example 2d: Cultivation of Pichia pastoris KM71H / pSM54.2-l / l to -1/3 and induction of the plasminogen gene

The preparation of the precultures and of the main cultures and the induction with methanol were carried out analogously to the conditions described in Example 1d.

Example 2e: Measurement of the plasminogen activity in the samples of the main cultures after activation with streptokinase

The plasminogen activity after activation with streptokinase was determined as described in example le for KM71H / pMHS476.1-l / l to -1/3. The following activity values were obtained for the samples taken after 72 h induction: KM71H / pSM54.2-l / l: 2 U / 1; KM71H / pSM54.2-l / 2: 8 U / 1; KM71H / pSM54.2-l / 3: 6 U / 1. After 118 h of induction, the following activity values could be determined: KM71H / ρSM54.2-l / l: 8 U / 1; KM71H / pSM54.2-1 / 2: 17 U / 1; KM71H / pSM54.2-l / 3: 13 U / 1. Example 3a: Amplification of the plasminogen gene with its own signal sequence and cloning into the vector pPICZA; Transformation of Pichia pastoris

The amplification of the plasminogen gene including the sequence coding for the own signal peptide (pre-plasminogen) for cloning into the vector pPICZA was carried out with the two oligonucleotide primers N034 and N037 (Seq. ID No. 4) using the ones described in Example la Conditions carried out.

The cloning of the pre-plasminogen gene into the vector pPICZA was carried out analogously to the cloning described in Example 1b, with both the vector and the PCR product being cut with the restriction endonucleases Sful and Kspl. The plasmid obtained was designated pSM49.8 (Fig.3). The correct sequence (Seq. ID No. 11) was confirmed by sequence analysis.

As described in Example lc for pMHS476.1, Pichia pastoris KM71H was transformed with the plasmid pSM49.8. The colonies obtained were referred to as Pichia pastoris KM71H / pSM49.8-l / a, where “a” in turn stands for the consecutive numbering of the colonies starting with 1.

Example 4a: Amplification of the human GIu plasminogen gene with incorporation of the codons of a Kex2 interface and cloning into the expression vector pPICZα (alpha) A; Transformation of Pichia pastoris

The amplification of the glu-plasminogen gene for cloning into the vector pPICZαA with incorporation of the codons for a Kex2 interface was carried out with the two oligonucleotide primers N034 and N035 (Seq. ID No. 5) using the conditions described in Example la , In addition to the bases complementary to the glu-plasminogen gene, the oligonucleotide primer N035 bears the codons for the Kex2 interface.

The cloning of the glu-plasminogen gene in the vector pPICZαA for the production of a fusion gene from the gene of the prepropeptide of the alpha factor of the yeast S. cerevisiae and the human glu-plasminogen gene with incorporation of the codons for the interfaces of the protease Kex2 was analogous to the cloning described in Example 1b.

The plasmid obtained was designated pSM82.1 (Fig.4). The correct sequence (Seq. ID No. 13) was confirmed by sequence analysis. As described in Example lc for pMHS476.1, Pichia pastoris KM71H was transformed with the plasmid pSM82.1. The colonies obtained were referred to as Pichia pastoris KM71H / pSM82.1 / a, where “a” again stands for the consecutive numbering of the colonies starting with 1.

Example 5a: Amplification of the human glu-plasminogen gene with incorporation of the codons of a Kex2 site and two Stel3 sites at the 5 'end and cloning into the expression vector pPICZαA; Transformation of Pichia pastoris

The amplification of the glu-plasminogen gene for cloning into the vector pPICZαA with incorporation of the codons for a Kex2 interface and two Stel3 interfaces was carried out with the two oligonucleotide primers N034 and N056 (Seq. ID No. 6) using the in Example la conditions described performed. In addition to the bases complementary to the glu-plasminogen gene, the oligonucleotide primer N056 bears the codons for the Kex2 and Stel3 interfaces.

The cloning of the glu-plasminogen gene in the vector pPICZαA to produce a fusion gene from the gene of the prepropeptide of the alpha factor of the yeast S. cerevisiae and the human glu-plasminogen gene with incorporation of the codons for the interfaces of the proteases Kex2 and Stel3 was carried out analogously to the cloning described in Example 1b. The plasmid obtained was designated pSM58.1 (Fig.5). The correct sequence (Seq. ID No. 15) was confirmed by sequence analysis.

As described in Example lc for pMHS476.1, Pichia pastoris KM71H was transformed with the plasmid pSM58.1. The colonies obtained were designated Pichia pastoris KM71H / pSM58.1 / a, where “a” in turn stands for the consecutive numbering of the colonies starting with 1.

Example 6a: Insertion of the Lys plasminogen gene from pSM54.2 into the vector pPIC9K

150 ng of the vector pPIC9K (Invitrogen, Groningen, Netherlands) were cut with 10 U each of the restriction endonucleases Sαcl and Notl (both Röche Diagnostics, Mannheim). 300 ng of the plasminogen expression plasmid pSM54.2 (see Example 2b) were also cut with the enzymes Sßd and Notl. The DNA treated in this way was separated by gel electrophoresis with a 0.9% agarose gel. The larger fragment was extracted from the gel using the QIAgen Gel Extraction Kit (Qiagen, Hilden). The two fragments were combined and ligated with 1 U T4 DNA ligase overnight at 4 ° C. The transformation of E. coli DH5α, the isolation and the characterization of the resulting plasmid was carried out analogously to the description in Example 1b, the antibiotic ampicillin being used instead of the antibiotic Zeocin for the selection of transformants. The plasmid constructed in this way was designated pAC37.1 (FIG. 6).

Example 6b: Transformation of Pichia pastoris with the plasmid pAC37.1

As described in Example 1c for the transformation of Pichia pastoris KM71H with pMHS476.1, Pichia pastoris KM71 was transformed with the plasmid pAC37.1 linearized with the restriction endonuclease SaR. The transformed cells were plated out on the histidine-free medium MD-agar (Multi-Copy Pichia Expression Kit Instruction Manual) and incubated. The colonies obtained were referred to as Pichia pastoris KM71 / pAC37.1-3 / a, where “a” in turn stands for the consecutive numbering of the colonies starting with 1.

Example 6c: Cultivation of Pichia pastoris KM71 / pAC37.1-3 / l and induction of the plasminogen gene

The preparation of the precultures and of the main cultures and the induction with methanol were carried out analogously to the conditions described in Example 1d. Induction took place over 216 h. It started with a methanol concentration of 0.5%, after 24 h and then at 48 h intervals, 2% methanol was added.

Example 6d: Measurement of the plasminogen activity in the samples of the main cultures after activation with streptokinase

The plasminogen activity after activation with streptokinase was determined as described in example le for KM71H / ρMHS476.1-l / l to -1/3. Obtain 120 U / 1 activity for the sample taken after 120 h induction. After 216 h induction, 190 U / 1 activity could be measured.

Example 6e: Induction of Pichia pastoris KM71 / pAC37.1-3 / l in minimal medium (BSM) and measurement of the plasminogen activity in the samples of the main cultures after activation with streptokinase

After cultivating Pichia pastoris KM71 / pAC37.1-3 / l in BMGY complex medium (see example ld), 80 g of the centrifuged cells were placed in 100 ml of minimal BSM medium resuspended for the induction phase. The BSM (Basal Salts Medium) minimal medium is composed as follows:

H3PO4, 85%: 26.0 mL / L; CaC12-2H2O: 0.6 g / L; K2SO4: 18.0 g / L; MgSO4-7H2O: 14.0 g / L;

KOH: 4.0 g / L; Glycerin: 20 mL / L; Anti-foam: 1.0 mL / L; Trace solution: 8.0 mL / L;

Biotin solution (0.2 g / L): 8.0 mL / L.

Composition of the trace solution: H2SO4: 5.0 mL / L; CuSO4-5H2O: 6.0 g / L; KI: 0.08 g / L;

MnSO4-H2O: 3.0 g / L; Na2MoO4: 0.2 g / L; H3BO3: 0.02 g / L; CoC12: 0.5 g / L; ZnC12: 20.0 g / L; FeSO4-7H2O: 65.0 g / L.

2% methanol was added daily for induction. The plasminogen activity after

Activation with streptokinase was determined as described in example le for KM71H / pMHS476.1-l / l to -1/3. After 120 hours of induction, 193 U / 1 plasminogen activity was determined, after 168 h 289 U / L could be measured.

Example 6f: Detection of the plasminogen activity in the samples of the main cultures after activation with streptokinase in the fibrinolysis clarification test

In preparation for the fibrinolysis-Klärhof test (Stack, MS, Pizzo, SV, and Gonzalez-Gronow, M. (1992): Effect of desialylation on the biological properties of human plasminogen. Biochem. J. 284, 81-86) ( 13) 1.5 g of GTG low-melting agarose in 75 ml of 50 mM sodium phosphate buffer pH 7.4 were melted by boiling. 35 ml of a fibrinogen solution (225 mg / 37.5 ml 50 mM sodium phosphate buffer pH 7.4) were bubble-free with 350 μl thrombin solution (10 U / ml in 50 mM sodium phosphate buffer pH 7.4) mixed, stirred into the agarose solution and poured into a petri dish. After the fibrin agar solidified, 1 mm holes were made in the agar.

In order to demonstrate the fibrinolysis activity of the recombinantly produced plasminogen after streptokinase activation, 20 μl of the following solutions were pipetted into the holes and incubated at 37 ° C. for 20 h:

1: 0.5 mg / ml plasminogen (Röche, Mannheim)

2: Culture supernatant KM71 / ρAC37.1-3 / l from example 6e

3: 0.5 mg / ml plasminogen, streptokinase activated

4: Culture supernatant KM71 / pAC37.1-3 / 1 from example 6e, streptokinase-activated

5: 0.25 mg / ml plasminogen, streptokinase activated

6: Culture supernatant KM71 / pAC37.1-3 / l from Example 6e, diluted 1: 2, streptokinase activated

7: 0, 125 mg / ml plasminogen, streptokinase activated

8: Culture supernatant KM71H, as described in Example 6e for KM71 / pAC37.1-3 / l, streptokinase activated For activation with streptokinase, 2 μl streptokinase were added to 40 μl of the respective solutions

(100 U / μl, Sigma, Deisenhofen) pipetted and incubated at 37 ° C for 60 min.

The clarifications caused by fibrinolytic activity are shown in Fig. 8.

Example 6g: Purification of the plasminogen recombinantly produced in Pichia pastoris KM71 / pAC37.1-3 / l by affinity chromatography

50 ml of the culture supernatant of Pichia pastoris KM71 / pAC37.1-3 / 1 from Example 6c / 6d was dialyzed at 4 ° C. against 4 L of 50 mM sodium phosphate buffer pH 7.5. After 24 hours the dialysis buffer was changed and dialyzed for a further 24 hours. The dialysate was then pressed through a 0.02 μm filter and then applied to a Lysine-Sepharose ™ 4B column (diameter: 16 mm, height: 95 mm) (Amersham Biosciences) equilibrated with 50 mM sodium phosphate buffer pH 7.5. Proteins bound non-specifically bound were washed with 50 mM sodium phosphate buffer, pH 7.5, 0.5 M NaCl. The bound plasminogen was eluted with 50 mM sodium phosphate buffer pH 7.5, 0.01 M ε-aminocaproic acid. Individual samples were analyzed by 7.5% SDS-PAGE with subsequent silver staining (FIG. 11). The recombinant plasminogen contained in the fractions is in the gel at the level of the human plasminogen applied as a reference (see FIG. 11).

11 shows a 7.5% SDS-PAGE of the cleaning fractions from example 6g. The abbreviations used in FIG. 11 have the following meanings:

M: size standard (from top to bottom: 116 kDa, 66 kDa, 45 kDa, 35 kDa.)

D: dialysate,

N: non-binding fraction,

W: wash fraction,

F1-F5 plasminogen-containing elution fractions,

Plg: plasminogen (American Diagnostica, Pfungstadt).

Example 6h: Fermentation of Pichia pastoris KM71 / pAC37.1-3 / l to evaluate the pH and the influence of the substrate

50 ml of YEP-G medium (10 g / 1 yeast extract, 20 g / 1 casein peptone, 20 g / 1 glycerol) in a 1 1 wide-necked flask without baffles were mixed with 2 ml glycerol cryoculture Pichia pastoris KM71 / ρAC37.1-3 / l inoculated and incubated for 9 h at 30 ° C and 300 rpm. 5 ml of this culture was used to make 50 ml MG medium (5 g / 1 yeast nitrogen base w / o amino acids, 20 g / 1 glycerol, 2.5 ml / 1 biotin solution (0.2 g / l)) in a 1 1 wide neck shake flask without inoculating. This second preculture was incubated at 30 ° C. and 300 rpm for 16 h. The main culture was fermented in the "stirrer-pro" multi-fermenter system (DASGIP, Jülich), which allows the parallel fermentation of four cultures under different conditions. For this purpose, 150 ml of BSM medium (see Example 6e) were inoculated with 15 ml of the second preculture The fermentations were started at pH 6, the target pH was activated after the substrate dosing had started, and the different conditions and results of the parallel fermentations are shown in Table 1.

Sheetl

Experiment pH substrate feed rate OD ₆₀₀ plasminogen concentration

I 6 methanol profile 187 1.4 mg / 1

II 7 methanol profile 160 6.1 mg / 1

III 6 methanol / glycerol 1 ml / h 270 10.1 mg / 1

TV 6 methanol profile 130 3.4 mg / 1

In experiment IV, 30 g / 1 peptone was added to the medium. Before starting the methanol metering, glycerol feed medium (500 g / l anhydrous glycerol, 10 ml / l trace solution, 10 ml / l biotin solution [see Example 6e]) was metered in for 4 h at a constant rate. For the profile in experiments I, II and IN, the following term was entered as a dosing function: f (x) = Pl + (P2 / l + exρ (-P3 (t-P4)))) + (P5 / (l + exρ (-P6 (t-P7)))) with P1 = 0; P2 = 0.7; P3 = 0.2; P4 = 5; P5 = P6 = P7 = 0th It can be seen from Table 1 that the plasminogen concentrations are highest at neutral pH or mixed glycerol / methanol dosing.

Example 7a: Inserting the Lys plasminogen gene from pAC37.1 into the vector pGAPZαA

150 ng of the vector pGAPZαA (Invitrogen, Groningen, Netherlands) were cut with 10 U each of the restriction endonucleases Xhol and Notl (both Röche Diagnostics, Mannheim). 300 ng of the plasminogen expression plasmid pAC37.1 (see Example 6a) were also cut with the enzymes Xhol and Notl. The DNA treated in this way was separated by gel electrophoresis with a 0.9% agarose gel. The 2715 bp plasminogen Gene fragment from ρAC37.1 and the 3073 bp vector fragment from pGAPZαA were extracted from the gel using the QIAgen Gel Extraction Kit (Qiagen, Hilden). The two fragments were combined and ligated with 1 U T4 DNA ligase overnight at 4 ° C. The transformation of E. coli DH5α, the isolation and characterization of the resulting plasmid was carried out analogously to the description in Example 1b. The plasmid constructed in this way was designated pJW9.1 (Fig.7).

Example 7b: Transformation of Pichia pastoris with the plasmid pJW9.1

As described in Example 1c for the transformation of Pichia pastoris KM71H with pMHS476.1, Pichia pastoris KM71H was transformed with the plasmid pJW9.1 linearized with the restriction endonuclease Blnl. The transformed cells were plated on YPDS agar with 100 μg / ml Zeocin (EasySelect ™ Pichia Expression Kit Instruction Manual) and incubated. The colonies obtained were designated Pichia pastoris KM71H / JW9.1-a, where "a" in turn stands for the consecutive numbering of the colonies starting with 1.

Example 7c: Fermentation of Pichia pastoris KM71H / pJW9.1-3 to evaluate the pH value and the glycerol feed rate

The precultures and the fermentation in the "Stirrer-pro" were carried out as described in Example 6i. The results are shown in Tagelle 2.

Table 2

Experiment pH substrate feed rate OD ₆₀₀ plasminogen concentration

I 6.5 glycerol l ml / h 220 18.6 mg / 1

II 7.0 glycerol 1 ml / h 203 22.2 mg / 1

III 6.5 glycerol 0.5 ml / h 142 10.1 mg / 1

IV 7.0 glycerol 0.5 ml / h 99 3.8 mg / 1

Even with glycerol feeding, the best yields were obtained by fermentation at neutral pH, the influence of the substrate dosage (feed rate) on the product formation being clearly visible.

Example 7d: Fermentation of Pichia pastoris KM71H / pJTW9.1-3

50 ml of YEP-G medium (10 g / 1 yeast extract, 20 g / 1 casein peptone, 20 g / 1 glycerol) in a 1 1 wide neck flask without baffles were inoculated with Pichia pastoris KM71H / pJW9.1-3 and added for 9 h Incubated at 30 ° C and 300 rpm. 10 ml of this culture was used to make 40 ml MG medium (5 g / 1 yeast nitrogen base w / o amino acids, 20 g / 1 glycerol, 2.5 ml / 1 biotin solution (0.2g / l)) in a 1 1 wide-mouth shake flask without inoculation. This culture was incubated at 30 ° C and 300 rpm for 16 h.

3 1 BSM medium (see Example 6e) were inoculated with 30 ml of this culture in a 7.5 1 laboratory fermenter (type Labfors, ors AG, CH). The fermentation was carried out at 25 ° C. and a constant gassing rate of 3.2 l / min. After 24 h, glycerol solution (500 g / l glycerol, 10 ml / 1 trace solution, 10 ml / 1 biotin solution [see Example 6e]) was metered in. The dosage rate was gradually increased from 10 ml / h during the fermentation to 45 ml / h. After 250 h, 1375 U / 1 plasminogen activity after streptokinase activation could be measured.

Example 8: Identification of plasminogen activators

24 commercially available proteases were tested for their suitability for plasminogen activation. The experiments for this were carried out in 100 mM sodium phosphate buffer pH 8, 0.36 mM CaCl ₂ , 0.9% NaCl.

The proteases supplied in powder form were dissolved in buffer, the proteases supplied in solution were used directly or, if necessary, diluted with buffer. 25 ul of the protease solutions were mixed with 25 ul plasminogen according to the present invention (20 mg / ml) and incubated at 37 ° C for 10 min. The plasmin activity with respect to the substrate N-tosyl-Gly-Pro-Lys-pNA was then determined. For this purpose, 200 μl substrate solution (9.5 mg N-tosyl-Gly-Pro-Lys-pNA, dissolved in 75 mg glycine / 10 ml, 2% Tween® 20) were pipetted into 850 μl buffer, with the 50 μl of the pre-incubated plasminogen -Protease mixture added and incubated further at 37 ° C. The increase in absorbance was measured photometrically at 405 nm. To measure the increases in extinction caused by the proteases, controls were carried out in which a likewise pre-incubated buffer-protease mixture was used instead of the pre-incubated plasminogen-protease mixture.

The proteases protease from S. griseus, protease VIII, protease XXIII, protease XLX, protease XVIII, ficin, metalloendopeptidase, clostripain, Glu-C, protease XIII, chymopapain, chymotrypsin, protease X, bromelain, kallikrein and proteinase A were from Sigma, Related to Deisenhofen; Trypsin, papain, Asp-N, Dispase I, Lys-C, thrombin and elastase were from Röche, Mannheim, proteinase K was supplied by QIAGEN, Hilden. The protease stock solutions produced had the protein concentrations given in the table. The dilution factor F indicates the ratio in which the stock solutions were diluted for the measurements.

The following plasmin activities could be determined after activation (1 U / mg = 1 μmol N-tosyl-Gly-Pro-Lys-pNA conversion per minute per mg protein): Table 3

Protease plasmin activity n cprotein rmg / ml] F

Protease from S., griseus 613.3 U / mg 0.77 1000

Protease VIII 9 U / mg 3.58 1000

Protease XXIII * 7.8 50000

Protease XLX * 2.78 100

Protease XVIII 0.7 U / mg 1.79 100

Ficin 0.01 U / mg 0.81 1

Metalloendopeptidase 8.9 U / mg 0.01 1

Clostripain 1.7 U / mg 0.25 1

Endopoteinase 'Glu-C 0.6 U / mg 0.81 1

Protease XIII 0.01 U / mg 0.43 1

Chymopapain * 2.02 1

Chymotrypsin * 0.14 1

Protease X * 2.01 1

Bromelain * 0.81 1

Kallikrein * 0.56 1

Proteinase A 0.02 U / mg 0.36 1

Trypsin l l kU / mg 3.40 100000

Papain * 0.64 10

Endoproteinase Asp-N 4.3 U / mg 0.004 1

Dispase I * 0.2 1

Endoproteinase Lys-C * 0.01 1

Thrombin 83.0 U / mg 0.59 500

Elastase 0.63 U / mg 0.36 5

Proteinase K * 3.60 100

* No plasminogen activation could be detected for the proteases Protease XXIII, Protease XLX, Chymopapain, Endoproteinase Lys-C, Chymotrypsin, Papain, Dispase I, Protease X, Bromelain, Kallikrein and Proteinase K. Example 9: Pharmaceutical Formulations

The recombinant functional plasminogen used in the following examples was obtained using the inventive production process. Here, the term "plasminogen" refers to both recombinant micro, mini, Lys or glu plasminogen and the term "plasmin" to plasmin, which by proteolytic cleavage of recombinant micro, mini, Lys or glu plasminogen was won. The activation of micro, mini, Lys or Glu plasminogen can be obtained using the same plasminogen activators, in particular plasminogen activating proteases, as described above, but is not limited to this, the ratio of units activator to units plasminogen (micro , Mini, Lys or Glu plasminogen) is approximately 1: 1000.

The plasminogen can be proteolytic, i.e. H. activated by the proteases tissue plasminogen activator, urokinase or the proteases VIII or protease from S. griseus described in the patent, and also by complexation with streptokinase or staphylokinase.

Example 9a: Pharmaceutical formulations

hydrogels

Basic formulation for hydrogels (100g)

Plasminogen 100 U

Plasminogen activators) 0.1 U

Hydroxyethyl cellulose 10,000 3.5 g optional preservation (sorbic acid / potassium sorbate 0.1-0.4% », PHB ester 0.1%). purified water ad 100.0

The hydroxyethyl cellulose or instead hypromellose or methyl cellulose can alternatively be used in an amount of 0.5-15.0 g.

gel

Plasminogen 1000 U

Plasminogen activators) 1 U

Glycerol (85%) 150.0 g

Hydroxyethyl cellulose 10,000 32.5 g optional preservation (sorbic acid / potassium sorbate 0.1-0.4%, PHB ester 0.1%)

Ringer's solution without lactate to 1000.0g

alternatively: 100g contain:

Plasminogen 100 U

Plasminogen activator (s) 0.1 U

Hydroxyethyl cellulose 30,000 2.5 g

Glycerol 85% 10.0 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2%, PHB ester 0.1%) purified water ad 100.0

alternatively:

100g gel contain:

Plasminogen 100 U

Plasminogen activator (s) 0.1 U

Polyacrylic acid 1 g

Propylene glycol 8 g

Medium chain triglycerides 8 g

Diethylamine (for pH adjustment) q.s. optional preservation (sorbic acid / potassium sorbate 0.1-0.2%, PHB ester 0.1%)

2-propanol 0-1 g

100g of water

Hydrophilic ointment (macrogol ointment)

50g included

Plasminogen 50 U.

Plasminogen activator (s) 0.05 U

Macrogol 400 30.0 g

Macrogol 4000 10.0 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2%, PHB ester 0.1%) purified water ad 50.0 g

alternatively:

anhydrous macrogol ointment

100g contain:

Plasminogen 100 U

Plasminogen activators) 0.1 U

Macrogol 300 50 g

Macrogol 1500 to 100 g alternative

Water-absorbing ointment

Plasminogen 100 U

Plasminogen activator (s) 0.1 U

Cetylstearyl alcohol 29 g

Paraffin, viscous 34 g

Vaseline, white to 100 g

Hydrophobic ointment

Plasminogen 100 u

Plasminogen activators) 0.1 u

Vaseline 80.0 g

Paraffin thin to 100.0 g

Hydrophobic paste

Plasminogen 100 U Plasminogen activator (s) 0.1 U Hypromellose 400 20 g Vaseline, white to 100 g

alternative

Plasminogen 100 U

Plasminogen activator (s) 0.1 U

Carbomer (e.g. Carbopol 974p) 15 g

Paraffin, viscous 40 g white vaseline to 100.0 g

Cream:

Plasminogen 100 U

Plasminogen activator) 0.1 U

Medium chain triglycerides 20 g

Emulsifying cetylstearyl alcohol 10 g

Lanolin 10 g

Sorbitol 10 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Water purified to 100 g Nonionic hydrophilic cream

Plasminogen 100 u

Plasminogen activator (s) 0.1 u

Cetyl alcohol 20 g

2-ethyl lauromyristate 10 g

Glycerol 85% 6 g

Potassium sorbate 0.14 g

Citric acid 0.07 g

Water ad 100 g

Nonionic cream

Plasminogen 100 U plasminogen activator (s) 0.1 U polysorbate 60 5 g cetylstearyl alcohol 10 g glycerol 85% 10 g petroleum jelly, white 25 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2% », PHB ester 0.1% ) Water ad 100 g

Liposomal formulation

Plasminogen 100 U

Plasminogen activator (s) 0.1 U

Soy lecithin, chicken lecithin 15 g optional preservation

(Sorbic acid / potassium sorbate 0.1-0.2%, PHB ester 0.1% »or diazodinyl urea l-2g)

Water to 100.0 g

Capsule one capsule with 0.25g powder / granules contains:

Plasminogen 5 U

Plasminogen activator (s) 0.005 U

Starch 0.1 g

Silicon dioxide 0.02 g

Magnesium stearate 0.002 g

Polymethacrylate copolymers / polymethacrylic acid 0.015 g

Triethyl citrate 0.0005 g

Talc 0.001 g

Cellulose, microcrystalline at 0.25 g alternative

one capsule with 0.25g powder / granules contains:

Plasminogen 5 U

Plasminogen activator) 0.005 U

Silicon dioxide 0.01 g

Magnesium stearate 0.002 g

Polymethacrylate copolymers / polymethacrylic acid 0.015 g

Triethyl citrate 0.0001 g

Talc 0.001 mg

Mannitol at 0.25 g

tablet

Contain 100mg tablet granules

Plasminogen 5 U

Plasminogen activator (s) 0.005 U

Strength 30 mg

Silicon dioxide 2 mg

Magnesium stearate 4 mg

Polymethacrylate copolymers / polymethacrylic acid 5 mg

Triethyl citrate 0-1 mg

Talc 0.0001 mg

Cellulose microcrystalline at 100 mg

pellets

100g pellets contain:

Plasminogen 2000 U

Plasminogen activator (s) 2 U

Starch 20 g

Sucrose stearate 20 g

Silicon dioxide 2 g

Magnesium stearate 3 g

Polyvinylpyrrolidone 0 - 1 g

Polymethacrylate copolymers / polymethacrylic acid 5 g

Talc 0.2 g

Triethyl citrate 0.1 g

Cellulose, microcrystalline at 100 g injection

Plasminogen 500 U.

Plasminogen activator (s) 0.5 U

Ethanol 0 - 1 g

Propylene glycol 10 g

Polyethylene glycol 0 - 1 g

Sodium chloride q.s. optional buffer (sodium hydrogen phosphate / sodium dihydrogen phosphate) purified water to 100 ml

Instead of micro, mini, Lys or Glu plasminogen, the same amount based on the activity of plasmin can also be used in the formulations listed. If plasmin is used directly, the plasminogen activator (s) need not be contained in the pharmaceutical formulation.

Example 9b: Pharmaceutical Formulations a) Hydrogels

Basic formulation for hydrogels (100 g) plasmin 100 U

Hydroxyethylcellulose 400 2.5 - 5.0 g of purified water to 100.0 g

The swelling time is 1 to 3 hours.

It is possible to use 1 - 1000 U plasmin per gram of hydrogel.

b) Hydrophilic ointment

Basic formulation of a hydrophilic ointment (1000 g):

Plasmin 1000 U anhydrous glycerol 85.0 g

Hydroxyethylcellulose O.000 32.5 g optional polyhexanide 0.2% by weight

Ringer's solution without lactate at 1000.0 g

Polyhexanide can optionally be used as an antimicrobial agent in a concentration of up to 0.2

% By weight are added.

Instead of 10,000 hydroxyethyl cellulose (Natrosol 250 ^® HX PHARM) can also

Hydroxyethylcellulose 400 (e.g. Tylose ^® H 300 or Natrosol 250 ^® HX PHARM)

It is possible to use 1 - 10000 U plasmin per gram of ointment. c) ointment

Basic formulation for ointment (50 g)

Plasmin 50 U.

Macrogol 400 30.0 - 32.5 g

Macrogol 4000 12.5 - 7.5 g of purified water to 50.0 g

Preparation:

12.5 g Macrogol 4000 and 30.0 g Macrogol 400 (7.5 g Macrogol 4000 and 32.5 g Macrogol 400 for soft ointments) are heated in an ointment dish in a water bath until the macrogol has melted. After cooling, the corresponding amount of plasmin, which was produced with the aid of the inventive method, is added dissolved in 7.5 g of purified water and then homogenized.

Φ capsules

Basic formulation for 0.5 g

Plasmin 5 U

Lactose 0.42 g

Thickness 0.06 g

Magnesium stearate 0.02 g

It is possible to use 0.1-100 U plasmin per capsule.

e) Solution for injection / infusion basic formulation for 100 ml

Plasmin 500 U.

Ethanol 0.01 g

Propylene glycol 30 ml of purified water to 100 ml.

It is possible to use 1 - 500 U plasmin per ml solution.

Instead of plasmin, micro-, mini-, lys- or glu-plasminogen can also be used in the amounts specified for the plasmin based on the activity in units, if at least one plasminogen activator is added at the same time, in an amount of 1: 10,000 to 1 : 100, preferably in an amount of 1: 1000 based on the plasminogen activity. Example 10a: Amplification and cloning of various forms of the mini and microplasminogen gene and cloning into the vector pPICZαA; Transformation of Pichia pastoris

Mini and microplasminogen are shortened plasminogen derivatives which lack N-terminal domains, but which can nevertheless be activated to active plasmin. The amplification of the mini and microplasminogen genes for cloning into the vector pPICZαA was carried out with the oligonucleotide primer N034 for the 3 'end and one of the primers N036a-j (Seq. ID No. 19 to 28) for the respective 5' end using the conditions described in Example la. In addition to the bases complementary to the plasminogen gene, the oligonucleotide primers N036a, c, e, g, i carry the codons for the Kex2 interface, and the primers N036b, d, f, hj also carry the codons for the Kex2- Interface the codons for two Stel3 interfaces. The primer N034 also carries a Kspϊ interface, the primers N036 a-j carry a J ^ ol interface.

The cloning of the mini and microplasminogen genes into the vector pPICZαA was carried out analogously to the cloning described in Example 1b, both the vector and the respective PCR product with the and Kspl were cut. In summary, the primers used, the names of the plasminogen derivative, the encoded protease cleavage sites, the name of the plasmids obtained and the N-terminal amino acid of the secreted plasminogen derivative can be found in the following table.

5 'primer 3' primer name protease plasmid name N-terminal interface amino acid *

N036a N034 miniplasminogen Kex2 pPLGl.l A463

N036b N034 mini plasminogen Kex2, 2xStel3 pPLG2.1 A463

N036c N034 Microplasminogen Kex2 pPLG3.2 K550

N036d N034 Microplasminogen Kex2, 2xStel3 pPLG4.2 K550

N036e N034 Microplasminogen Kex2 pPLG5.3 L551

N036f N034 Microplasminogen Kex2, 2xStel3 pPLG6.1 L551

N036g N034 Microplasminogen Kex2 pPLG7.1 A562

N036h N034 Microplasminogen Kex2, 2xStel3 pPLG8.3 A562

N036i N034 Microplasminogen Kex2 pPLG9.1 S564

N036J N034 Microplasminogen Kex2, 2xStel3 pPLGlO.l S564

* The numbering refers to the 810 amino acid long pre-plasminogen (Seq. ID No. 12) The plasmid pPLGl. 1 is shown as an example in FIG.

As described in Example lc for pMHS476.1, Pichia pastoris KM71H was used with the

Plasmid pPLGl.l transformed. The resulting colonies were called Pichia pastoris

KM71H / pPLGl.l-l / a, where "a" in turn for the consecutive numbering of

Colonies starting at 1.

The generation of strains based on the plasmids pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLGό.l, pPLG7.1, pPLG8.3, pPLG9.1 and pPLGlO.l was carried out in accordance with the

Production of the strain KM71H / pPLGl.l-l / a.

Oligonucleotide primer N036a-j

N036a AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG

N036b AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG

N036c AAAAACTCGAGAAAAGAAAACTTTACGACTACTG

N036d AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG

N036e AAAAACTCGAGAAAAGACTTTACGACTACTGTG

N036f AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG

N036g AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG

N036h AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG

N036i AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC

N036j AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC.

Example 10b: Amplification and cloning of various forms of the mini and microplasminogen gene and cloning into the vector pGAPZαA; Transformation of Pichia pastoris

The amplification of the mini and microplasminogen genes for cloning into the vector pGAPZαA was carried out with the oligonucleotide primer N034 for the 3 'end and one of the primers N036a-j (Seq. ID No. 19 to 28) for the respective 5th 'End performed using the conditions outlined in Example la. In addition to the bases complementary to the plasminogen gene, the oligonucleotide primers N036a, c, e, g, i carry the codons for the Kex2 interface, and the primers N036b, d, f, h, j also carry the codons for the Kex2 interface the codons for two Ste 13 interfaces. The primer N034 also carries an i & pI interface, the primers N036 a-j carry a J ^ ol interface.

The cloning of the mini and microplasminogen genes in the vector pGAPZαA was carried out analogously to the cloning described in Example 1b, both the vector and the respective PCR product with the restriction endonucleases Xhol and Kspl were cut. In summary, the primers used, the names of the plasminogen derivatives, the encoded protease cleavage sites, the name of the plasmids obtained and the N-terminal amino acid of the secreted plasminogen derivative can be found in the following table.

5 'primer 3' primer name protease plasmid name N-terminal interface amino acid *

N036a N034 mini plasminogen Kex2 pPLG11.2 A463

N036b N034 mini plasminogen Kex2, 2xStel3 pPLG12.1 A463

N036c N034 Microplasminogen Kex2 pPLGB.l K550

N036d N034 Microplasminogen Kex2, 2xStel3 pPLG14.2 K550

N036e N034 Microplasminogen Kex2 pPLG15.1 L551

N036f N034 Microplasminogen Kex2, 2xStel3 pPLG16.3 L551

N036g N034 Microplasminogen Kex2 pPLG17.2 A562

N036h N034 Microplasminogen Kex2, 2xStel3 pPLGIS.l A562

N036i N034 Microplasminogen Kex2 pPLG19.2 S564

N036J N034 Microplasminogen Kex2, 2xStel3 pPLG20.1 S564

* The numbering refers to the 810 amino acid long pre-plasminogen (Seq. ID

No. 12)

The plasmid pPLGl 1.2 is shown by way of example in FIG. 9.

As described in Example 7a for pJW9.1, Pichia pastoris KM71H was obtained using the

Restriction endonuclease Blnl linearized plasmid pPLG11.2 transformed. The resulting colonies were named Pichia pastoris KM71H / pPLG11.2-l / a, where

"A" again stands for the consecutive numbering of the colonies starting with 1.

In order to generate strains based on the plasmids pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 and pPLG20.1, the KM71H strain was produced in accordance with the preparation /pPLGl.ll/a.

sequence Listing

Sequence 1: oligonucleotide primer N034

AAAAACCGCGGTCAATTATTTCTCATCACTCCC

Sequence 2: oligonucleotide primer N036

AAAAACTCGAGAAAAGAAAAGTGTATCTCTCAGAGTG

Sequence 3: oligonucleotide primer N057

AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTG

Sequence 4: oligonucleotide primer N037

AAAAATTCGAAAAATGGAACATAAGGAAGTGG

Sequence 5: oligonucleotide primer N035

AAAAACTCGAGAAAAGAGAGCCTCTGGATGACTAT

Sequence 6: oligonucleotide primer N056

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTAT

Sequence 7: human Lys-plasminogen fusion gene with the codons for the Kex2

Interface and the gene of the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCGTCCGCATTAGCTGCT CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTAC AGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCT CCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTAC TGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAG AGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAA AACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCT CAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAG AATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAAC AAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCC ACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTT TCCGGGCÄCACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCA GAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGG GCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCC TGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACC CCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACC ACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAG ACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCC GATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTG AAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCA GATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGC AAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCAT AGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGC CGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTT TACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCA TCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA ATAGAAGTGTCTAGGCTGTTCTTG GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA AGCAGTCCTGCCGTCΆTCACTGACAAAGTAΆTCCCAGCTTGTCTGCCATCCCCAAΆTTAT GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTT GGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGT GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 8: human Lys plasminogen with Kex2 cleavage and the signal peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSAAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTKNGLLFINTTIASIAAKEEGVS EKR VYLSECKTGNGK Y RGT SKTK GITCQKWSSTSPHRPRFSPATHPSEG EE YCRNPDNDPQG PWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQA DS QSPffi GYIPSKFPNKNLKKNYCRNPDRELRP CFTTDPNKR ELCDIPR CTTPPPSSGPTYQCLKGTGEKTYRGNVÄVTVSGHTCQH SAQTPHTHNRTP ENFPCKN DE YCRNPDGKRAP CHTTNSQVRWEYCKI SCDSSPVSTEQ LAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGKKCQS SSM PHRHQK TPENYPNAG T YCRNPDADKGP CFTTDPSVR EYCN KCSGTEASV VAPPPWL PDVETPSEEDCMFGNGKGYRG RATTV GTPCQD AAQEPH RHSIFTPETNPRAGLEK YCRNPDGDVGGP CYTTNPRKLYDYCDVPQCA APSFDCGKPQVEPKKCPGRWGGCVAHPHSWP QVSLRTRFGMHFCGGTL ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSR FLEP TRKDIAL KLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTF GAGLLKEAQLPVIEN VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG GP VCFE DKYI QGVTS GLGCARPNKPGVYVRVSR VT IEGVMKN *

Sequence 9: human Lys-plasminogen fusion gene with the codons for the Kex2 site and two Stel3 sites and the gene for the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae _A T _GAGATTTCCTT C _AATTTT TA _CT GCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT _CCAGTC A _ACA C _TA C _AA C _A G _A AG _A TGAΆACGGCACAAATTCCGGCTGAAGCTGTCATCGGT _TACT C _A G _A T _TT AG _A AGGGGATT _T CGATGTTGCTGTTTTGCCATTTTCCAΆCAGCACAAAT _AACG GG _TTATT G _T T _TATA A _A TACTACTATTGCCΆGCATTGCTGCTAAAGAAGAAGGGGTA _CT CTCG _A GAAA _A GAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTGCAAGACTGGGAAT _A GGAΆAG ACTACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGG AGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTG G _A G _A TCC GGAG _A ACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACΤ _A GAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCAT TGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAG GCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAG AACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACC ACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCA TCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTG GCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACAT AACAGGACACCAGAAAACTTCCCCTG CAAAAATTTGGATGAAAACTACTGCCGCAATCCT GACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGT AAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCA CCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGC ACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACAC CGGCACCAGAAGACCCCAGAAAACTACCCÄAATGCTGGCCTGACAATGAACTACTGCAGG AATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAG TACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTT GTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGATGGGAAA GGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCC CAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAA AAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAAT CCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCC CCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAA TCTCGAACCG CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA TCCCCAAATTATGTGGTCGCTGΆCCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAA GTGTGCAΆTCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG CΆTTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTC GAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG AGAAATAATTGA

Sequence 10: human Lys-plasminogen with Stel3 and Kex2 interfaces and the signal peptide of the alpha-factor of the yeast Saccharomyces cerevisiae RFPSIFTAVLFAASSALAAPVN TTEDETAQIPAEAVIGYSD EGDFDV AV PFSNSTNNGL FI TTIASIAAKEEGVSLEKREAEAKVY SECKTGN GK1 -TRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEG EENYCRNPDN DPQGPWCYTTDPEKRYDYCDILECEEEC HCSGENYDGKISKTMSG ECQ ATiTOSQSPHAHGYIPSKFPNKl r KKNYCRNPDRELRP CFTTDPN R ELC DIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQH SAQTPHTH KKTPENFPC N DE YCRNPDG RAP CHTTNSQVR EYC IPSCDSSPV STEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTG CQSWSSMTPH RHQKTPEJSTYPNAG TMNYCRNPDADKGP CFTTDPSVRWEYCN KKCSGT EAS APPPWLLPDVETPSEEDCMFGNGKGYRGK_RATTWGTPCQDWAA QEPHRHS I FTPETNPRAGLEKNYCRNPDGDVGGP CYTTNPRK YDYCDV PQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHS P QVSLRTRFGMHFC GGTLISPΞWV TAAHCLEKSPRPSSYKVI GAHQEVNLEPHVQEIEVSRL FLΞPTRKDIA LKLSSPAVITDKVIPAC PSPNYWADRTECFITG GET QGTFGAGL KEAQLPVIENKVCNRYEF NGRVQSTE CAGHLΆGGTDSCQ GDSGGP VCFEKD YILQGVTSWG GCARPNKPGVYVRVSRFVTWIEGV

RNN *

Sequence 11: human pre-plasminogen gene

_AT GGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAGAG CCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAGCAG CTGGGAGCAGGAAGTATAGAΆGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACC TGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGG AAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGΆAAAGAAAGTGTATCTC TCAGAGTGCΆAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAAT GGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGΆCCTAGATTCTCACCTGCT ACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAG GGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAG TGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACC ATGTCTGGACTGGAΆTGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATT CCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAG CTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCC CGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGT GAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGT GCACAGACGCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGAT GAAΆACTACTGCCGCAA TCCTGΆCGGAΆAAAGGGCCCCATGGTGCCATACAACCAACAGC CAAGTGCGGTGGGAGTACTGTAΆGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAA CAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGT GATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCT TGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGC CTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACA GACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGT GTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGAC TGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACG CCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACA AATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGT CCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGG GTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCT GCCCACTGCTTGGAGAAGTCCCCAΆGGCCTTCATCCTACAΆGGTCATCCTGGGTGCACAC CAAGAAGTGAATCTCGAACCGCATGTTCAGGAΆAT AGAAGTGTCTAGGCTGTTCTTGGAG CCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAA GTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTC A _TC A _C T _GGCTGGG G _A G _AA ACCC _A AGG _TA CTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG _{CTCCCTGTGATTGAGAAT} A _AA GTG _T GC _AA TCGCTATGAGTTTCTGAATGGAAGAGTCCAA _TCCACCG A _ACTCT G _T GC _T GGGCAT _T TGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT _GG A _G G _T CC _T C _T GGT _T TGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG _GGT C _TT GGC _T G _T GCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT ACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 12: human pre-plasminogen

EHKEVV L FLKSGQGEP DDYVNTQGASLFSVTKKQ GAGSIEECA AKCΞEDEEFTCRAFQYHSKEQQCVIMAENRKSS11IRMRDW FEKKVYL SΞC TGNGKNYRGTMSKTKNGITCQ SSTSPHRPRFSPATHPSEGLEEN YCRPDKIDPQGPWCYTTDPEKRYDYCDI ECEEECMHCSGENYDGKISKT SGLECQADSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDP NKRE CDIPRCTTPPPSSGPTYQC KGTGENYRGNVAVTVSGHTCQH S AQTPHTHNRTPENFPC DEKTYCRNPDGKRAP CHTTNSQVR EYCKIP SCDSSPVSTEQLAPTAPPE TPWQDCYHGDGQSYRGTSSTTTTGKKCQS WSSM PHRHQKTPE YPNAGLT NYCRPDAD GP CFTTDPSVREYCN KCSGTEASWAPPPW LPDVETPSEEDC FGNGKGYRGKRATTV GT PCQDAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGP CYTTNPRK LYDYCDVPQCAAPSFDCGKPQVEP KCPGRWGGCVAHPHSWP QVSLRT RFG HFCGGTLISPE VTAAHCLEKSPRPSSYKVI GAHQEV LEPHVQ EIEVSRLF EPTRDIA KLSSPAVITDKVIPACLPSPWYWADRTECF ITG GETQGTFGAGLL EAQ PVIENKVCRYEFLNGRVQSTELCAGHLA GGTDSCQGDSGGPLVCFE D YILQGV S GGCARPNKPGVYVRVSRFV TWIEGVMRNN *

Sequence 13: human glu-plasminogen fusion gene with the codons for the Kex2-

Interface and the gene for the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAΆΆGAGAGCCTCTGGΆTGACTATGTGAATACCCAGGGGGCTTCACTGTTC AGTGTCACTAAGAAGCAGCTGGGAGCAGGAΆGTATAGAAGAATGTGCAGCAAAATGTGAG GAGGACGAAGAATTCACCTGCAGGGCATΤCCAΑTATCACAGTAAAGAGCAACAATGTGTG ATAATGGCTGAAΆACAGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTT GAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACG ATGTCCAAAACAAAAAΆTGGCATCACCTGTCAAAAΆTGGAGTTCCACTTCTCCCCACAGA CCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAAT CCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGAC TACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGAC GGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCA CACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAΆCCTGAAGAAGAATTACTGT CGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGG GAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAG TGTCTGAAGGGAAC AGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCAC A _CCTGTCAGCA C _T G _GAG TGCA _CA G _A CCCCTCACACACATAACAGGACACCAGAAAACTTC _CCCTGC A _AAAATTTG G _A TG _AAAA CT _A CTGCCGCAATCCTGACGGAAAAAGGGCCCCATGG _{TGCCATACAACCA} AC _AG CC _AA G _T GCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCC _TCCCCA G _TATCCA CGG _AA CA _ATT GGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTC _CAGGACTGCTAC C _AT G _GT G _A TGG _A CAGAGCTACCGAGGCACATCCTCCACCACCACCACA _GG A _AAG A _A G _{T G T} C _A G _T GG _TCTT CATCTATGACACCACACCGGCACCAGAAGACCCCAGAA _A AC _TA CCCA GCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGC _AAT CCC _{T T} GGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGC CAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAG ACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCG ACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGC ATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCT GATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTAC TGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCG AAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG CAAGTCAGTCTTAGAACAAGGT TTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCA GAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG TCTAGGCΓGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCT GACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTA CAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 14: human Glu plasminogen with Kex2 cleavage and the signal peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSD EGDFDV AVIIPFSNSTLWGLFINTTIASIAAKEEGVSLEKREPLDDYVNTQGAS F SVKKQ GAGSIΞECAACEEDEEFTCRAFQYHSKEQQCVIMAENRKSSI IIRMRDVV FEKKVYLSEC TGNGKNYRGTMSKTKNGITCQ SSTSPHR PRFSPATHPSEG EE YCRNPDNDPQGPWCYTTDPE RYDYCDILECEEE CFFLCSGENYDGKISKMSGECQAWDSQSPHAHGYIPS FPNNIJKKYC RNPDRE RPWCFTTDPNKRWE CDIPRCTTPPPSSGPTYQCLKGTGENYR GNVAVVSGHTCQH SAQTPHTHNRTPENFPCKLTTDENYCRNPDGKRAP CHTTNSQVR EYCKIPSCDSSPVSTEQLAPTAPPE TPWQDCYHGDGQS YRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKG P CFTTDPSVR EYCNLKKCSGTEASWAPPPWL PDVETPSEEDCMFG NG GYRGRATTVTGTPCQDAAQEPHRHSIFTPETNPRAG EKNYCRNP DGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGG CVAHPHS P QVS RTRFGHFCGGTLISPEWVLTAAHC EKSPRPSSYK VI GAHQEVN EPHVQEIEVSRLF EPTRDIALLK SSPAVITDKVIPA CLPSPISΓYVVADRTECFITGGETQGTFGAGLL EAQLPVIENVCNRYEF L ^NG RV ^Q STELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGV S GLGC ARPNKPGVYVRVSRFVWIEGV RNN * Sequence 15: human glu-plasminogen fusion gene with the codons for the Kex2 site and two Stel3 sites and the gene for the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae

A _TG AGATTT _C CTT _C A _A TTT _T T _A CTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT _CC A _GTC AA _C ACT _AC AA _{C AA} AG GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT _TACT C _AGATT DAY _AAGGGGATTT CGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT _{AACGGGTTATTGTTTATAA} ATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA _TC T _CTCGAGA A _AA G _A G _A GGCTGAAGCTGAGCCTCTGGATGACTATGTGAATACCCAGGGG

GC _TTCACTGTT C _A GTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCA GC _{AAAAT TGA} GG G _A GG _A CGAAGAATTCACCTGCΆGGGCATTCCAATATCACAGTAAAGΆG CAAC _AAT G _T G _T G _{A A} TAΆTGGCTGAAΆACAGGAΆGTCCTCCATAΆTCATTAGGATGAGAGAT GTAGTTTTATTTGAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC TACAG GGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACT TCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAAC TACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAA AAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGA GAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGAC TCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAG AAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCC AACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGT CCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACC GTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACA CCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAA AGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATA CCG TCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTA ACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCC ACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAG AAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGAT GCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCΆGGTGGGAGTACTGCAAC CTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTT CCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGA GGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCC CATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAΆAAAAATTAC TGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAΆ CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG CTAAGCAGTCCTGCCGT CATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAAT TATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT CGCTΆTGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCT GGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT

TGA Sequence 16: human Glu-plasminogen with Stel3 and Kex2 interfaces and the signal peptide of the alpha-factor of the yeast Saccharomyces cerevisiae RFPSIFTÄVLFAASSA AAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNG LFINTTIASIAAKEEGVSLEKREAEAEPLDDYVNTQG ASLFSV KKQ GAGSIEECAAKCEEDΞEFTCRAFQYHSKEQQCVIMAENR KSS11IR RDVVLFEKKVY SEC TGNGKNYRGTMSKT NGITCQKWSST SPHRPRFSPATHPSEGLEΞNYCRNPDNDPQGP CYTTDPEKRYDYCDILE CEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLK KNYCRNPDRELRP CFTTDPN R ELCDIPRCTTPPPSSGPTYQCLKGTG ENYRGNVAVTVSGHTCQH SAQTPHTHNRTPENFPCK DE YCRNPDGK RAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQ APTAPPELTPWQDCYHG DGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPD AD GPWCFTTDPSVR EYCN KKCSGTEASWAPPPW LPDVETPSEED CMFGNGKGYRG RATTVTGTPCQD AAQEPHRHSIFTPETNPRAGLEK Y CR PDGDVGGP CYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR WGGCVAHPHS P QVSLRTRFG HFCGGTLISPEWVLTAAHCLE SPRP SSYKVILGAHQEVN EPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDK VIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQ PVIENKVCN RYEFLNGRVQSTE CAGHLAGGTDSCQGDSGGPLVCFEKDKYIL QGVTSW GLGCARPNKPGVYVRVSRFVTWIEGVMRNN *

Sequence 17: sequence of the glu-plasminogen secreted into the medium (pSM49.8, pSM58.1 and pSM82.1)

EPLDDYVNTQGAS FSVTKKQLGAGSIEECAAKCEΞDEEFTCRAFQYHSK EQQCVI AE RKSS11IRMRDVV FEKKVY SECKTGNGKNYRGTMSKT NGITCQK SSTSPHRPRFSPATHPSEGLEENYCR PDNDPQGP CYTTDP EKRYDYCDILECEEECiyiHCSGENYDGKISKTMSG ECQAWDSQSPHAHGY IPSKFPNINLKK YCRNPDRELRP CFTTDPN RWE CDIPRCTTPPPSS GPTYQC KGTGElvrYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNL DE YCRNPDGKRAP CHTTNSQVR EYCKIPSCDSSPVSTΞQLAPTAPPE LTPWQDCYHGDGQSYRGTSSTTTTGKKCQSWSS TPHRHQKTPENYPNA GLT lSrYCRNPDAD GPWCFTTDPSVR EYCiN KKCSGTEASVVAPPPVVL LPDVETPSEEDC FGNGKGYRGKRATTVTGTPCQD AAQEPHRHSIFTPE TNPRAGLEKNYCR PDGDVGGP CYTTNPRK YDYCDVPQCAAPSFDCGK PQVEPK CPGRWGGCVAHPHSWP QVS RTRFGMHFCGGTLISPE VT AAHCLE SPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALL KLSSPAVITDKVIPACLPSPNYWADRTECFITG GETQGTFGAG lkea QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEK DKYILQGVTS G GCARPNKPGVYVRVSRFVT IEGVMRHN *

Sequence 18: sequence of the Lys plasminogen secreted in the medium (pMHS476.1, pSM54.2, pAC37.1 and pJW9.1)

KVY SECKTGNGK YRGT SKTK GITCQKWSSTSPHRPRFSPATHPSEG LEE YCRNPDNDPQGP CYTTDPE RYDYCDI ECEEECMHCSGENYDGK ISKTMSGLECQA DSQSPHAHGYIPSKFP KNLKKNYCR PDRE RPWCF TTDPNKRWE CDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTC ^Q H SAQTPHTHNRTPENFPCKIJLDElSrϊCRNPDGKRAP CHTTNSQVR ΞY CKIPSCDSSPVSTEQLAPTAPPE TPWQDCYHGDGQSYRGTSSTTTTG KCQS SSMTPHRHQ TPENYPNAGTMNYCRNPDADKGP CFTTDPSVR EYCNK CSGTEASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATT VTGTPCQDAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGP CYTT NPR LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHS PWQV S RTRFGHFCGGT ISPE VLTAAHCLEKSPRPSSYKVILGAHQEVN E PHVQEIEVSRLFLEPTRKDIAL KLSSPAVITDKVIPACLPSPNYWADR TECFITGWGETQGTFGAG LKEAQLPVIEN VCNRYEFLNGRVQSTE CA GHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTS G GCARPNKPGVYVRV SRFVTWIΞGVMRNN *

Sequence 19: oligonucleotide primer N036a

AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG

Sequence 20: oligonucleotide primer N036b

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG

Sequence 21: oligonucleotide primer N036c

AAAAACTCGAGAAAAGAAAACTTTACGACTACTG

Sequence 22: oligonucleotide primer N036d

AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG

Sequence 23: oligonucleotide primer N036e

AAAAACTCGAGAAAAGACTTTACGACTACTGTG

Sequence 24: oligonucleotide primer N036f

AAΆΆACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTΆCTGTG

Sequence 25: oligonucleotide primer N036g

AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG

Sequence 26: oligonucleotide primer N036h

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG

Sequence 27: oligonucleotide primer N036i

AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC Sequence 28: oligonucleotide primer N036j

_AAAAA C _TC G _Ά G _A AA _A GAGAGGCTGAΆGCTTCATTTGATTGTGGGAAGCC

Sequence 29: mini-plasminogen (pPLGl.l and pPLG2.1)

APPPW PDVETPSΞEDCMFGNGKGYRG RATTVTGTPCQDWAAQEPHR HS I FTPETNPRAGLE YCRNPDGDVGGP CYTTNPRKLYDYCDVPQCAA PSFDCG PQVEPKKCPGRWGGCVAHPHS P QVSLRTRFG HFCGGTLI SPE VLTAAHCLEKSPRPSSYKVI GAHQEVNLEPHVQEIEVSR FLEPT RKDIAL K SSPAVITDKVIPAC PSPNYWADRTECFITGWGETQGTFG AGL EAQ PVIENKVC RYEF NGRVQSTELCAGHLAGGTDSCQGDSGG PLVCFEKDKYILQGVTS GLGCARPN PGVYVRVSRFVT IEGVMRNN *

Sequence 30: micro-plasminogen (ρPLG3.2 and pPLG4.2)

KLYDYCDVPQCAAPSFDCGKPQVEP KCPGRWGGCVAHPHS P QVS R TRFG HFCGGTLISPEWVTAAHC EKSPRPSSY VILGAHQEVNLEPHV QEIEVSRLFLΞPTRDIALKLSSPAVITDKVIPACLPSPNYVVADRTEC FITGWGETQGTFGAG KEAQLPVIENKVCNRYEFNGRVQSTELCAGHL AGGTDSCQGDSGGPLVCFEKDKYI QGVTS GLGCARPNKPGVYVRVSRF VTWIEGVMRNN *

Sequence 31: micro-plasminogen (pPLG5.3 and pPLGό.l)

LYDYCDVPQCAAPSFDCGKPQVEPK CPGRWGGCVAHPHS P QVSLRT RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNEPHVQ EIEVSR F EPTRKDIALL SSPAVITDKVIPACLPSPNYWADRTECF ITGGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTΞ CAGHLA GGTDSCQGDSGGPVCFEKDKYILQGVTSWGLGCARPN PGVYVRVSRFV TWIΞGVMRNN *

Sequence 32: micro-plasminogen (pPLG7.1 and pPLG8.3)

APSFDCGKPQVEP KCPGRWGGCVAHPHS PWQVS RTRFGMHFCGGTL ISPEWVLTAAHCLE SPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP TRKDIALL SSPAVITDKVIPACLPSPNYWADRTECFITG GETQGTF GAG KEAQLPVIEN VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG GPVCFEKDKYILQGVTSWGLGO PNIPGVYVRVSRFVTWIEGvMRlTO

Sequence 33: micro-plasminogen (pPLG9.1 and pPLGlO.l)

SFDCGKPQVEP KCPGRWGGCVAHPHS PWQVSLRTRFG HFCGGTLIS PE VLTAAHCLEKSPRPSSY VILGAHQEVNLEPHVQEIEVSR FLEPTR IΦIA L SSPAVITDKVIPAC PSPlSrYV ÄDRTECTITGGDLGCVFGGGLQG LVCFEi KYILQGVTS G Gα ^ PNKPGVYVRVSRFVT IEGVMRNN *

Sequence 34: DNA sequence of the alpha factor from the yeast Saccharomyces cerevisiae in pPICZαA up to the Kex2 interface.

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAΆATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC

TCGAG

Sequence 35: amino acid sequence of the alpha factor from the yeast Saccharomyces cerevisiae in pPICZαA up to the Kex2 interface.

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNST N G FINTTIASIAAKEEGVSLE

Sequence 36: DNA sequence of the Kex2 interface

AAAAGA

Sequence 37: DNA sequence of the Stel3 interfaces

GAGGCTGAAGCT

Sequence 38: amino acid sequence of the Kex2 interface

KR

Sequence 39: amino acid sequence of the Stel3 sites

EAEA

Sequence 40: amino acid sequences of the human mini-plasminogen as in pPLGl.l with Kex2 cleavage and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSD EGDFDV AVLPFSNSTNNGLLFI TTIASIAAKEEGVSLEKRAPPPW PDVETPS EEDQ FGNGKGYRGKRATTVTGTPCQD AAQEPHRHSIFTPETNPRAGLE KNYCR PDGDVGGP CYTTNPRK YDYCDVPQCAAPSFDCGKPQVEPKKC PGRWGGCVAHPHS QVSLRTRFGMHFCGGTLISPE P V ^Q TAAHCLEKS PRPSSYKVILGAH EVNLEPHVQEIEVSR F EPTRKDIAL K SSPAVI TDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQ PVIENK VCNRYEFLNGRV ^Q STELCAGHLAGGTDSCQGDSGGPLVCFEKDKYI QGV TSWGLGCARPNKPGVYVRVSRFVTWIEGVMRSTN * Sequence 41: amino acid sequence of the human Mmi plasminogen as in pPLG2.1 with Kex2 cleavage and two Stel cleavage sites and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSA AAPVOTTTEDETAQIPAEAVIGYSDLEGDFDV AV PFSNSTNNG LFINTTIASIAAKEEGVS EKREAEAAPPPWLLPDV ETPSEEDCMFGNGKGYRGKRATTVTGTPCQD AAQEPHRHSIFTPETNPR AGLEKNYCRNPDGDVGGP CYTTNPRKLYDYCDVPQCAAPSFDCGKPQVE PKKCPGRWGGCVAHPHS P QVS RTRFG HFCGGTLISPE VLTAAHC LEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLF EPTRKDIALLKLSS PAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGL KEAQLPV IENKVCNRYEF NGRVQSTE CAGH AGGTDSCQGDSGGP VCFΞKDKYI LQGVTS GLGCARPNKPGVYVRVSRFVT IΞGV RNN *

Sequence 42: amino acid sequence of the human micro-plasminogen as in pPLG3.2 with Kex2 cleavage and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GL FINTTIASIAAKEEGVSLΞKRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCV AHPHS P QVSLRTRFG HFCGGTLISPEWVLTAAHC EKSPRPSSYKVILGAHQEVN EP HVQEIEVSRLFLEPTRKDIAL KLSSPAVITDKVIPACLPSPNYWADRTECFITG GETQ GTFGAGLLKEAQLPVIENKVC RYEFLNGRVQSTE CAGH AGGTDSCQGDSGGPLVCFEK DKYILQGVTS G GCARPNKPGVYVRVSRFVT IEGVMRNN *

Sequence 43: amino acid sequence of the human micro-plasminogen as in pPLG4.2 with Kex2 cleavage and two Stel3 cleavages and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

^ FPSIFTAVLFAASSA AAPV TTEDETAQIPAEAVIGYSDLEGDFDVAV PFS NSTNNGLLFINTTIASIAAKEEGVSLEKREAEAKLYDYCDVPQCAAPSFDCGKPQV EPKKCPGRWGGCVAHPHS P QVSLRTRFGMHFCGGT ISPE VLTAAHCLEKSP RPSSYKVILGAHQEVNLEPHVQEIEVSRLFLΞPTRKDIALLKLSSPAVITDKVIPA CLPSPNYWADRTECFITG GETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQ STE CAGH AGGTDSCQGDSGGPLVCFEKDKYI QGVTS GLGCARPNKPGVYVRV SRFVTWIEGVMR N *

Sequence 44: amino acid sequence of the human micro-plasminogen as in pPLG5.3 with Kex2 cleavage and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GLLFINTTIASIAAKEEGVS EKRLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVA HPH PW ^Q VS RTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVN EPH V ^Q EIEVSRLF EPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITG GETQG TFGAGLKEA _Q LPVIENKV _C NRY _E FLNG _R VQ _S TELCAGHAGGTDSCQGDSGGPLVCFEKD

KYI QGVTS GLGCARPNKPGVYVRVSRFVT IEGVRNN *

Sequence 45: amino acid sequence of the human micro-plasminogen as in pPLG6.1 with Kex2 cleavage and two Stel3 cleavages and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAV PFS NS _T NNG LFINTTIASIAAKEEGVSLEKREAEALYDYCDVPQCAAPSFDCGKPQVE PKKCPGRVVGGCVAHPHS P QVS RTRFGHFCGGTLISPE VLTAAHCLEKSPR PSSYVILGAHQEVNLEPHVQEIΞVSRLFLEPTRKDIALLKLSSPÄVITDKVIPAC PSPNYWADRTECFITG GETQGTFGAG LKEAQLPVIENKVCNRYEFLNGRVQS TE CAGHLAGGTDSCQGDSGGPLVCFEKDKYI QGVTS GLGCARPNKPGVYVRVS RFVTWIEGVMRNN *

Sequence 46: Ammo acid sequence of the human micro-plasminogen as in pPLG7.1 with Kex2 cleavage and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAV FAASSA AAPVNTTTEDETAQIPAEAVIGYSD EGDFDVAVLPFSNSTNN GL FINTTIASIAAKEEGVSLEKRAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVS RTRFGMHFCGGTLISPE VLTAAHC EKSPRPSSYKVILGAHQEVN EPHVQEIEVSRLFL EPTRKDIAL K SSPAVITD VIPAC PSPKΓYWADRTECFITG GETQGTFGAGLLKEAQ LPVIENKVC RYEFLNGRVQSTE CAGHIIAGGTDSCQGDSGGP VCFEKDKYI QGVTS G LGCÄRPNKPGVYVRVSRFVTWIEGV RNN *

Sequence 47: amino acid sequence of the human micro-plasminogen as in pPLG8.3 with Kex2 cleavage and two Stel3 cleavages and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN G FIKFTTIASIAAKEEGVS EKREAEAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPW QVS RTRFGMHFCGGT ISPE V TAAHCLEKSPRPSSYKVI GAHQΞVNLEPHVQEIEVS R FLEPTRKDIAL KLSSPÄVITDKVIPACLPSPNYWADRTECFITG GETQGTFGAG KEAQ PVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGP VCFE DKYILQGV TS glGo PNKPGVYVRVSRFVTWIEGVMRNN *

Sequence 48: amino acid sequence of the human micro-plasminogen as in pPLG9.1 with Kex2 cleavage and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

KRFPSIFTAVLFAASSALAAPVlrrTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN G FINTTIASIAAKEEGVS EKRSFDCGKPQVEPKKCPGRWGGCVAHPHS PWQVS RT RFGMHFCGGTLISPEWV TAAHCLEKSPRPSSYKVI GAHQEVNLEPHVQEIEVSRLFLEP

TRKDIALLK SSPAVITDKVIPAC PSPNYWADRTECFITG GETQGTFGAGL KEAQLP VIENKVCNRYEF NGRVQSTE CAGHLAGGTDSCQGDSGGP VCFEKDKYI QGVTS G G (^ ARPNKPGVYVRVIR

Sequence 49: amino acid sequence of the human micro-plasminogen as in pPLGlO.l with Kex2 cleavage and two Stel3 cleavages and the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

Π ^ FPSIF AV FAASSAIJ PVN T EDETAQIPAEA IGYSD EGDFDVAV PFSNSTNN G LFINTTIASIAAKEEGVS EKREAEASFDCGKPQVΈPKKCPGRWGGCVAHPHSWPWQV SLRTRFGMHFCGGTLISPEWV TAAHCLEKSPRPSSYKVILGAHQEV LEPHVQEIEVSRL F EPTRKDIAL K SSPAVITDKVIPAC PSPNYWADRTECFITGWGETQGTFGAGLLKE AQ PVIENKVCNRYΞFLNGRVQSTELCAGH AGGTDSCQGDSGGPLVCFEKDKYI QGVTS G GCARPNKPGVYVRVSRFVT IEGVMRNN *

Sequence 50: nucleic acid sequence of the human mini-plasminogen gene as in pPLGl.l with the codons for the Kex2 site and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAΆTTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAΆCACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAΆGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGA AGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCΆCTCCAGAGA CAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGG TCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGG TTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAG GTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCC CACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAG AAGTGAATCTCGAACCGCATGTTCAGGAΆATAGAΆGTGTCTAGGCTGTTCTTGGAGCCCAC ACGAAAAGATATTGCCTTGCTAAAGCTAΆGCAGTCCTGCCGTCATCACTGACAAAGTAATC CCΆGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTG GCTGGGGAGΆAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGΆAGCCCAGCTCCCTGT GATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAA CTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTC TGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTG GCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGΆTTGAG GGAGTGATGAGAAATAATTGA

Sequence 51: nucleic acid sequence of the human mini-plasminogen gene as in pPLG2.1 with the codons for the Kex2 interface and the Stel3 interfaces and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAΆCAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA _C TCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC G _GG TTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGAC TCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACC ACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTT TCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAΆAAAATTACTGCCGTAACCCTGATGG TGATGTAGGTGGTCCCTGGTGCTACACGACAAΆTCCAAGAAAACTTTACGACTACTGTGAT GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAAT GTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAG TCTTAGAACAΆGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTG TTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGG GTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTT CTTGGAGCCCACACGAΆAAGATATTGCCTTGCTAAAGCTAΆGCAGTCCTGCCGTCATCACT GACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAAT GTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGC CCAGCTCCCTGTGATTGAGAATAAΆGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACA GTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTG GGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAΆGGTTTGTT ACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 52: Nuclear acid sequence of the human micro-plasminogen gene as in pPLG3.2 with the codons for the Kex2 site and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGA TTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTG GCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCT GTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTC CCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCT TGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATC CCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAA ^G GTA ^C TTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGT GCAΆT GCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTT ^C GG ^CC G GA ^G AGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAG ^C AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATA AGC CTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAA

TTGA

Sequence 53: nucleic acid sequence of the human micro-plasminogen gene as in pPLG4.2 with the codons for the Kex2 and Stel3 sites and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGC CCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTG GAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTG CTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTG AATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAA AAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGC TTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGG GGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTG AGAATAAΆGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTG TGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTT TGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCAC GCCCCAA TAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGT GATGAGAAATAATTGA

Sequence 54: nucleic acid sequence of the human micro-plasminogen gene as in pPLG5.3 with the codons for the Kex2 interface and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC

CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTG TGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTG GAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCC AAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCAT GTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGC TAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCC AAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCA ATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGC CGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATÄAGCCTG GTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTG

A Sequence 55: nucleic acid sequence of the human micro-plasminogen gene as in pPLG6.1 with the codons for the Kex2 site and the Stel3 sites and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

AT _{G T} AGATTTCCT CAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTC ACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA _A C _T C _A G _{A AGA} TTT AGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGT _T ATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCC TTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGG GGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAA TGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTT GGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAAT CTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAG ATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTG TCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGA GAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGA ATAAAGTGTGCÄATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGC TGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGC TTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTG TGCACGCC CCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGAT GAGAAATAATTGA

Sequence 56: nucleic acid sequence of the human micro-plasminogen gene as in pPLG7.1 with the codons for the Kex2 site and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCC TGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTT AGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGA CTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGC ACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGΆAATAGAAGTGTCTAGGCTGTTCTTG GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACA AAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTT CATCACTGGCTGGGGAGAAΆCCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAΆGAGTCCAAT CCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGG AGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGT CTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTT GGATT GAGGGAGTGATGAGAAATAATTGA

Sequence 57: Nuclear acid sequence of the human microplasminogen gene as in pPLG8.3 with the codons for the Kex2 interface and the Stel3 cleavage sites and the gene of the prepro peptide of the alpha Factor of the yeast Saccharomyces cerevisiae

_ATG A _G ATT _T C T _{C T} C _{A T} ATTT TACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC _CAG TC _A ACACTA _CAA CAGA _A GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA _C T _CAG A _TTT AG _AA GGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGG _T TATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCC GAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG CAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAG AGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGT CATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCT AGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCG TCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCG GACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATG GAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCA GGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTC ACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGG TGTCTATGTTCGTGTTTCAA GGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 58: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG9.1 with the codons for the Kex2 site and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAΆGGGGTATCTC TCGAGAAAAGATCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAG GGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTG CCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCA AGAAGTGAATCTCGAΆCCGCATGTTCAGGAAATAGAΆGTGTCTAGGCTGTTCTTGGAGCCC ACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAA TCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCAC TGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCT GTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAΆTGGAAGAGTCCAATCCACCG AACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCC TCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGC TGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTG AGGG AGTGATGAGAAATAATTGA

Sequence 59: Nuclear acid sequence of the human micro-plasminogen gene as in pPLGlO.l with the codons for the Kex2 interface and the Stel3 interfaces and the gene of the prepro peptide of the alpha factor of the yeast Saccharomyces cerevisiae

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTA CTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTC TCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAA ATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC AGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGG TGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCT GGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTG TTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCA CTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGA ATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAA GCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAG TCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGA CAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTG TTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 60: nucleic acid sequence of the human mini-plasminogen gene as in pPLGl.l and pPLG2.1

GCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATG TTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGC CAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCA CGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGG TGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCC CCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTT GGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCAC TGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAA GTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACA CGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACT GGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCT GTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACC GAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGT CCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTT GGCTGTGCACGCCCCAATAAGCCT GGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGG ATTGAGGGAGTGATGAGAAATAATTGA

Sequence 61: nucleic acid sequence of the human micro-plasminogen gene as in pPLG3.2 and pPLG4.2

AAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGC CTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACA TTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTT

CATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGA AATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA AGCAGTCCTGCCGTCATCACTGACAAAGTAΆTCCCAGCTTGTCTGCCATCCCCAAΆTTATG TGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGG AGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTAT GAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCA CTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACAT TTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTAT GTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 62: nucleic acid sequence of the human micro-plasminogen gene as in pPLG5.3 and pPLG6.1

CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTC AAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTC CTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCAT CCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAAT AGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGC AGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGG TCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGC TGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAG TTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTG ACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTT ACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 63: nucleic acid sequence of the human micro-plasminogen gene as in pPLG7.1 and pPLG8.3

GCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTG TGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTT TGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCAC TGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAG TGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACG AAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCA GCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCT GGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGAT TGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGΆGTCCAATCCACCGAACTC TGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGG TTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGC ACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGA GTGATGAGAAATAATTGA

Sequence 64: Nuclear acid sequence of the human micro-plasminogen gene as in pPLG9.1 and pPLGlO.l

TCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGG GGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAAT GCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTG GAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATC TCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGA TATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGT CTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAG AAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAA TAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAΆGAGTCCAATCCACCGAACTCTGTGCT GGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCT TCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCC CAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG AGAAATAATTGA

Sequence 65: nucleic acid sequence of the human glu-plasminogen gene

GAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAG CAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTC ACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAAC AGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAΆAGTGTAT CTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAA AATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCT GCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCG CAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTT GAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAG ACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATAC ATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGG GAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATC CCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACA GGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGG AGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTG GATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAAC AGCCAAGTGCGGTGGGAGTACTG TAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACG GAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCAT GGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAG TCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCT GGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACC ACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCG AGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAA GACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAG ACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGT GGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAG TGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAΆGTGGAGCCGAAGAAATGTCCTGGA AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGA ACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT GCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCA CACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG GAGCCCACACGAAAΆGATATTGCCTTGCTAAΆGCTAAGCAGTCC TGCCGTCATCACTGAC AAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGT TCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCC CAGCTCCCTGTGATTGAGAATAΆAGTGTGCAATCGCTATGAGTTTCTGAΆTGGAAGAGTC CAATCCACCGAΆCTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAΆGGAGTCACTTCT TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTT GTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 66: nucleic acid sequence of the human lys-plasminogen gene

AΆAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCC AAAACAAΆAAATGGCATCACCTGTCAΆAAATGGAGTTCCACTTCTCCCCACAGACCTAGA TTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGAC AACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGC GACATTCTTGAGTGTGAΆGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAΆ ATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCT CATGGATACATTCCTTCCAAATTTCCAACAΆGAACCTGAAGAAGAATTACTGTCGTAAC CCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTT TGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTG AAGGGAACAGGTGAAAACTATCGCGGGAΆTGTGGCTGTTACCGTTTCCGGGCACACCTGT CAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGC AAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCAT ACAACCAACAGCCAΆGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCA GTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGAC TGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAG AAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTAC CCAAATGCTGGCCTGAC AATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGG TGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGA ACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCT TCCGAAGAΆGACTGTATGTTTGGGAΆTGGGAAAGGATACCGAGGCAAGAGGGCGACCACT GTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTC ACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGT GATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAΆGAAAACTTTACGACTACTGTGAT GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAA TGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC AGTCTTAGAΆCAΆGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGG GTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGG CTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAAT GGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCAT TTGGCCGGAGGCACTGACAGTTGC CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAΆTACATTTTACAAGGA GTCACTTCTTGGGGTCTCTTGGCTGTGCACGCCCCAATAAGCCTGGTAGGTGTGTGAGAGAGGTGTGTGTAGT bibliography

(1) = Desire Collen, Thrombosis and Haemostasis, 82, 1999

(2) = Forsgren et al., FEBS Lett. 213, 1987

(3) = Petersen et al., J. Biol. Chem., 265, 1990

(4) = Duman et al., Biotechnol. Appl. Biochem. 28; 39-45, 1998

(5) = Guan et al., Sheng Wu Gong Cheng Xue Bao, 17, 2001

(6) = Gonzalez-Gronow et al., Biochimica et Biophysica Acta, 1039, 1990

(7) = Whitefleet-Smith et al., Arch. Biochem. Biophys., 271, 1989

(8) = Nilsen and Castellino, Protein Expression and Purification, 16, 1999

(9) = Busby et al., J. Biol. Chem., 266, 1991

(10) = Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor press, 1989

(11) = Gassen & Schrimpf, genetic engineering methods, spectrum academic publisher,

Heidelberg, 1999

(12) = Malinowski et al., Biochemistry, 23, 1984

(13) = Stack et al., Biochem. J. 284, 1992

Claims

claims

1. A recombinant production process for a functional plasminogen in microorganisms, which comprises at least the following step:

a) Fusion of a nucleic acid sequence coding at least for the functional part of the plasminogen with a nucleic acid sequence which codes for at least one signal peptide, the nucleic acid sequence coding for the functional plasminogen and the nucleic acid sequence coding for at least the signal peptide being linked to codons for interfaces of proteases, which ensure the cleavage of the signal peptide.

2. Recombinant production method according to claim 1, wherein the nucleic acid sequence coding for at least the functional part of the plasminogen contains the proteolytic domain of plasminogen or a mutant or a fragment thereof.

3. A recombinant production method according to claim 1 or 2, wherein the nucleic acid sequence coding for at least the functional part of the plasminogen codes for glu or Lys plasminogen.

4. Recombinant production method according to one of claims 1 to 3, wherein the nucleic acid sequence coding for at least one signal peptide codes for a prepropeptide, a prepeptide or a propeptide and / or wherein the codons for interfaces of proteases for the protease Kex 2 and / or Ste 13 encode.

5. Recombinant production method according to claim 4, characterized in that the nucleic acid molecule coding for at least one signal peptide encodes the signal peptide of the alpha factor or of SUC2, PHA-E or PH01 from the yeast Saccharomyces cerevisiae.

6. Recombinant production method according to one or more of the preceding claims, characterized in that at least 20 mg / 1 functional glu or Lys plasminogen are produced.

7. Recombinant manufacturing method according to one or more of the preceding claims, characterized in that at least 120 U / 1 functional Lys plasminogen are produced after a manufacturing process of 120 hours.

8. Recombinant production method according to one or more of the preceding claims, wherein two oligonucleotide primers selected from the group comprising N036a (Seq. ID No. 19), N036b (Seq. ID No. 20), N036c ( Seq. ID No. 21), N036d (Seq. ID No. 22), N036e (Seq. ID No. 23), N036f (Seq. ID No. 24), N036g (Seq. ID No. 25), N036h ( Seq. ID No. 26), N036i (Seq. ID No. 27) and N036J (Seq. ID No. 28).

9. Recombinant production method according to one or more of the preceding claims, wherein two oligonucleotide primers selected from the group comprising N034 (Seq. ID No. 1), N036 (Seq. ID No. 2), N057 ( Seq. ID No. 3), N037 (Seq. ID No. 4), N035 (Seq. ID No. 5) and N056 (Seq. ID No. 6), and from the group comprising N036a (Seq. ID No. 6) 19), N036b (Seq. ID No. 20), N036c (Seq. ID No. 21), N036d (Seq. ID No. 22), N036e (Seq. ID No. 23), N036f (Seq. ID No. 21) 24), N036g (Seq. ID No. 25), N036h (Seq. ID No. 26), N036i (Seq. ID No. 27) and N036J (Seq. ID No. 28).

10. Recombinant production method according to one or more of the preceding claims, wherein the fusion product formed in step a) is inserted into an expression vector suitable for microorganisms.

11. Recombinant manufacturing method according to claim 10, wherein the expression vector is suitable for fungi.

12. A recombinant production method according to claim 10 or 11, wherein the expression vector contains an inducible or constitutive promoter.

13. Recombinant production method according to claim 10 or 11, wherein the expression vector contains the constitutive GAP promoter from P. pastoris.

14. Recombinant production method according to claim 12, characterized in that the nucleic acid thus obtained is a plasmid, preferably selected from the group pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17. 2, pPLG18.1, pPLG19.2, pPLG20.1, pAC37.1, pJW9.1, pMHS476.1, pSM54.2, pSM49.8, pSM 82.1 and pSM58.1.

15. Recombinant production method according to one or more of the preceding claims, characterized in that the codons for the interface of the protease Encode Kex2 and the plasminogen fusion gene described in Seq. ID No. 7, 13, 50, 52, 54, 56 or 58 nucleic acid sequence shown.

16. Recombinant production method according to one or more of the preceding claims, characterized in that the codons code for the interface of the protease Kex2 and the plasminogen fusion protein which is described in Seq. ID No. 8, 14, 40, 42, 44, 46 or 48 amino acid sequence shown.

17. Recombinant production method according to one or more of the preceding claims, characterized in that the codons for the interface of the protease Kex2 and the protease Stel3 code and the plasminogen fusion gene described in Seq. ID. No. 9, 15, 51, 53, 55 or 57 nucleic acid sequence shown.

18. Recombinant production method according to one or more of the preceding claims, characterized in that the codons for the interface of the protease Kex2 and the protease Stel3 code and the plasminogen fusion protein described in Seq. ID No. 10, 16, 41, 43, 45, 47 or 49 amino acid sequence shown.

19. Recombinant production method according to one or more of the preceding claims, wherein a host belonging to the microorganisms is transformed with the plasmid obtained in claim 14.

20. Recombinant production method according to one or more of the preceding claims, wherein the host organism used is a eukaryotic microorganism.

21. Recombinant production method according to claim 20, characterized in that the host organism used belongs to the department of fungi.

22. Recombinant production method according to claim 21, characterized in that the host organism used belongs to the genus Pichia, Saccharomyces, Hansenula, Candida or Aspergillus.

23. Recombinant production method according to claim 22, characterized in that the host organism used belongs to the species Pichia pastoris, Pichia methanolica, Hansenula polymorpha, Aspergillus niger, Aspergillus oryzae or Aspergillus nidulans.

24. Recombinant production method according to one or more of the preceding claims, wherein from a microbial host organism transformed with the fusion product formed in step a), the nucleic acid sequence coding for at least the functional part of the plasminogen is overexpressed and at least the functional part of plasminogen is secreted.

25. Recombinant production method according to one or more of the preceding claims, wherein the functional part of the nucleic acid sequence of plasminogen is one of the sequences Seq. ID No 60, 61, 62, 63, 64, 65 or 66.

26. Recombinant production method according to one or more of the preceding claims, characterized in that a human functional plasminogen is produced.

27. Recombinant production method according to one or more of the preceding claims, characterized in that a human-homologous functional plasminogen is produced.

28. Plasmid pPLG11.2, plasmid pPLG12.1, plasmid pPLG13.1, plasmid pPLG14.2, plasmid pPLG15.1, plasmid pPLG16.3, plasmid pPLG17.2, plasmid pPLG18.1, plasmid pPLG19.2 or plasmid pPLG20. 1.

29. Plasmid pMHS476.1, plasmid pSM54.2, plasmid pSM49.8, plasmid pSM82.1

30. Plasmid pSM58.1, plasmid pAC37.1, plasmid pJW9.1

31. Nucleic acid sequence obtainable by the recombinant production method according to one or more of claims 1 to 27, characterized in that the nucleic acid sequence which codes at least for the functional part of plasminogen with a promoter which is active in yeast and further with a nucleic acid sequence which is coded for at least one signal peptide, is operatively linked, the nucleic acid sequence coding for the functional plasminogen and the nucleic acid sequence coding for at least the signal peptide being linked to codons for the interfaces of the proteases Kex2 and Stel3.

32. Plasminogen obtainable by the recombinant production process according to one or more of claims 1 to 27.

33. Plasminogen according to agreement 32, characterized in that it is microplasminogen, miniplasminogen, Lys-plasminogen, Glu-plasminogen or a plasminogen derivative.

34. Plasminogen derivative according to claim 33, characterized in that it contains the functional proteolytic domain of the plasminogen and contains at least one deletion and / or at least one amino acid exchange and / or is fused with at least one further amino acid or at least one peptide or at least one protein.

35. Plasminogen derivative according to claim 33 or 34, characterized in that it can be activated to at least one plasminogen activator to active plasmin.

36. Plasminogen derivative according to any one of claims 33-35, characterized in that it contains at least the functional proteolytic domain of plasminogen and a sequence homology of over 80%, preferably over 90% and particularly preferably over 95% of micro, mini -, Lys or Glu plasminogen.

37. Plasmin obtainable by activating plasminogen or a plasminogen derivative according to any one of claims 32-36 using at least one plasminogen activator.

38. Microbial host organism containing the fusion product obtained in step a) according to one of claims 1 to 27 or a nucleic acid sequence derived therefrom.

39. Microbial host organism according to claim 38, characterized in that it is selected from the group Pichia pastoris, Pichia methanolica, Saccharomyces cerevisiae, Hansenula polymorpha, Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans.

40. Use of a functional plasminogen produced according to one of claims 1 to 27 and / or the resulting plasmin for the manufacture of a medicament.

41. Use of a functional plasminogen produced according to one of claims 1 to 27 and / or the resulting plasmin for the treatment of wounds, thrombotic events or in the prevention of thrombotic events.

42. Use of a functional plasminogen produced according to one of claims 1 to 27 and / or the plasmin resulting therefrom as anti-thrombotic and anti-coagulant active substances.

43. Use in particular according to claim 42 for the prophylaxis and / or treatment of heart attack, stroke, thrombosis, venous thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammation of the blood vessels, acute pulmonary embolism, acute and subacute arterial thrombosis, fresh or older clots in venous thrombosis, deep vein thrombosis of the pelvis and extremities, early thrombosis in the area of desobliterated vessels, acute central vascular occlusion on the eye, conjunctivitis with plasminogen type I deficiency, burns, burns and frostbite, disseminated intravascular coagulation in shock, acute arterial occlusion of the extremities, chronic occlusive thrombosis, chronic occlusive arterial thrombosis, chronic occlusive arterioscopy of arteriovenous shunts and for treatment after a heart attack, after a bypass operation, after angioplasty and after balloon dilatation, for thrombolytic therapy for acute heart attacks, for recanalizing arteriovenous shunts and for reperfusion of closed coronary arteries in acute myocardial infarction.

44. Use according to one or more of claims 40 to 43 in combination with an anticoagulant.

45. Use according to claim 44, characterized in that the anticoagulant is heparin, heparin derivatives or acetylsalicylic acid.

46. Use of a functional plasminogen produced according to one of claims 1 to 27 and / or the plasmin resulting therefrom for introduction into dressing materials, plasters or for use in combination with wound healing agents.

47. Dressing materials, wound dressings and plasters containing functional plasminogen produced according to one of claims 1 to 27 and / or plasmin obtained therefrom.

48. Dressing materials, wound dressings and plasters according to claim 47, containing 0.01-500 U of plasminogen and / or a plasmin resulting therefrom per cm ^{2 of} pharmaceutical formulation, preferably 0.1-250 units and particularly preferably 1-150 units of plasminogen and / or a resulting plasmin per cm of pharmaceutical formulation.

49. Use of the dressing materials, wound dressings and plasters according to claim 47 or 48 for the treatment of burns, frostbite, burns, injuries and / or wounds.

50. A pharmaceutical composition comprising a plasminogen and / or a resulting plasmin, produced according to one or more of claims 1 to 27, and a pharmaceutically acceptable carrier, additive and / or a solvent and optionally anticoagulant active ingredients.

51. Pharmaceutical composition according to claim 50, suitable for oral, topical or parenteral, intravascular, in particular intravenous, intraperitoneal, subcutaneous or intramuscular use.

52. Pharmaceutical composition suitable for topical application according to spoke 51, containing 0.01-500 U of plasminogen and / or a resulting plasmin per gram of the pharmaceutical composition, preferably 0.1-250 units and particularly preferably 1-150 units of plasminogen and / or a resulting plasmin per gram of the pharmaceutical composition.

53. Pharmaceutical composition suitable for oral application according to spoke 51, containing 0.1-100,000 U of plasminogen and / or a plasmin resulting therefrom per gram of the pharmaceutical composition, preferably 100-80,000 units and particularly preferably 1,000-50,000 units of plasminogen and / or a resulting plasmin per gram of the pharmaceutical composition.

54. Pharmaceutical composition suitable for injection or infusion according to claim 51, containing 0.1-100 million U of plasminogen and / or a plasmin resulting therefrom per 10 ml of solution, preferably 1-10 million units and particularly preferably 3-5 million units of plasminogen and / or a resulting plasmin per 10 ml of the solution for injection or infusion.

55. Use of the pharmaceutical composition according to any one of claims 50 to 54 for the prophylaxis and / or treatment of heart attack, stroke, thrombosis, venous thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammation of the blood vessels, acute pulmonary embolism, acute and subacute arterial thrombosis, fresh or older clots for venous thrombosis, deep vein thrombosis of the pelvis and extremities, early thrombosis in the area of desobliterated vessels, acute central vascular occlusion on the eye, conjunctivitis with plasminogen type I deficiency, burns, burns and frostbite, disseminated intravascular coagulation in shock, acute arterial occlusion of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts as well as for treatment after a heart attack, after a bypass operation, after angioplasty and after balloon dilatation, for thrombholytic therapy for acute heart attack, for recanalizing arteriovenous arterios for reperfusion of closed coronary arteries in acute myocardial infarction.

56. Vector, comprising the fusion product obtained in step a) according to spoke 1 or a nucleic acid sequence derived therefrom.

57. DNA molecule comprising the fusion product obtained in step a) according to approach 1 or a nucleic acid sequence derived therefrom.

58. RNA molecule, comprising the fusion product obtained in step a) according to approach 1 or a nucleic acid sequence derived therefrom.

59. Screening method for the identification of plasminogen activators using the functional plasminogen according to claim 36 or 37.

60. Screening method according to claim 59, characterized in that after preincubation of the proteases with the functional plasminogen according to claim 35 or 36, the resulting plasmin activity is measured.

61. Screening method according to claim 59 or 60, characterized in that the resulting plasmin activity is measured with a synthetic peptide substrate.

62. Screening method according to spoke 61, characterized in that the resulting plasmin activity is measured with N-Tosyl-Gly-Pro-Lys-pNA.