WO2023099927A1 - Method for the preparation of a recombinant type ii transmembrane protease serine fragment, said fragment, and its use - Google Patents

Abstract

Method for the preparation of a recombinant type II transmembrane protease serine fragment, said fragment, and its use The present invention relates to a method for producing a fragment of a mammalian TMPRSS2 protein in a recombinant manner, wherein i) said mammalian TMPRSS2 fragment comprises the mammalian TMPRSS2 ectodomain or a part thereof, comprising at least the SP domain of the mammalian TMPRSS2 protein; ii) said mammalian TMPRSS2 fragment is expressed in a protein expression system, iii) said protein expression system contains an inhibitor of said mammalian TMPRSS2 protein at least during step ii). The present invention further relates to a mammalian TMPRSS2 fragment of a mammalian TMPRSS2 protein and to its use, where the mammalian TMPRSS2 fragment is the mammalian TMPRSS2 ectodomain or a part thereof, comprising at least the SP domain of the mammalian TMPRSS2 protein. The present invention relates to transfection vectors and cell lines suitable for the recombinant production of said mammalian TMPRSS2 fragment, and to use thereof.

Description

Method for the preparation of a recombinant type II transmembrane protease serine fragment , said fragment , and its use

Field of the Invention

The obj ect of the present invention relates to a method for the recombinant production of an enzymatically active type I I transmembrane protease serine 2 fragment . Especially, the present invention provides a method for the preparation of the ectodomain of TMPRSS2 or part thereof , which is useful in the research and development of inhibitors of TMPRSS2 . These inhibitors can be used in the prophylaxis and treatment of TMPRSS2-related diseases such as viral infections and prostate cancer .

The present invention further relates to the protein produced by said method, i . e . , to the ectodomain fragment of the TMPRSS2 protein or part thereof .

Background of the Invention

The type I I transmembrane protease serine 2 (hereinafter referred to as TMPRSS2 ) is a type I I transmembrane protease serine (hereinafter referred to as TTSP ) . The TTSPs form the largest group of membrane-anchored serine proteases comprising 17 members (Antalis et al . , 2011 ) . A general TTSP is built up by the following parts : an N-terminal cytoplasmic domain, a single-pass transmembrane domain, an extracellular stem region consisting of di f ferent non-catalytic domains and a C-terminal serine protease domain with chymotrypsin ( S I ) fold . The schematic structure of the native TMPRSS2 protein can be seen in Fig. 1. Here, the numbering of the amino acid sequence is shown from 1 to 492, i.e., from the N-terminus towards the C-terminus of the human TMPRSS2 protein. TMPRSS2 comprises an intracellular domain, a transmembrane domain (TM) penetrating through the membrane (the membrane is represented by a double dotted line) , a low-density lipoprotein receptor type-A domain (LDLRA) , a scavenger receptor cysteine-rich domain (SRCR) and a chymotrypsin fold, trypsin-like specificity serine protease (SP) domain at the C-terminus ( Paoloni-Giacobino et al., 1997; Shen et al., 2017) . The LDLRA, the SRCR and the SP domains form the biologically active ectodomain of TMPRSS2.

TTSPs play important roles in human physiology from developmental processes to regulation of the blood pressure. The typical substrates of the TTSPs are peptide hormones, growth- and differentiation factors, receptors, enzymes, adhesion molecules. The uncontrolled activity of the TTSPs can contribute to the development of diseases, especially to different types of cancer (Tanabe and List, 2017) .

In the cell membrane, there are two broad-specificity inhibitors of the TTSPs: hepatocyte growth factor activator inhibitor-1 and hepatocyte growth factor activator inhibitor-2 (hereinafter referred to as HAI-1 and HAI-2) , that are encoded by the SPINT1 and SPINT2 genes, respectively (Faller et al., 2014) . These serine protease inhibitors are type I transmembrane proteins, having an N-terminal extracellular region and a C-terminal intracellular part. HAI-1 and HAI-2 reside on the cell surface of a number of tissues, including those, lining the respiratory tract. The N-terminal extracellular regions of HAI-1 and HAI-2 contain two Kunitz- type inhibitor domains that have been found to be potent inhibitors of a number of trypsin-like serine proteases, including the hepatocyte growth factor activator. HAI-1 is mainly found complexed with the TTSP matriptase on the extracellular surface of the plasma membrane (Friis et al., 2011) . Although it is not fully characterized yet, HAI-2 is also an important inhibitor of different members of the TTSP network .

Another function of HAI-1 and HAI-2 has also been described. In the case of matriptase and TMPRSS13 (both are TTPS proteins) it has been shown that both HAI-1 and HAI-2 may act as chaperones in the secretory pathway helping the correct folding and membrane positioning of these two proteins (Godiksen et al., 2008; Nonboe et al., 2017; Murray et al., 2017) .

Evolutionary conservation of the TTSPs, including TMPRSS2, suggests that all have important physiological functions. In fact, most TTSPs have known vital physiological functions and accordingly, the removal of their genes is lethal or results in the development of severe disease condition. In contrast, no normal physiological function has been revealed for TMPRSS2 yet, and the TMPRSS2 knock-out mice (Kim et al., 2006) and pigs (Whitworth et al., 2017) do not show any abnormal phenotype; they are viable and fertile. On the other hand, TMPRSS2 has been implicated in the pathophysiology of viral infections and prostate cancer; therefore, it has been identified as a potential drug target in both contexts.

In the case of prostate cancer, it has been shown that overexpression of HAI-2 suppresses the TMPRSS2-mediated matrix degradation and prostate cancer invasion. The expression levels of TMPRSS2 are inversely correlated with HAI-2 levels during prostate cancer progression (Ko et al., 2020) . HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. It is very likely that HAI-2 functions as a cognate inhibitor of TMPRSS2 in human prostate cancer cells. It has been suggested that forced expression of HAI-2 could decrease TMPRSS2-induced cell invasion, tumour growth and metastasis; consequently, it could be an alternative therapeutic option to treat prostate cancer.

Viruses use host proteins to enter the human cells (Millet and Whittaker, 2015) . The enzymatic activity of TMPRSS2 is essential for influenza- (A and B strains) and coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2) to rapidly and efficiently infect the human tissues, especially that of the respiratory system (Evans and Liu, 2021; Laporte and Naesens, 2017) . The global pandemic COVID-19 further enhanced the impact of TMPRSS2 in human diseases and highlighted the medical need for efficient and selective inhibitors against this enzyme.

During infection, the SARS coronaviruses bind to their receptor, namely to the angiotensin-converting enzyme 2 (ACE2) , on the host cell surface. This virus-ACE2 interaction is mediated by the spike protein (hereinafter referred to as S protein) that is displayed on the surface of the virus particles. The S protein-ACE2 interaction alone is not sufficient for the virus-host membrane fusion and infection. Priming of the S protein by host cell proteases is essential for viral entry into the cell (Heurich et al., 2014) . For the infection, the S protein must be cleaved at two distinct sites, namely at the S1/S2 and the S2' sites. TMPRSS2 seems to be the dedicated enzyme for proteolytic processing of the S protein (Hoffmann et al., 2020) . The S1/S2 (multibasic) cleavage site of SARS-CoV-2 S protein harbours several arginine residues, it can be efficiently cleaved by TMPRSS2 or by a proprotein convertase (e.g., furin) . The S2' site, however, can only be cleaved by TMPRSS2 (Papa et al., 2021) . The S2 ' cleavage is a prerequisite for membrane fusion (Bestle et al . , 2020 ) . Inhibiting the proteolytic activity of TMPRSS2 can be an attractive therapeutic option for preventing and treating coronavirus infection, especially infection of SARS- CoV-2 (Ragia and Manolopoulos , 2020 ) . HAI-2 , the cognate endogenous inhibitor of TMPRSS2 itsel f could alleviate SARS- CoV-2 infection ( Tomita et al . , 2021 ; Ramirez Alvarez et al . 2021 ) .

According to the published scienti fic literature above , there is a high medical need for ef ficient and selective TMPRSS2 inhibitors and for the purpose of this inhibitor development correctly folded enzymatically active and homogeneous TMPRSS2 protein preparation is also needed . However, there is currently no available method for the production of a TMPRSS2 protein preparation in a suf ficient amount and quality .

Therefore , the aim of the present invention is to provide a method for producing enzymatically active TMPRSS2 or its fragment , which is suitable for the research aimed at identi fying ef fective and selective TMPRSS2 inhibitors .

Summary of the Invention

The present invention was made in view of the prior art described above , and the obj ect of the present invention is to provide a method for the recombinant expression and puri fication of enzymatically active TMPRSS2 or its fragment . The TMPRSS2 preparation obtained in this way is preferably homogenous . This TMPRSS2 preparation will be suitable for screening inhibitors of the native TMPRSS2 protein . The inhibitors obtained by the use of said TMPRSS2 preparation will be useful in drug development against TMPRSS2-related diseases such as viral infections and prostate cancer .

To solve the problem, the present invention provides a method for the production of a TMPRSS2 protein preparation in suf ficient amount and quality . Briefly, we surprisingly found that while expressing the wild type TMPRSS2 ectodomain alone did not yield any detectable amount of recombinant enzyme , coexpressing the wild type TMPRSS2 ectodomain with HAI -2 resulted in high-yield secretion of correctly folded, enzymatically active protein . Based on this recognition, we developed an ef ficient method for the production of wild type TMPRSS2 ectodomain ( i . e . , the LDLRA-SRCR-SP fragment ) or a part thereof comprising at least the SP domain in recombinant , secreted, enzymatically active form in mammalian cells .

Based on the above , the present invention relates to a method for producing a fragment of a mammalian TMPRSS2 protein in a recombinant manner, wherein i ) said mammalian TMPRSS2 fragment comprises the mammalian TMPRSS2 ectodomain or a part thereof , comprising at least the SP domain of the mammalian TMPRSS2 protein; ii ) said mammalian TMPRSS2 fragment is expressed in a protein expression system, iii ) said protein expression system contains an inhibitor of the serine protease activity of said mammalian TMPRSS2 fragment at least during step ii ) .

According to a preferred embodiment , said mammalian TMPRSS2 protein is the human TMPRSS2 protein .

According to another preferred embodiment , said mammalian TMPRSS2 fragment has no transmembrane domain . According to a further preferred embodiment , said mammalian TMPRSS2 fragment comprises the SP domain of said mammalian TMPRSS2 protein and the LDLRA domain and/or the SRCR domain of a mammalian TMPRSS2 protein, preferably said LDLRA domain and the SRCR domain originate from the same mammalian species where said mammalian TMPRSS2 protein originates from .

According to another preferred embodiment , the inhibitor of said mammalian TMPRSS2 protein is a protein, preferably a protein with at least one transmembrane region, more preferably the inhibitor is selected from the HAI- 1 or HAI-2 proteins , most preferably the inhibitor is the HAI -2 protein .

According to a further preferred embodiment , said mammalian TMPRSS2 fragment and said mammalian TMPRSS2 inhibitor protein originate from the same mammalian species , preferably said mammalian TMPRSS2 fragment and said mammalian TMPRSS2 inhibitor protein originate from Homo sapiens .

According to still another preferred embodiment , said mammalian TMPRSS2 fragment and said mammalian TMPRSS2 inhibitor protein are coexpressed in said protein expression system .

According to another preferred embodiment , said protein expression system is selected from the list consisting of : cell-based expression system, eukaryotic cell-based expression system, eukaryotic cell-based expression system comprising HEK cells , eukaryotic cell-based expression system comprising HEK293F cells , cell- free expression system, transgenic animal , or transgenic plant .

According to a further preferred embodiment , either said mammalian TMPRSS2 fragment comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:1; or said SP domain comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 fragment consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:1; or said SP domain consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:2.

According to a further preferred embodiment, either said mammalian TMPRSS2 fragment comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO:1; or said SP domain comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 fragment consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO:1; or said SP domain consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 2.

According to a further preferred embodiment, either said mammalian TMPRSS2 fragment comprises the polypeptide sequence according to SEQ ID NO:1; or said SP domain comprises the polypeptide sequence according to SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 fragment consists of the polypeptide sequence according to SEQ ID NO:1; or said SP domain consists of the polypeptide sequence according to SEQ ID NO: 2.

According to a further preferred embodiment, said mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO: 3.

According to another preferred embodiment, said mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO: 3.

According to another preferred embodiment, said mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 3.

According to another preferred embodiment, said mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 3.

According to another preferred embodiment, said mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence according to SEQ ID NO: 3.

According to another preferred embodiment, said mammalian TMPRSS2 inhibitor protein consist of a polypeptide sequence according to SEQ ID NO: 3.

According to another preferred embodiment, it comprises the steps of a ) providing a first DNA construct suitable for the expression of the protein encoded by the first DNA construct in said protein expression system, where the first DNA construct is encoding said mammalian TMPRSS2 fragment ; b ) providing the inhibitor of said mammalian TMPRSS2 protein of step iii ) ; c ) introducing said first DNA construct and said inhibitor into said protein expression system; d) producing said mammalian TMPRSS2 fragment by said protein expression system .

According to another preferred embodiment , in step b ) of the above mentioned method, a second DNA construct suitable for the expression of the protein encoded by the second DNA construct in said protein expression system is provided, where the second DNA construct is encoding a mammalian TMPRSS2 inhibitor protein according to any of claims 5 and claims 15 to 20 ; and step c ) comprises the introduction of said second DNA construct into said protein expression system.

According to another preferred embodiment , said mammalian TMPRSS2 fragment is the mammalian TMPRSS2 fragment according to any of claims 9 to 14 .

According to another preferred embodiment , said protein expression system is a cell-based expression system, preferably a eukaryotic cell-based expression system, more preferably a eukaryotic cell-based expression system comprising HEK cells , most preferably a cell-based expression system comprising HEK293F cells ; and step c ) involves a trans fection with said first DNA construct .

According to another preferred embodiment , said protein expression system is a cell-based expression system, preferably a eukaryotic cell-based expression system, more preferably a eukaryotic cell-based expression system comprising HEK cells , most preferably a cell-based expression system comprising HEK293F cells ; and step c ) involves a trans fection with said first DNA construct and said second DNA construct , and the trans fections with said first and second DNA constructs are carried out simultaneously .

According to another preferred embodiment , said first DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 9, most preferably it comprises the nucleic acid sequence of

SEQ ID NO : 9 .

According to another preferred embodiment , said first DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 4, even more preferably it comprises the nucleic acid sequence of SEQ ID NO : 4 , most preferably it consists of SEQ ID NO : 4 .

According to another preferred embodiment , said second DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 5 , even more preferably it comprises the nucleic acid sequence of SEQ ID NO : 5 , most preferably it consists of SEQ ID NO : 5 .

The present invention further relates to a mammalian TMPRSS2 fragment of a mammalian TMPRSS2 protein, where the mammalian TMPRSS2 fragment is the mammalian TMPRSS2 ectodomain or a part thereof , comprising at least the SP domain of the mammalian TMPRSS2 protein . According to a preferred embodiment, said mammalian TMPRSS2 ectodomain is the human TMPRSS2 ectodomain.

According to another preferred embodiment, either said mammalian TMPRSS2 ectodomain comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:1; or said SP domain comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 ectodomain consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:1; or said SP domain consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 ectodomain comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO:1; or said SP domain comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 ectodomain consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO:1; or said SP domain consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 2. According to another preferred embodiment, either said mammalian TMPRSS2 ectodomain comprises the polypeptide sequence according to SEQ ID NO:1; or said SP domain comprises the polypeptide sequence according to SEQ ID NO: 2.

According to another preferred embodiment, either said mammalian TMPRSS2 ectodomain consists of the polypeptide sequence according to SEQ ID NO:1; or said SP domain consists of the polypeptide sequence according to SEQ ID NO: 2.

According to another preferred embodiment, said mammalian TMPRSS2 ectodomain is produced according to the method of any of claims 1 to 28.

The present invention further relates to a transfection vector comprising a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 4, even more preferably it comprises the nucleic acid sequence of SEQ ID NO: 4, most preferably it consists of the nucleic acid sequence of SEQ ID NO: 4.

The present invention further relates to another transfection vector comprising a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 5, even more preferably it comprises the nucleic acid sequence of SEQ ID NO: 5, most preferably it consists of the nucleic acid sequence of SEQ ID NO: 5.

The present invention also relates to a combination of two transfection vectors, wherein one of the transfection vectors comprises a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 4, more preferably it comprises the nucleic acid sequence of SEQ ID NO: 4, most preferably it consists of the nucleic acid sequence of SEQ ID NO : ; and where the other one of the trans fection vectors comprises a nucleic acid sequence having at least 95% sequence identity, preferably 98 % sequence identity with SEQ ID NO : 5 , more preferably it comprises the nucleic acid sequence of SEQ ID NO : 5 , most preferably it consists of the nucleic acid sequence of SEQ ID NO : 5 .

The present invention further relates to a cell line , which comprises eukaryotic cells , and the cells of the cell line are trans fected with the combination of said two trans fection vectors .

According to a preferred embodiment , said eukaryotic cells are preferably HEK cells , more preferably HEK293F cells .

According to another preferred embodiment , the cell line is a permanent cell line .

The present invention further relates to a method for determining whether a compound is an inhibitor of a mammalian TMPRSS2 protein, which method comprises the following steps : a ) providing the mammalian TMPRSS2 fragment of the present invention, originating from the same species as said mammalian TMPRSS2 protein; b ) contacting said mammalian TMPRSS2 fragment with said compound; c ) identi fying whether said compound acts as an inhibitor of said mammalian TMPRSS2 protein .

According to a preferred embodiment , said mammalian TMPRSS2 protein is the human TMPRSS2 protein .

The present invention further relates to the use of the mammalian TMPRSS2 fragment according to the present invention in screening methods , where said screening method is suitable for identi fying inhibitors of a mammalian TMPRSS2 protein, preferably said mammalian TMPRSS2 fragment is originating from the same species as said mammalian TMPRSS2 protein .

According to a preferred embodiment , said mammalian TMPRSS2 fragment is the human TMPRSS2 fragment and said mammalian TMPRSS2 protein is the human TMPRSS2 protein .

The present invention relates also to the use of the above- mentioned combination of trans fection vectors in trans fecting cells , where said cells after trans fection are suitable to produce the mammalian TMPRSS2 fragment according to the present invention .

Brief Description of the Drawings

Fig . 1 is the schematic structure of the native TMPRSS2 protein .

Fig . 2 is the schematic representation of the pSecTag2c - human TMPRSS2 ectodomain DNA construct .

Fig . 3 is the schematic representation of the pcDNA3 . 1 - human HAI-2 DNA construct .

Fig . 4 is an SDS-PAGE picture showing the human TMPRSS2 ectodomain content of the cell culture supernatant from the protein production step .

Fig . 5 is an SDS-PAGE picture showing the human TMPRSS2 ectodomain content of samples after puri fication and concentration, where "A" chain refers to the LDLRA-SRCR domains , and the "B" chain refers to the SP domain .

Fig . 6 depicts the enzymatic activity of the recombinant human TMPRSS2 ectodomain on the Z-GGR-AMC substrate . Fig. 7. depicts the enzymatic activity of the recombinant human TMPRSS2 ectodomain compared to that of trypsin.

Detailed Description of the Invention

Recombinant protein expression seemed to be a straightforward method to produce active TMPRSS2. However, there is no reproducible method described in the art that could yield active, wild type TMPRSS2. For drug development screening purposes the TMPRSS2 ectodomain of the membrane protein, or at least its SP domain, which is part of the ectodomain are the optimal choices. Bacterial expression of the TMPRSS2 ectodomain (with or without renaturation) yielded improperly folded, aggregated protein with very low enzymatic activity (Meyer et al., 2013) . Expression of the wild type protein in insect (e.g. Spodoptera frugiperda) cells failed to yield secreted ectodomain. Recently, it has been reported that a zymogen mutant of TMPRSS2, in which the wild type SRQSR255 activation peptide segment of the SP domain has been replaced with an enterokinase cleavage site (DDDDK) , has been expressed in Sf9 cells successfully (Fraser et al., 2021) . After isolation, the zymogen protease was activated by enterokinase and exhibited significant enzymatic activity. Importantly, the resulted protein does not contain a wild type TMPRSS2 SP domain. There are several scientific papers that report recombinant expression of wild type TMPRSS2 in mammalian (e.g., HEK, CHO) cells (Ko et al., 2015; Afar et al., 2001; Chen YW et al., 2010) . These are well-known traditional expression systems, and the corresponding publications imply that these can be used without any modification or supplementation with additional components. However, we could not reproduce these protocols. The TMPRSS2 ectodomain or the SP domain alone could not be detected in the cell culture medium in our experiments, and only misfolded, degraded protein could be detected inside the cells. Fusion proteins containing the maltose-binding protein (MBP) and either the TMPRSS2 ectodomain, or the SP domain were secreted into the medium, however, the protein had a strong tendency for aggregation, and it displayed hardly detectable enzymatic activity. The commercially available recombinant TMPRSS2 products also show low level enzymatic activity in in vitro experiments, compared to the efficiency of virus infection in cell culture (Shrimp et al., 2020; Hoffmann et al., 2021) .

To our greatest surprise, while expressing the wild type TMPRSS2 ectodomain alone did not yield any detectable amount of recombinant enzyme, coexpressing the TMPRSS2 ectodomain with HAI-2 resulted in high-yield secretion of correctly folded, enzymatically active protein. According to the state of the art, HAI-2 coexpression is either not necessary for active TMPRSS2 production (Ko et al., 2015; Afar et al., 2001; Chen et al., 2010) or it would actually decrease the amount of active TMPRSS2 in the system (Ko et al., 2020) . The fact that the cognate inhibitor, HAI-2 promoted recombinant expression of a catalytically active TMPRSS2 fragment, and the overwhelming extent of this effect completely contradicts the state of the art. For the expression we used the full-length human HAI-2 gene, consequently, as the HAI-2 protein has a transmembrane region, the recombinant HAI-2 remained in the cell membrane. The TMPRSS2 ectodomain did not contain the transmembrane region of the wild type TMPRSS2 protein, consequently, it was secreted into the cell culture medium. With this expression method of the present invention, the enzyme (i.e., the mammalian TMPRSS2 fragment, which was the human TMPRSS2 ectodomain in our mentioned experiments) and its inhibitor (HAI-2) became spatially separated: after centrifugation, the cells bearing recombinant HAI-2 were removed, and the supernatant contained the secreted recombinant human TMPRSS2 ectodomain . The human TMPRSS2 ectodomain could be easily puri fied from the cell culture using chromatographic methods well known by the person skilled in the art . The puri fied recombinant protein produced by the method of the present invention exhibits higher enzymatic activity than those described in the literature ( Shrimp et al . 2020 , Hof fmann et al . 2021 ) .

Under the term " correctly folded enzyme" we understand an enzyme derivative ( or the original protein) that has a correct fold, namely it maintains the original level of catalytic ef ficiency and speci ficity, and can perform all of its original catalytic functions , including auto-activation . A correctly folded enzyme can be a truncated enzyme compared to its wildtype form, and/or can have additional protein parts ( even other domains ) from other proteins .

A native , correctly folded TMPRSS2 protein, or a fragment thereof that comprises its ectodomain, or at least its SP domain, all have auto-activation and auto-degradation capacity . Based on the above , the inventive idea behind the present invention is that a TMPRSS2 protein, or a fragment thereof that comprises its ectodomain, or at least its SP domain, can be ef fectively expressed in a protein expression system, i f an inhibitor of the TMPRSS2 protein is also present in the protein expression system . Namely, an inhibitor of the TMPRSS2 protein shall be present in the protein expression system to be able to physically contact the TMPRSS2 protein and thereby exerting its inhibitory ef fect .

Under the term " ectodomain" , the extracellular fragment of the TMPRSS2 protein is to be understood . The extracellular fragment is built up by the LDLRA-SRCR-SP fragments , as shown in Fig. 1. It is apparent for the person skilled in the art that different mammals can have slightly different TMPRSS2 ectodomain amino acid sequences. It is also obvious for a person skilled in the art that the N-terminal and C-terminal chain termini of the TMPRSS2 ectodomain and the linker parts between said LDLRA, SRCR, and SP fragments can vary within a certain limit without significantly modifying the function and effectiveness of the TMPRSS2 ectodomain, especially the serine protease function of the SP domain. It is also obvious for a person skilled in the art that the amino acid sequence of LDLRA-SRCR-SP fragments can also vary within a certain limit without significantly modifying the function and effectiveness of the TMPRSS2 ectodomain. The preferred TMPRSS2 ectodomain according to the present invention is the human TMPRSS2 ectodomain. The most preferred TMPRSS2 ectodomain according to the present invention is the human TMPRSS2 ectodomain having the amino acid sequence according to SEQ ID NO:1. SEQ ID NO:1 corresponds to the sequence from positions 106 to 492 of the wild type human TMPRSS2 (see Fig. 1) . SEQ ID NO:1 has different numbering, namely amino acid No. 1 in SEQ ID NO:1 corresponds to position 106 of the wild type human TMPRSS2 shown in Fig. 1. The serine protease domain (i.e., the SP domain) of human TMPRSS2 protein begins with the Ile242 residue and ends with the Gly492 residue of the wild type human TMPRSS2, the sequence of this human SP domain is shown under SEQ ID NO: 2.

As mentioned above, the mammalian TMPRSS2 fragment of the present invention comprises the ectodomain of the TMPRSS2 protein or a part thereof, at least the SP domain (i.e., the serine protease domain) . The term "or part thereof" in this context refers to any parts of the ectodomain of the TMPRSS2 protein, provided that it includes the SP domain. Namely, any mammalian TMPRSS2 fragment falls within the scope of the present invention that comprises its SP domain and optionally it comprises any building blocks from the following, non- exhaustive list : the LDLRA domain, the SRCR domain, the LDLRA and the SRCR domain together ( i . e . , the whole ectodomain) , other domains even from other proteins , oligopeptides , prosthetic groups for various purposes , etc . It is important to note that said building blocks are not necessarily originating from the same species as the SP domain . As can be seen from the above listed examples , the mammalian TMPRSS2 fragment of the present invention involves also a protein construct that additionally comprises fragments or domains from the TMPRSS2 protein or from other proteins .

Theoretically, the domains of the TMPRSS2 protein can be fused to any soluble protein domain, and such a construct can be recombinantly expressed in any protein expression system . One example is the maltose-binding protein (MBP ) -TMPRSS2 fusion protein ( for details , see Example 3 ) . Obviously the fusion partner of the MBP can be the SRCR-SP and the SP domains of the TMPRSS2 protein, as well . It is obvious for a person skilled in the art that for developing inhibitors that ef ficiently inhibit a wild type TMPRSS2 protein through binding to its SP domain, the protein used in the inhibitordevelopment process as target , should most preferably contain a wild type SP domain .

The type of the protein expression system used can be any of the known systems , i f such a protein expression system is suitable for the expression of the TMPRSS2 protein fragment in the presence of an inhibitor of the TMPRSS2 protein . As the mammalian TMPRSS2 fragment of the present invention in its native , correctly folded form comprises disulphide bridged parts , the protein expression system shall be suitable to express proteins with disulphide bridges ( for details see Example 2 ) . Several suitable protein expression systems are known according to the state of the art, e.g., cell-based expression systems, cell-free expression systems, cell extracts, protein mixtures, transgenic organisms etc.

According to the present invention, the presence of an inhibitor of the TMPRSS2 protein, i.e., an inhibitor which is acting on the SP domain, shall be present, at least during the expression, in the protein expression system used.

Under the term "inhibitor" or "inhibitor of the TMPRSS2 protein", we understand any chemical compound that inhibits the serine protease activity of the TMPRSS2 protein, i.e., the activity of the SP domain. Such inhibitors can be low molecular weight organic compounds, oligopeptides or even larger proteins. Examples for low molecular inhibitors can be selected from the following list: benzamidine, NPGB (4- nitrophenyl 4-guanidinobenzoate hydrochloride) , PMSF (phenylmethylsulfonyl fluoride) pefabloc, camostat mesylate, nafamostat mesylate. The membrane-bound HAI-1 or HAI-2 proteins are the natural inhibitors of the TMPRSS2 protein, which are preferred inhibitors in the sense of the present invention. Also fragments of the HAI-1 or HAI-2 proteins can be inhibitors, provided that these fragments contain those parts of the HAI proteins that are responsible for the inhibitory activity of the serine protease function of the mammalian TMPRSS2 fragment. For example, a HAI protein that lacks the transmembrane domain can be an inhibitor of the TMPRSS2 protein, as the inhibitory activity of the HAI protein is exerted by its extracellular domain. However, more preferred inhibitors are HAI proteins with a transmembrane domain if a cell-based expression system is used. This preference is due to the fact that the transmembrane domain keeps the HAI protein bound to the membrane of the cells of the cell-based expression system, thereby making the later isolation of the mammalian TMPRSS2 fragment easier, as the latter preferably does not comprise a transmembrane domain and becomes secreted from the cell . Note , that in the case of a eukaryotic cell-based expression system, an ER signal is necessary on the HAT protein to make sure that it goes through the same path as the also ER signal containing mammalian TMPRSS2 fragment . As mentioned, while the transmembrane domain free TMPRSS2 fragment becomes secreted, the transmembrane domain containing inhibitor remains on the cell . Nevertheless , it is also possible that the mammalian TMPRSS2 fragment has an ER signal and a transmembrane domain, and the HAT protein ( or any other coexpressed inhibitory protein) has an ER signal , but lacks any transmembrane domain . Thereby the cell surface density of the active mammalian TMPRSS2 fragment of the present invention can be increased .

I f the mammalian TMPRSS2 fragment of the present invention comprises other protein domains that are di f ferent from the SP domain and which can have inhibitors , those inhibitors are always clearly identi fied in the text of the present description . Therefore , i f the term " inhibitor" is used and no other protein or protein domain is identi fiable in that context , this term " inhibitor" shall be understood as the inhibitor of the SP domain of the mammalian TMPRSS2 fragment of the present invention .

The role of the inhibitor is to inhibit the activation of the mammalian TMPRSS2 fragment during the expression . Therefore , it is essential that the inhibitor is inhibiting the serine protease activity of the mammalian TMPRSS2 fragment produced by the method of the present invention . Namely, the inhibitor shall inhibit the auto-activation once the mammalian TMPRSS2 fragment is produced by the ribosome . The inhibitor has to prohibit the binding of the substrate in the substrate binding site of the SP domain, therefore the inhibitor preferably binds into the substrate binding site . According to our finding, for the expression of a zymogen which cannot be activated, no inhibitor is needed ( for details , see Example 2 ) .

An inhibitor can be introduced into a protein expression system in several ways that are known for a person skilled in the art such that it can provide continuous presence around the TMPRSS2 protein . For instance , for cell- free protein expression systems that inhibitor compound can simply be mixed into the solution of the protein expression system, or mixed beforehand into one of the buf fers of the protein expression system . The inhibitor can also be prepared in si tu in the protein expression system by adding the necessary ingredient ( s ) to the protein expression system beforehand . One of the preferred way for introducing the inhibitor into a protein expression system is , where the inhibitor is a protein, and this inhibitor protein is coexpressed with the TMPRSS2 protein fragment . It is also within the scope of the present invention, i f combination of inhibitors is used, e . g . , an inhibitor protein coexpressed with the TMPRSS2 protein fragment together with a low molecular weight inhibitor . The inhibitor can be present in the protein expression system in an immobili zed form, for example low molecular weight inhibitors can be immobili zed on beads , which construct is easy to handle in e . g . , cell- free expression systems . I f the protein expression system is a eukaryotic cell-based expression system, it is important that the inhibitor follows the full path of the mammalian TMPRSS2 fragment produced in order to avoid the activation . In the case of low molecular weight inhibitors one of the several options is when that inhibitor can freely penetrate through all eukaryotic membranes . The TMPRSS2 protein is a conserved protein among the mammals. Therefore, the inventive idea behind the present invention can be applied to any mammalian TMPRSS2 proteins. Indeed, several non-human mammals can have diseases, for which drugs could be developed with the help of the mammalian TMPRSS2 fragment of the present invention. For example, coronavirus infection of pets (e.g., cats) and influenza virus infection of pigs are known problems in the veterinary. However, the major health issue of the human mankind today is the worldwide spread of the COVID-19 disease, caused by the infection of the SARS-CoV- 2 virus. The human TMPRSS2 fragment of the present invention is therefore a preferred embodiment, i.e., the method for producing a fragment of a human TMPRSS2 protein is a major achievement of the present invention.

The mammalian TMPRSS2 fragment of the present invention preferably lacks the transmembrane domain compared to the wild type TMPRSS2 protein. It is preferred not to have the transmembrane domain to keep the mammalian TMPRSS2 fragment of the present invention soluble for purpose of the efficient isolation, and also for easier later use in e.g., drug development tests. However, as obvious for a person skilled in the art, a mammalian TMPRSS2 fragment comprising its original or another transmembrane domain can be prepared by the method of the present invention, i.e., by having an inhibitor in the cell-based expression system.

According to another preferred embodiment, the mammalian TMPRSS2 fragment comprises not only the SP domain, but also the LDLRA domain and/or the SRCR domain of a mammalian TMPRSS2 protein. In other words, in addition to the SP domain, any, or even all the other domains of the ectodomain part of the TMPRSS2 protein (see Fig. 1) can be present in the protein construct of the present invention . It is not necessary that these other domains of the ectodomain part are originating from the same species , as under certain circumstances hybrid constructs ( e . g . , one domain from a sheep TMPRSS2 protein, an other one from the human TMPRSS2 protein) could also be useful . However, according to the preferred embodiment , all protein parts of the mammalian TMPRSS2 fragment originate from the same species , which helps to avoid compatibility problems .

As detailed above , any inhibitor that inhibits the serine protease activity of the mammalian TMPRSS2 fragment can be used during the expression . However, the preferred inhibitors are the inhibitor proteins due their high speci ficity and the possibility for their coexpression . The inhibitor proteins according to the present invention can be soluble or can have one or more transmembrane domains . It is more preferred, i f the inhibitor protein has at least one transmembrane domain, as this inhibitor would remain in the membrane of the cells of the cell-based expression system and thereby making the isolation of the mammalian TMPRSS2 fragment easier . The HAI- 1 and HAI-2 proteins are natural inhibitors of the TMPRSS2 protein, which makes any of these proteins an obvious choice for being the inhibitor according to the present invention . HAI-2 is the most preferred inhibitor protein in the sense of the present invention .

I f inhibitor proteins are applied for the purpose of the present invention, it is preferred i f the inhibitor protein originates from the same mammalian species as the mammalian TMPRSS2 protein, as the speci ficity and inhibition ability are probably higher in this case . As our main purpose is to provide a drug development tool against human diseases , it is preferred i f both the mammalian TMPRSS2 fragment and the mammalian TMPRSS2 inhibitor protein originate from Homo sapi ens, i . e . , both are of human origin .

The term " coexpression" in the context of the present invention means that the two genes , i . e . , the gene encoding the mammalian TMPRSS2 fragment of the present invention, and the gene of the inhibitor protein are expressed at the same time in the same expression system . It can be done for example via the simultaneous transient trans fections with the corresponding two DNA constructs . It is also possible that the two genes in question are present in the same DNA construct and therefore the trans fection is done only with one DNA construct . A further possibility is to make a cell that permanently contains one of the two genes , while the DNA encoding the other gene is transiently trans fected into the cell . Another possibility is where both genes are inserted into the genome of the host cell and this permanent cell line can thereafter be used for the coexpression of the two proteins . To summari ze , the requirement of the coexpression is that the genes of both proteins are present at the same time in the cell and both genes can be transcribed and the resulted is mRNA molecules translated by the same cell .

I f the inhibitor is a protein, the method of the present invention is easier to carry out i f both the mammalian TMPRSS2 fragment and said TMPRSS2 inhibitor protein are coexpressed in the protein expression system .

As mentioned above , several suitable protein expression systems are known . The protein expression system is preferably a cell-based expression system, i . e . , where living cells are expressing the mammalian TMPRSS2 fragment in the presence of an inhibitor . The gene of the mammalian TMPRSS2 fragment coupled to a suitable promoter shall be either already present in the genome or transported into the cell with any appropriate technique ( e . g . , virus vector, liposomal trans fection, CRISPR-Cas 9 etc . ) , thereafter the cell will produce the mammalian TMPRSS2 fragment . Generally, a protein will be expressed by the cell-based expression system i f the protein is not lethal to the cells . In the present case , we could not express the recombinant human TMPRSS2 ectodomain (which is a mammalian TMPRSS2 fragment in the sense of the present invention) , as the protein was capable for autoactivation and the active protein was harmful for the cells and also to itsel f , therefore it degraded before the secretion and was stuck in the Golgi . This is why our inventive idea, i . e . , applying an inhibitor during the expression, worked, namely, the mammalian TMPRSS2 fragment could not exert its harmful auto-activating and auto-degrading activity in the presence of its inhibitor .

Prokaryotic expression systems capable of producing correctly folded disulphide containing recombinant proteins either in the cytoplasm or the periplasm are known and can be useful for the purpose of the present invention . In the case of a prokaryotic cell-based expression system the mammalian TMPRSS2 fragment and the inhibitor ( e . g . , a protein inhibitor ) can be similarly coexpressed and yet finally separated . For example , the protease inhibitor can remain in the cytoplasm, while the TMPRSS2 fragment is directed into the periplasm, or becomes exported, or the protease inhibitor can be directed into the periplasm, while the TMPRSS2 fragment is secreted .

Eukaryotic cell-based expression systems are preferred, as they are well characteri zed, readily available and besides assisting disulphide bridge formation needed for the correct folding and biological activity of many extracellular or membrane proteins of both prokaryotic and eukaryotic organisms, also perform all post-translational protein modifications (e.g., glycosylation, phosphorylation etc.) , that are specific for eukaryotic proteins. More preferred are those eukaryotic cell-based expression systems, where the cells are HEK cells, and the most preferred system is the one containing HEK293F cells. HEK cells are routinely used for recombinant protein expression in the laboratory, as they are easy to handle and usually provide a good yield for a broad spectra of recombinant proteins. The special advantage of HEK293F cells is that they can be used in suspension cell culture, which enables higher cell number and higher recombinant protein yield in the same volume of cell culture medium compared to the simple cell culture flask technique (where the cells are attached to the bottom surface of the flask) .

Cell-free expression systems are also known according to the state of the art (Nakano and Yamane, 1998; Gregorio et al., 2019; Garenne et al., 2021) , which systems are also suitable for expressing the TMPRSS2 protein fragment in the presence of an inhibitor, e.g., by coexpressing the TMPRSS2 protein fragment with an inhibitor protein, or adding an isolated protein inhibitor or low molecular weight inhibitor to the cell-free system. In the latter case the purification of the product can be enhanced if the inhibitor is immobilized on a surface. In case of the use of a cell-based expression system, the purification of the product can be enhanced if the cells are bound to a surface.

As apparent for a person skilled in the art, transgenic animals and transgenic plants may also be useful for the expression of the TMPRSS2 protein fragment in the presence of an inhibitor of the TMPRSS2 protein. Protein expression in animals (Echelard, 1996; Hunter, 2019; Houdebine, 2000) and in plants (Werner et al . , 2011 ; Burnett and Burnett , 2020 ) are well described in the art . In these cases , the TMPRSS2 protein fragment is preferably expressed into a secretion fluid of said organism, as the later isolation of the TMPRSS2 protein fragment is easier to carry out from a separable fluid . Such a secretion fluid can be for example the milk . A transgenic animal can be for example the rabbit or the sheep . I f a transgenic animal or transgenic plant is used as the protein expression system, it is preferred i f the inhibitor of the TMPRSS2 protein is a protein, more preferred i f said two proteins are coexpressed by said transgenic animal or transgenic plant . However, the use of small molecules as the inhibitor of the TMPRSS2 protein is not excluded in cases a transgenic animal or transgenic plant is used as the protein expression system, as small molecules are usually easy to administer to such organisms .

The human TMPRSS2 ectodomain has the amino acid sequence according to SEQ ID NO : 1 . The SP domain of the human TMPRSS2 ectodomain has the amino acid sequence according to SEQ ID NO : 2 .

According to a preferred embodiment , the mammalian TMPRSS2 fragment of the present invention comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 1 . The term " comprise" refers here to the possibility that the mammalian TMPRSS2 fragment has also further polypeptide parts or prosthetic groups . The SP domain, which is the minimally essential part of the mammalian TMPRSS2 fragment comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 2 . The term " comprise" refers here to the possibility that the SP domain has also further polypeptide parts or prosthetic groups . The term " similarity" in the context of amino acid sequences within the framework of the present invention allows conservative substitutions of amino acid residues having similar physicochemical properties over a defined length of a given alignment . The defined length in this context is determined by the sequence identi fied with its SEQ ID NO to which the sequence defined by its similarity is aligned . For the determination of the percentage of similarity, substitution scoring matrices can be used . As the similarity defined in the present description is over 90% , those similarity scoring matrices are suitable in this context that work for closely related protein sequences , i . e . , matrices that target high similarities , such as BLQSUM90 , PAM100 , and VTML10 . The similarity percentages given in the present description are preferably defined by the VTML10 similarityscoring matrix .

According to another preferred embodiment , the mammalian TMPRSS2 fragment of the present invention consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 1 . The term " consist of" refers here to that the mammalian TMPRSS2 fragment has no other parts . The SP domain, which is the minimally essential part of the mammalian TMPRSS2 fragment consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 2 . The term " consist of" refers here to that the SP domain has no other parts .

According to a further preferred embodiment , the mammalian TMPRSS2 fragment of the present invention comprises a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 1 . The term " comprise" refers here to the possibility that the mammalian TMPRSS2 fragment has also further polypeptide parts or prosthetic groups . The SP domain which is the minimally essential part of the mammalian TMPRSS2 fragment comprises a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 2 . The term " comprise" refers here to the possibility that the SP domain has also further polypeptide parts or prosthetic groups .

The term " identity" in the context of amino acid sequences within the framework of the present invention does not allow any substitutions of amino acid residues . The percentage of identity determines the number of identical residues over a defined length in a given alignment . The defined length in this context is determined by the sequence identi fied with its SEQ ID NO to which the sequence defined by its similarity is aligned .

According to a further preferred embodiment , the mammalian TMPRSS2 fragment of the present invention consists of a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 1 . The term " consist of" refers here to that the mammalian TMPRSS2 fragment has no other parts . The SP domain which is the minimally essential part of the mammalian TMPRSS2 fragment consists of a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 2 . The term " consist of" refers here to that the SP domain has no other parts .

According to another preferred embodiment , the mammalian TMPRSS2 fragment of the present invention comprises the polypeptide sequence according to SEQ ID NO : 1 . The term " comprise" refers here to the possibility that the mammalian TMPRSS2 fragment has also further polypeptide parts or prosthetic groups . The SP domain which is the minimally essential part of the mammalian TMPRSS2 fragment comprises the polypeptide sequence according to SEQ ID NO : 2 . The term " comprise" refers here to the possibility that the SP domain has also further polypeptide parts or prosthetic groups .

According to another preferred embodiment , the mammalian TMPRSS2 fragment of the present invention consists of the polypeptide sequence according to SEQ ID NO : 1 . The term " consist of" refers here to that the mammalian TMPRSS2 fragment has no other parts . The SP domain which is the minimally essential part of the mammalian TMPRSS2 fragment consists of the polypeptide sequence according to SEQ ID NO : 2 . The term " consist of" refers here to that the SP domain has no other parts .

As mentioned above , according to a preferred embodiment of the present invention, the inhibitor of the mammalian TMPRSS2 fragment is a protein . According to a more preferred embodiment , this inhibitor protein comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 3 . The term "comprise" refers here to the possibility that the inhibitor protein has also further polypeptide parts or prosthetic groups . SEQ ID NO : 3 is the amino acid sequence of the human HAI-2 protein . The human HAI-2 protein has inhibitory ef fect also on nonhuman mammalian TMPRSS2 proteins , therefore , the use of the human HAI-2 protein as the inhibitor is not limited to the human TMPRSS2 fragment .

According to a further preferred embodiment , the mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO: 3. SEQ ID NO: 3 is the amino acid sequence of the human HAI-2 protein. The term "consist of" refers here to that the inhibitor protein has no other parts. Namely, according to one of the most preferred embodiments, the inhibitor protein is very similar to the human HAI-2 protein in its entirety.

According to a further preferred embodiment, the mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 3. SEQ ID NO: 3 is the amino acid sequence of the human HAI-2 protein. The term "comprise" refers here to the possibility that the inhibitor protein has also further polypeptide parts or prosthetic groups.

According to another preferred embodiment, the mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 3. SEQ ID NO: 3 is the amino acid sequence of the human HAI-2 protein. The term "consist of" refers here to that the inhibitor protein has no other parts.

According to still a further preferred embodiment, mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence according to SEQ ID NO: 3. SEQ ID NO: 3 is the amino acid sequence of the human HAI-2 protein. The term "comprise" refers here to the possibility that the inhibitor protein has also further polypeptide parts or prosthetic groups.

According to another preferred embodiment, mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence according to SEQ ID NO: 3. SEQ ID NO: 3 is the amino acid sequence of the human HAI-2 protein. The term "consist of" refers here to that the inhibitor protein has no other parts. According to this one of the most preferred embodiments , the inhibitor is the human HAI-2 protein .

According to a preferred embodiment , the method of the present invention further comprises the following steps : a ) providing a first DNA construct suitable for the expression of the protein encoded by the first DNA construct in said protein expression system, where the first DNA construct is encoding said mammalian TMPRSS2 fragment ; b ) providing the inhibitor of said mammalian TMPRSS2 protein of step iii ) ; c ) introducing said first DNA construct and said inhibitor into said protein expression system; d) producing said mammalian TMPRSS2 fragment by said protein expression system .

Accordingly, the mammalian TMPRSS2 fragment of the present invention is encoded by said first DNA construct , which DNA construct is suitable for the expression of the mammalian TMPRSS2 fragment , i f introduced into an appropriate protein expression system . Suitable protein expression systems are described above . Beside this first DNA construct , we need also the inhibitor which inhibits the serine protease activity of the mammalian TMPRSS2 fragment . Once we have these two components , we have to introduce them into the protein expression system . It is obvious for a person skilled in the art how to introduce a DNA construct and an inhibitor into a protein expression system . Once these components were introduced into the protein expression system, the protein synthesis can start , namely the protein expression system will produce the mammalian TMPRSS2 fragment in the presence of the inhibitor . As the inhibitor binds immediately after the protein synthesis into the binding site of the SP domain, the synthesi zed mammalian TMPRSS2 fragment will not be auto- activated . It is obvious for a person skilled in the art that an inhibitor that binds only to the activated SP domain, cannot perfectly prevent , but can signi ficantly slow down the auto-activation process , and can potently inhibit the already activated TMPRSS2 enzymes . This way such inhibitor can similarly protect the protein expressing system ( i . e . , the cell or the protein components of the cell- free system) from TMPRSS2-driven degradation . The invention is related to both inhibitor types .

According to a preferred embodiment , a second DNA construct is also provided, which second DNA construct is encoding a mammalian TMPRSS2 inhibitor protein . Usefulness of inhibitor proteins were detailed above . I f the inhibitor is provided in the form of this second DNA construct , then this second DNA construct is introduced also into the protein expression system . The introduction of these two DNA constructs can be done together or one after the other . In the latter case the inhibitor providing DNA construct should be introduced first . It is also within the scope of the present invention when said two DNA constructs are located on the same entity, like e . g . , in the same plasmid .

The protein expression system is preferably a cell-based expression system . The cell-based expression system can be for example a eukaryotic cell-based expression system, a eukaryotic cell-based expression system comprising HEK cells , or a eukaryotic cell-based expression system comprising HEK293F cells ( for details , see above ) . The trans fection with the DNA constructs can be carried out in a transient way, followed by a transient expression . Transient trans fection means that the constructed plasmids are introduced into host cells in some ways , but the foreign genes of the plasmid are not integrated into the genome of the host cell . In this case the host cells produce the recombinant mammalian TMPRSS2 fragment (and optionally the inhibitor protein if said second DNA construct is also used) until there is the transfected DNA in the cell. It is possible to carry out the method of the present invention in a permanent cell line, where the cells into the genome of which the transfected DNA was integrated shall be selected e.g., via an antibiotic resistance selection method. Such a permanent cell line can be used for a longer time period of time. Gene editing with the CRISPR-Cas9 system can also be useful in the context of the present invention. In summary, any transfection method can be useful for the purpose of the present invention, wherein the mammalian TMPRSS2 fragment can be expressed, even if the inhibitor is a protein (like e.g., the HAI proteins) and the inhibitor is coexpressed .

The above mentioned first DNA construct encodes the mammalian TMPRSS2 fragment of the present invention. As discussed above in detail, the mammalian TMPRSS2 fragment comprises at least the SP domain of the of the mammalian TMPRSS2 protein. Furthermore, as also discussed above, the mammalian TMPRSS2 protein is preferably the human TMPRSS2 protein, as the use of the present invention in the human medicine is of special interest. Therefore, we provide in the present description a modified nucleic acid sequence of the human SP domain under SEQ ID NO: 9, which sequence codes for an identical, original human SP domain amino acid sequence, but differs from the original human SP domain nucleic acid sequence such that it is optimised for the codon usage of the E. Coll translation machinery. Surprisingly, this sequence optimised for E. Coll performed well in mammalian (i.e., HEK) cells. Namely, according to a preferred embodiment, the first DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO : 9 , most preferably it comprises the nucleic acid sequence of SEQ ID NO : 9 . The term " comprise" refers here to the fact that the first DNA construct has other coding parts .

We provide the sequence of the pSecTag2c expression vector with the human TMPRSS2 ectodomain coding region under SEQ ID NO : 4 ( for details see Step 1 of Example 1 ) . This vector was used in some of our experiments , therefore , it is one of the preferred first DNA construct in the sense of the present invention . Namely, according to a preferred embodiment of the present invention, the first DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 4 , even more preferably it comprises the nucleic acid sequence of SEQ ID NO : 4 , most preferably it consists of SEQ ID NO : 4 . The term " comprise" refers here to the fact that the first DNA construct has other coding parts . The term " consists of" refers here to that the first DNA construct has the sequence as shown in SEQ ID NO : 4 .

Similarly, we provide the sequence of the pcDNAS . 1 expression vector with the human HAI-2 protein coding region under SEQ ID NO : 5 ( for details see Step 1 of Example 1 ) . This vector was used in some of our experiments , therefore , it is one of the preferred second DNA construct in the sense of the present invention . Namely, according to a preferred embodiment of the present invention, the second DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 5, even more preferably it comprises the nucleic acid sequence of SEQ ID NO : 5 , most preferably it consists of SEQ ID NO : 5 .

The present invention further relates to the protein itsel f , which is a mammalian TMPRSS2 fragment of a mammalian TMPRSS2 protein, and which mammalian TMPRSS2 fragment is the mammalian TMPRSS2 ectodomain or a part thereof , comprising at least the SP domain of the mammalian TMPRSS2 protein . The terms ectodomain, SP domain etc . were detailed above when describing and explaining the method of the present invention . The protein of the present invention is preferably the human TMPRSS2 ectodomain . The mammalian TMPRSS2 fragment of the present invention is catalytically fully functional , namely, it is inherently capable of auto-activation and autodegradation .

The explanations in relation to the sequence similarities and sequence identities with the amino acid sequences SEQ ID NO : 1 and SEQ ID NO : 2 described above in relation to the method of the present invention are also valid to the protein of the present invention, therefore we do not detail them here again .

According to a preferred embodiment , the protein of the present invention, i . e . , the mammalian TMPRSS2 fragment described above , is prepared by the method of the present invention . The method for the production of the protein of the present invention is detailed above and exempli fied in Example 1 below .

The present invention further relates to the trans fection vectors that are suitable for trans fecting cell-based expression systems in order to make such a cell-based expression system able to produce the protein according to the present invention with the method of the present invention . Namely, one of the trans fection vectors encodes the human TMPRSS2 ectodomain, the other one encodes the human HAI-2 protein . SEQ ID NO : 4 shows the nucleic acid sequence of the pSecTag2c expression vector with the human TMPRSS2 ectodomain encoding region, and SEQ ID NO : 5 shows the nucleic acid sequence of the pcDNA3 . 1 expression vector with the human HAI - 2 protein encoding region. The transfection vectors of the present invention have at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 4 and SEQ ID NO: 5, respectively. More preferably the transfection vectors of the present invention comprise a nucleic acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5, respectively. Most preferably, the transfection vectors of the present invention consist of a nucleic acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The term "comprise" refers here to the fact that the transfection vector in question has other coding parts. The term "consists of" refers here to that the transfection vector in question has the sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

The present invention also relates to the combination of the above mentioned two transfection vectors, as these two transfection vectors together form a product which is suitable for carrying out the method of the present invention and thereby producing the protein of the present invention.

The present invention further relates to cell lines, the cells of which are transfected with the combination of two transfection vectors mentioned above. This cell line of the present invention is suitable for preparing the mammalian TMPRSS2 fragment of the present invention with the method of the present invention. As the cell line is transfected with the combination of the transfection vectors, the cells will produce both the mammalian TMPRSS2 fragment and the inhibitor protein at the same time. As the inhibitor protein is present in the cell when the mammalian TMPRSS2 fragment is synthesized, it can bind immediately into the binding pocket of the serine protease domain, and the mammalian TMPRSS2 fragment will not be auto-activated. Alternatively, an inhibitor that binds only to the activated SP domain, while cannot perfectly prevent , but can signi ficantly slow down the auto-activation process , and can potently inhibit the already activated TMPRSS2 enzymes . This way such inhibitor can similarly protect the protein expressing system ( i . e . , the cell or the protein components of the cell- free system) from TMPRSS2-driven degradation . Both inhibitor types fall within the scope of the present invention . The cell line of the present invention is thereby suitable for producing a mammalian TMPRSS2 fragment , which can thereafter be puri fied and concentrated and used e . g . for drug development purposes . The cell line of the present invention comprise HEK cells , preferably HEK293F cells , which cell type was proved to be suitable in our experiments . According to another preferred embodiment , the cell line is a permanent cell line , namely it is a stable cell line and can produce the mammalian TMPRSS2 fragment of the present invention over a long period of time .

The present invention further relates to a method for determining whether a compound is an inhibitor of a mammalian TMPRSS2 protein, wherein the method comprises the following steps : a ) providing the mammalian TMPRSS2 fragment of the present invention, originating from the same species as said mammalian TMPRSS2 protein; b ) contacting said mammalian TMPRSS2 fragment with said compound; c ) identi fying whether said compound acts as an inhibitor of said mammalian TMPRSS2 protein .

The term " inhibitor" in this context refers to any compound that inhibits the mammalian TMPRSS2 protein, and thereby can be a potential lead compound for the research of drugs that can be used for the prophylaxis or treatment of diseases that can be cured by inhibiting the TMPRSS2 protein . Namely, the term "inhibitor" in this context is different from the term "inhibitor" that is used during the expression of the mammalian TMPRSS2 protein and described in detail above.

In this method, which aims at searching new inhibitory compounds against the mammalian TMPRSS2 protein, the mammalian TMPRSS2 fragment of the present invention is used. Here, obviously, the mammalian TMPRSS2 fragment shall be of the same origin (i.e., from the same species) as the targeted mammalian TMPRSS2 protein. The compound to be tested is contacted with the mammalian TMPRSS2 fragment, and if the compound inhibits the serine protease activity of the mammalian TMPRSS2 fragment, one can identify this compound as an inhibitor of the mammalian TMPRSS2 protein. Such testing methods and test systems for searching serine protease inhibitors are well known by the person skilled in the art.

According to a preferred embodiment, this method, i.e., the method for searching new mammalian TMPRSS2 protein inhibitors, is directed for searching human TMPRSS2 protein inhibitors, as human diseases are of special interest according to the present invention. For example, this method is suitable for drug development purposes against the COVID-19 disease.

Furthermore, the mammalian TMPRSS2 fragment of the present invention can be used in screening methods outlined above.

Furthermore, the above detailed combination of transfection vectors of the present invention can be used in transfecting cells, thereby one can obtain a cell line which is able to produce the mammalian TMPRSS2 fragment according to the present invention. The method of the present invention is suitable for the preparation of any mammalian TMPRSS2 fragments. It is known by the person skilled in the art that several mammalian diseases exist, where the TMPRSS2 protein of said mammal could be a potential drug target. Although, the preferred object of the method of the present invention is to be able to identify drugs against human diseases related to the TMPRSS2 protein, the present invention also relates to other mammalian TMPRSS2 proteins, like e.g., cats, dogs, horses, sheep etc., for example for the purpose of identifying drugs against coronavirus related diseases of said mammals.

As detailed above, the mammalian TMPRSS2 fragment of the present invention is prepared in the presence of its inhibitor, i.e., the enzyme is inactive in this form. If the whole ectodomain is produced, during its purification and concentration, the mammalian TMPRSS2 fragment, which has a serine protease activity, is auto-activated and goes through an autolysis. Various cleavages occur in the LDLRA and SRCR domains, however, the ectodomain does not fall apart after these cleavages due to the presence of disulphide bridges, and it remains fully functional. It dissociates into polypeptide fragments only under reducing and denaturing conditions.

Examples

Hereinafter, the present invention is described in more detail and specifically with reference to the Examples, which however are not intended to limit the present invention.

According to the method of the present invention, the wild type TMPRSS2 ectodomain (i.e., the LDLRA-SRCR-SP fragment) in recombinant, secreted, enzymatically active form in mammalian cells could be expressed.

We used the cognate TMPRSS2 inhibitor HAI-2 as a coexpression partner during transient expression in mammalian cells. The human HAI-2 protein has the amino acid sequence according to SEQ ID NO: 3.

Example 1: Preparation of the human TMPRSS2 ectodomain

In this example, the steps of the preparation of the human TMPRSS2 ectodomain is described in detail. Based on this example, a skilled person can prepare other mammalian TMPRSS2 ectodomains, as well.

Step 1: Preparation of DNA constructs

All synthetic genes were purchased from Life Technologies Corporation (Regensburg, Germany) .

The gene constructs of the human TMPRSS2 ectodomain (i.e., the LDLRA-SRCR-SP extracellular fragment of the human TMPRSS2) and the full-length human HAI-2 protein (i.e., having the Kunitzl- Kunit z2-transmembrane domain-cytoplasmic tail structure) were subcloned into appropriate expression vectors. SEQ ID NO: 4 shows the nucleic acid sequence of the pSecTag2c expression vector with the human TMPRSS2 ectodomain encoding region, and SEQ ID NO: 5 shows the nucleic acid sequence of the pcDNAS .1 expression vector with the human HAI-2 protein encoding region .

For the expression of the human TMPRSS2 ectodomain the pSecTag2c vector was used. The DNA corresponding to the human TMPRSS2 ectodomain region was ampli fied from the synthetic TMPRSS2 gene by means of PCR using the following primers : TMPS2 eml forward primer EcoRI SEQ ID NO : 6 , and TMPS2 eml reverse primer Xhol according to SEQ ID NO : 7 .

The ampli fied DNA was isolated, digested with EcoRI and Xhol restriction enzymes and ligated into the EcoRI-XhoI site of the pSecTag2c vector ( Fig . 2 ) (hereinafter referred to as pSecTag2c-TMPRSS2 ) . The recombinant construct obtained in this way contained the CMV enhancer and promoter, the IgG kappa chain leader sequence , the FLAG-tag before the human TMPRSS2 ectodomain sequence and Myc and His-tag at the C-terminus of the gene .

The full-length human HAI-2 synthetic gene inserted into the pcDNA3 . 1 expression vector (hereinafter referred to as pcDNA3 . 1-HAI2 ) was also purchased from the Li fe Technologies Corporation ( Fig . 3 ) .

The recombinant plasmid DNA of both constructs (pSecTag2c- TMPRSS2 and pcDNA3 . 1-HAI2 ) were isolated and puri fied from E . coli culture by means of the Macherey-Nagel™ NucleoBond™ Xtra Midi Kit .

Step 2 : Transi ent transfecti on and recombinant protein producti on

For recombinant protein production Freestyle 293-F (HEK293F) cells were transiently trans fected with 15pg pSecTag2c-TMPRSS2 and 15pg pcDNA3 . 1-HAI2 constructs . A transient trans fection step was made with the pSecTag2c-TMPRSS2 alone , and another transient trans fection step was made with the pSecTag2c- TMPRSS2 and 15pg pcDNA3.1-HAI2 constructs simultaneously. For the transient transfection procedure, we used the protocol of Freestyle 293 Expression System from Thermo Fisher Scientific. The Freestyle 293-F cells were cultivated in suspension culture in Freestyle 293 Expression Medium. Each transfection was carried out in 30 ml suspension cell culture. The viability of the Freestyle 293-F cells was over 90% before transfection. For each transfection sample lipid-DNA complexes were prepared using Opti-MEM I and 293fectin reagents. 30 pg plasmid DNA was diluted in 1 ml of Opti-MEM I. 60pl 293fectin reagent was diluted in 1 ml of Opti-MEM I. After 5 minutes the two solutions were combined and incubated for further 20-30 minutes at room temperature to allow the DNA- 293fectin complexes to form. In the next step the 2 ml of DNA-293fectin complex was added to 28 ml of Freestyle 293-F cell suspension to get the 30 ml total volume. The final cell density was approximately 1 x 10⁶ viable cells/ml. The cell culture was incubated in a 37 °C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm. The cell culture was harvested at 48h (2d) and 120h (5d) posttransfection and the recombinant protein content was analysed. After centrifugation at 3000 rpm the cellular fraction and the cell culture supernatant was analysed separately (Fig. 4) . Both the cellular fraction (pellet) and the supernatant (sn) were analysed on the SDS-PAGE immunoblot under reducing (red) and nonreducing (no red) conditions.

The immunoblot of 12.5% SDS-PAGE showed that the transfection with the pSecTag2c-TMPRSS2 expression construct alone did not result in any secreted recombinant protein. In the supernatant no TMPRSS2 protein was detected. The recombinant protein has got stuck inside the HEK293F cells. In the case of the cotransfection with the pSecTag2c-TMPRSS2 and 15pg pcDNAS .1-HAI2 constructs, the recombinant protein could be detected in both the cellular and the supernatant fractions. The secreted human TMPRSS2 ectodomain protein appears predominantly at a size of approximately 50 kDa, which corresponds to the uncleaved, unactivated serine protease. The activation of the serine protease, which involves the cleavage of the Arg255-Ile256 peptide bond, occurs during the purification and concentration steps by an autocatalytic mechanism.

Step 3: Purification of the recombinant human TMPRSS2 ectodomain from the cell culture supernatant

The recombinant human TMPRSS2 ectodomain was purified in two steps. The first purification step was a gel filtration, the second purification step was carried out on an IMAC column (i.e., immobilized metal chelate affinity chromatography) . The purified protein was concentrated and analysed on SDS-PAGE. During the purification and concentration, the human TMPRSS2 ectodomain auto-activated and the LDLRA and SRCR domains were digested by autolysis.

The starting material of the purification is the supernatant of HEK cells producing the human TMPRSS2 ectodomain, i.e., the supernatant from the above detailed Step 2. In the example presented here, 600 ml of HEK supernatant was used.

The purpose of the gel filtration (i.e., first purification step) is to remove media components, which interfere with the second purification step, and to exchange the buffer to one that is suitable for the second purification step (i.e., for the IMAC column purification) . The gel filtration step is summarized in detail in Table 1. Table 1: Gel filtration protocol (first purification step)

The purpose of the IMAC affinity purification step is to selectively isolate the His-tagged recombinant TMPRSS2 ectodomain from the solution. The IMAC column will selectively bind the recombinant TMPRSS2 ectodomain. The IMAC purification step is summarized in detail in Table 2.

Table 2: IMAC purification protocol (second purification step)

Step 4: concentration and freezing

The eluate from Step 3 (i.e., from the IMAC column) was concentrated and the buffer was partially exchanged.

The sample was concentrated to 2 ml using a Thermo-Pierce (cat. no. 88528, PES, 10 kDa cutoff, 2-20 mL capacity) concentrator. This concentrated sample was then diluted 10- fold with 145 mM NaCl, 20 mM Hepes, pH 7.4 and concentrated to approx. 2 ml again. The final buffer composition, which contains about 10% buffer from the column and 90% of added buffer, comprises approximately the following ingredients: 160 mM NaCl, 18 mM Hepes, 10 mM imidazol, 1 mM Tris, and has a pH of about 7.5.

The concentrated human TMPRSS2 ectodomain preparation was stored frozen in aliquots (200-400 ,L) at -20°C or lower temperature .

Step 5: SDS-PAGE analysis

Purified human TMPRSS2 ectodomain samples were analysed by SDS-PAGE on 12.5% gels under reducing conditions (Fig. 5) . Lanes of the SDS-PAGE are as follow:

Lane 1: Bio-Rad Precision Plus Kaleidoscope Marker (250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa) ;

Lane 2: Cytiva LMW-SDS Marker (97, 66, 45, 30, 20.1, and 14.4 kDa) ;

Lane 3: Purified concentrated human TMPRSS2 ectodomain stored for 2 days at 4 °C;

Lane 4: Purified human TMPRSS2 ectodomain directly after the second purification step (Ni-NTA column) ;

Lane 5: Purified concentrated human TMPRSS2 ectodomain.

During purification and concentration, the recombinant human TMPRSS2 ectodomain auto-activated and autolysed. The 50 kDa zymogen protein, which can be seen in Fig 4., disappeared and the 26-29 kDa band corresponding to the active serine protease domain (SP domain) emerged.

The results indicate degradation of the "A" chain (i.e., the LDLRA-SRCR domains) . Purified TMPRSS2 gets activated during the concentration step presumably by auto-activation. The catalytic "B" chain (i.e., the SP domain) remains stable after purification and concentration. During the purification and concentration, the human TMPRSS2 ectodomain auto-activated and the LDLRA and SRCR domains were digested by autolysis.

Step 6: Assay for the enzymatic activity of the recombinant TMPRSS2

After purification, the concentration of the recombinant human TMPRSS2 ectodomain was determined by SDS-PAGE and absorbance measurement at 280 nm. The activity was determined using the Z-Gly-Gly-Arg-AMC (Bachem; #1-1140.0025) synthetic peptide substrate .

The concentration of the recombinant human TMPRSS2 ectodomain in the enzymatic test was 20 nM, while the substrate was used in 50 pM concentration. Reactions were carried out at 30°C in 100 pL volumes on microtiter plates (Greiner, Non-binding FIA; black; #655900) . The assay buffer contained 20 mM HEPES, pH 7.4, 145 mM sodium chloride and 0.05% Triton-XIOO. Activity was followed via the generation of one of the products, the fluorescent AMC group, for 20 min by 10 s sampling at 360 nm/480 nm excitation/emission wavelengths, respectively. As Fig. 6 and Fig. 7 show, the recombinant human TMPRSS2 ectodomain protease has very high activity on the synthetic substrate, its activity is commensurable to that of trypsin. (In Fig. 6, the curved line (*, 20 nM TMPRSS2) represents the activity of 20 nM human TMPRSS2 ectodomain on 50 pM substrate, while black line (+, Background) represents the self-decay of the substrate without enzyme. In Fig. 7, the lowest line (Background) indicates the self-decay of the substrate without enzyme, the next curve upwards represents the activity of 90 nM TMPRSS2 enzyme measured on 50 pM Z-GGR-AMC substrate, the next curve upwards represents the activity of 8 nM trypsin under the same conditions, and the last line upwards represents the activity of 180 nM TMPRSS2. ) Example 2 : Preparation of the human TMPRSS2 R255Q mutant

The zymogen form of TMPRSS2 is a one-chain molecule . During activation the peptide bond between R255 and I le256 residues is cleaved by limited proteolysis . The activated TMPRSS2 consists of two polypeptide chains held together by a disulphide bridge . TMPRSS2 is capable of auto-activation : the zymogen form cleaves itsel f at the R255- I le256 bond . After puri fication and concentration the auto-activation is complete . In order to prevent auto-activation we changed the R255 to Q255 in the expression construct . Proteases having trypsin-like activity cannot cleave after the glutamine residue . The R255Q mutant is a stable zymogen which remains in the one-chain form . We expressed the R255Q mutant of the human TMPRSS2 ectodomain with and without HAI-2 . In both cases we got the same high expression level . Consequently, the stable zymogen mutant R255Q protein can be expressed without the help of the HAI-2 . The zymogen mutant showed no enzymatic activity on the synthetic substrate Z-Gly-Gly-Arg-AMC . From these experiments we can draw the conclusion that the proteolytic activity of the wild-type TMPRSS2 is harmful for the expression . The role of HAI-2 in the coexpression is to prevent the proteolytic activity of the wild-type TMPRSS2 protease . The R255Q mutant of TMPRSS2 must have negligible protease activity compared to the wild type enzyme .

Example 3 : MBP-TMPRSS2 fusion protein

Maltose-binding protein (MBP ) is a frequent fusion partner in recombinant protein expression (Kapust and Waugh, 1999 ) . MBP is a 42 . 5 kDa protein of E. coll , which is highly soluble and binds to amylose with high af finity . The MBP- fusion protein can be af finity puri fied on an amylose column . The fusion protein binds to the amylose column, and it can be eluted by using of a maltose solution . We expressed the MBP-TMPRSS2 LDLRA-SRCR-SP fusion protein in HEK293 cells with and without HAI-2 , where HAI-2 served as the serine protease inhibitor during the expression of the above protein construct (where the MBP was the E. coll protein, and the TMPRSS2 and HAI-2 were human proteins ) . Without HAI-2 we got some soluble recombinant protein, but it had a strong tendency for aggregation and it showed hardly detectable enzymatic activity . In contrast to this , when we used HAI-2 as a coexpression partner, we got stable soluble recombinant protein which showed enzymatic activity on synthetic substrate . By this experiment we proved that an inhibitor of the serine protease activity is necessary during the expression .

The sequence of the MBP-TMPRSS2 construct is shown under SEQ ID NO : 8 . The construct contains a WELQ protease cleave site ( from Trp378 to Gln381 ) which facilitates the liberation of the recombinant TMPRSS2 fragment from the MBP .

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tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag 3420 cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga 3480 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg 3540 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg 3600 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg 3660 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg 3720 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga 3780 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc 3840 acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct 3900 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 3960 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 4020 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 4080 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct 4140 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 4200 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 4260 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 4320 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4380 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4440 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4500 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4560 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4620 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4680 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4740 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 4800 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 4860 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 4920 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 4980 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 5040 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5100 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5160 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5220 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5280 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5340 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 5400 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 5460 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 5520 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 5580 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 5640 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 5700 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 5760 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 5820 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 5880 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 5940 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 6000 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 6060 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 6120 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 6180 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 6240 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 6300 cccgaaaagt gccacctgac gtc 6323

<210> 5

<211> 10262

<212> DNA

<213> Arti ficial Sequence

<220>

<223> expres sion vector with the human HAI-2 protein coding region <400> 5 atcgaaatta atacgactca ctatagggag acccaagctg gctagcatgg cgcagctgtg 60 cgggctgagg cggagccggg cgtttctcgc cctgctggga tcgctgctcc tctctggggt 120 cctggcggcc gaccgagaac gcagcatcca cgacttctgc ctggtgtcga aggtggtggg 180 cagatgccgg gcctccatgc ctaggtggtg gtacaatgtc actgacggat cctgccagct 240 gtttgtgtat gggggctgtg acggaaacag caataattac ctgaccaagg aggagtgcct 300 caagaaatgt gccactgtca cagagaatgc cacgggtgac ctggccacca gcaggaatgc 360 agcggattcc tctgtcccaa gtgctcccag aaggcaggat tctgaagacc actccagcga 420 tatgttcaac tatgaagaat actgcaccgc caacgcagtc actgggcctt gccgtgcatc 480 cttcccacgc tggtactttg acgtggagag gaactcctgc aataacttca tctatggagg 540 ctgccggggc aataagaaca gctaccgctc tgaggaggcc tgcatgctcc gctgcttccg 600 ccagcaggag aatcctcccc tgccccttgg ctcaaaggtg gtggttctgg cggggctgtt 660 cgtgatggtg ttgatcctct tcctgggagc ctccatggtc tacctgatcc gggtggcacg 720 gaggaaccag gagcgtgccc tgcgcaccgt ctggagctcc ggagatgaca aggagcagct 780 ggtgaagaac acatatgtcc tgtgagcggc cgctcgagtc tagagggccc gtttaaaccc 840 gctgatcagc ctcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca 900 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 960 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 1020 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 1080 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 1140 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 1200 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 1260 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 1320 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 1380 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 1440 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 1500 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 1560 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 1620 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta 1680 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc 1740 caagctggct agcatggcgc agctgtgcgg gctgaggcgg agccgggcgt ttctcgccct 1800 gctgggatcg ctgctcctct ctggggtcct ggcggccgac cgagaacgca gcatccacga 1860 cttctgcctg gtgtcgaagg tggtgggcag atgccgggcc tccatgccta ggtggtggta 1920 caatgtcact gacggatcct gccagctgtt tgtgtatggg ggctgtgacg gaaacagcaa 1980 taattacctg accaaggagg agtgcctcaa gaaatgtgcc actgtcacag agaatgccac 2040 gggtgacctg gccaccagca ggaatgcagc ggattcctct gtcccaagtg ctcccagaag 2100 gcaggattct gaagaccact ccagcgatat gttcaactat gaagaatact gcaccgccaa 2160 cgcagtcact gggccttgcc gtgcatcctt cccacgctgg tactttgacg tggagaggaa 2220 ctcctgcaat aacttcatct atggaggctg ccggggcaat aagaacagct accgctctga 2280 ggaggcctgc atgctccgct gcttccgcca gcaggagaat cctcccctgc cccttggctc 2340 aaaggtggtg gttctggcgg ggctgttcgt gatggtgttg atcctcttcc tgggagcctc 2400 catggtctac ctgatccggg tggcacggag gaaccaggag cgtgccctgc gcaccgtctg 2460 gagctccgga gatgacaagg agcagctggt gaagaacaca tatgtcctgt gagcggccgc 2520 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc 2580 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 2640 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 2700 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 2760 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta 2820 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 2880 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 2940 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 3000 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 3060 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 3120 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 3180 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 3240 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc 3300 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 3360 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3420 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3480 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3540 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 3600 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg 3660 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga 3720 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt 3780 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct 3840 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg 3900 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt 3960 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc 4020 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc 4080 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga 4140 tctggacgaa gagacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa 4200 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 4260 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca 4320 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata 4380 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 4440 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 4500 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 4560 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 4620 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 4680 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 4740 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 4800 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 4860 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 4920 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 4980 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 5040 aagctggcta gcatggcgca gctgtgcggg ctgaggcgga gccgggcgtt tctcgccctg 5100 ctgggatcgc tgctcctctc tggggtcctg gcggccgacc gagaacgcag catccacgac 5160 ttctgcctgg tgtcgaaggt ggtgggcaga tgccgggcct ccatgcctag gtggtggtac 5220 aatgtcactg acggatcctg ccagctgttt gtgtatgggg gctgtgacgg aaacagcaat 5280 aattacctga ccaaggagga gtgcctcaag aaatgtgcca ctgtcacaga gaatgccacg 5340 ggtgacctgg ccaccagcag gaatgcagcg gattcctctg tcccaagtgc tcccagaagg 5400 caggattctg aagaccactc cagcgatatg ttcaactatg aagaatactg caccgccaac 5460 gcagtcactg ggccttgccg tgcatccttc ccacgctggt actttgacgt ggagaggaac 5520 tcctgcaata acttcatcta tggaggctgc cggggcaata agaacagcta ccgctctgag 5580 gaggcctgca tgctccgctg cttccgccag caggagaatc ctcccctgcc ccttggctca 5640 aaggtggtgg ttctggcggg gctgttcgtg atggtgttga tcctcttcct gggagcctcc 5700 atggtctacc tgatccgggt ggcacggagg aaccaggagc gtgccctgcg caccgtctgg 5760 agctccggag atgacaagga gcagctggtg aagaacacat atgtcctgtg agcggccgct 5820 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca 5880 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 5940 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 6000 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 6060 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 6120 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 6180 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 6240 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 6300 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 6360 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 6420 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 6480 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 6540 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 6600 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg 6660 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 6720 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 6780 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 6840 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc 6900 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga 6960 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 7020 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 7080 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 7140 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 7200 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 7260 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 7320 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 7380 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 7440 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 7500 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 7560 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 7620 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 7680 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 7740 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga 7800 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 7860 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 7920 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata 7980 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 8040 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt 8100 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 8160 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 8220 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 8280 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 8340 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 8400 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 8460 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 8520 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 8580 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 8640 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 8700 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 8760 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 8820 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 8880 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 8940 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 9000 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca 9060 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 9120 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 9180 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 9240 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 9300 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 9360 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 9420 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 9480 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 9540 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 9600 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 9660 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 9720 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 9780 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 9840 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 9900 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 9960 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 10020 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 10080 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 10140 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 10200 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 10260 tc 10262

<210> 6

<211> 53

<212> DNA

<213> Arti ficial Sequence

<220>

<223> TMPS2 eml forward primer EcoRI

<400> 6 tagaattcga ttacaaggat gacgacgata agaagttcat gggctccaag tgc 53

<210> 7

<211> 25

<212> DNA

<213> Arti ficial Sequence

<220>

<223> TMPS2 eml reverse primer Xhol

<400> 7 aactcgagga ccgtcagcac gcatc 25

<210> 8

<211> 771

<212> PRT

<213> Arti ficial Sequence

<220>

<223> fusion protein of maltose binding protein and TMPRSS2 <400>

His Met Lys He Glu Glu Gly Lys Leu Vai He Trp He Asn Gly Asp 1 5 10 15

Lys Gly Tyr Asn Gly Leu Ala Glu Vai Gly Lys Lys Phe Glu Lys Asp

20 25 30

Thr Gly He Lys Vai Thr Vai Glu His Pro Asp Lys Leu Glu Glu Lys

35 40 45

Phe Pro Gin Vai Ala Ala Thr Gly Asp Gly Pro Asp He He Phe Trp

50 55 60

Ala His Asp Arg Phe Gly Gly Tyr Ala Gin Ser Gly Leu Leu Ala Glu 65 70 75 80

He Thr Pro Asp Lys Ala Phe Gin Asp Lys Leu Tyr Pro Phe Thr Trp

85 90 95

Asp Ala Vai Arg Tyr Asn Gly Lys Leu He Ala Tyr Pro He Ala Vai

100 105 110

Glu Ala Leu Ser Leu He Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro

115 120 125

Lys Thr Trp Glu Glu He Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys

130 135 140

Gly Lys Ser Ala Leu Met Phe Asn Leu Gin Glu Pro Tyr Phe Thr Trp

145 150 155 160

Pro Leu He Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly

165 170 175

Lys Tyr Asp He Lys Asp Vai Gly Vai Asp Asn Ala Gly Ala Lys Ala

180 185 190

Gly Leu Thr Phe Leu Vai Asp Leu He Lys Asn Lys His Met Asn Ala

195 200 205 Asp Thr Asp Tyr Ser lie Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr

210 215 220

Ala Met Thr lie Asn Gly Pro Trp Ala Trp Ser Asn lie Asp Thr Ser

225 230 235 240

Lys Vai Asn Tyr Gly Vai Thr Vai Leu Pro Thr Phe Lys Gly Gin Pro

245 250 255

Ser Lys Pro Phe Vai Gly Vai Leu Ser Ala Gly lie Asn Ala Ala Ser

260 265 270

Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr

275 280 285

Asp Glu Gly Leu Glu Ala Vai Asn Lys Asp Lys Pro Leu Gly Ala Vai

290 295 300

Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg lie Ala

305 310 315 320

Ala Thr Met Glu Asn Ala Gin Lys Gly Glu lie Met Pro Asn lie Pro

325 330 335

Gin Met Ser Ala Phe Trp Tyr Ala Vai Arg Thr Ala Vai lie Asn Ala

340 345 350

Ala Ser Gly Arg Gin Thr Vai Asp Glu Ala Leu Lys Asp Ala Gin Thr

355 360 365

Arg lie Thr Gly Gly Ser Gly Thr Gly Trp Glu Leu Gin Ala Ser Met

370 375 380

Thr Lys Phe Met Gly Ser Lys Cys Ser Asn Ser Gly lie Glu Cys Asp

385 390 395 400

Ser Ser Gly Thr Cys lie Asn Pro Ser Asn Trp Cys Asp Gly Vai Ser

405 410 415

His Cys Pro Gly Gly Glu Asp Glu Asn Arg Cys Vai Arg Leu Tyr Gly

420 425 430 Pro Asn Phe lie Leu Gin Vai Tyr Ser Ser Gin Arg Lys Ser Trp His

435 440 445

Pro Vai Cys Gin Asp Asp Trp Asn Glu Asn Tyr Gly Arg Ala Ala Cys

450 455 460

Arg Asp Met Gly Tyr Lys Asn Asn Phe Tyr Ser Ser Gin Gly lie Vai

465 470 475 480

Asp Asp Ser Gly Ser Thr Ser Phe Met Lys Leu Asn Thr Ser Ala Gly

485 490 495

Asn Vai Asp lie Tyr Lys Lys Leu Tyr His Ser Asp Ala Cys Ser Ser

500 505 510

Lys Ala Vai Vai Ser Leu Arg Cys lie Ala Cys Gly Vai Asn Leu Asn

515 520 525

Ser Ser Arg Gin Ser Arg lie Vai Gly Gly Glu Ser Ala Leu Pro Gly

530 535 540

Ala Trp Pro Trp Gin Vai Ser Leu His Vai Gin Asn Vai His Vai Cys

545 550 555 560

Gly Gly Ser lie lie Thr Pro Glu Trp lie Vai Thr Ala Ala His Cys

565 570 575

Vai Glu Lys Pro Leu Asn Asn Pro Trp His Trp Thr Ala Phe Ala Gly

580 585 590 lie Leu Arg Gin Ser Phe Met Phe Tyr Gly Ala Gly Tyr Gin Vai Glu

595 600 605

Lys Vai lie Ser His Pro Asn Tyr Asp Ser Lys Thr Lys Asn Asn Asp

610 615 620 lie Ala Leu Met Lys Leu Gin Lys Pro Leu Thr Phe Asn Asp Leu Vai

625 630 635 640

Lys Pro Vai Cys Leu Pro Asn Pro Gly Met Met Leu Gin Pro Glu Gin 645 650 655

Leu Cys Trp lie Ser Gly Trp Gly Ala Thr Glu Glu Lys Gly Lys Thr

660 665 670

Ser Glu Vai Leu Asn Ala Ala Lys Vai Leu Leu lie Glu Thr Gin Arg

675 680 685

Cys Asn Ser Arg Tyr Vai Tyr Asp Asn Leu lie Thr Pro Ala Met lie

690 695 700

Cys Ala Gly Phe Leu Gin Gly Asn Vai Asp Ser Cys Gin Gly Asp Ser

705 710 715 720

Gly Gly Pro Leu Vai Thr Ser Lys Asn Asn lie Trp Trp Leu lie Gly

725 730 735

Asp Thr Ser Trp Gly Ser Gly Cys Ala Lys Ala Tyr Arg Pro Gly Vai

740 745 750

Tyr Gly Asn Vai Met Vai Phe Thr Asp Trp lie Tyr Arg Gin Met Arg

755 760 765

Ala Asp Gly

770

<210> 9

<211> 756

<212> DNA

<213> Arti ficial Sequence

<220>

<223> Nucleic acid sequence encoding the human SP domain, optimised for

E . coli

<400> 9 attgcatgtg gtgttaatct gaatagcagc cgtcagagcc gtattgttgg tggcgaaagc 60 gcactgcctg gtgcatggcc gtggcaggtt agtctgcatg ttcagaatgt tcatgtttgt 120 ggtggcagca ttattacacc ggaatggatt gttaccgcag cacattgtgt tgaaaaaccg 180 ctgaataatc cgtggcattg gaccgcattt gcaggtattc tgcgtcagag ctttatgttt 240 tatggtgcag gttatcaggt ggaaaaagtt attagccatc cgaactatga cagcaagacc 300 aaaaataacg atatcgccct gatgaaactg cagaaacctc tgacctttaa tgatctggtt 360 aaaccggtgt gtctgccgaa tccgggtatg atgctgcagc cggaacagct gtgttggatt 420 agcggttggg gtgcaaccga agaaaaaggt aaaaccagcg aagttctgaa tgcagcaaaa 480 gttctgctga ttgaaaccca gcgttgtaat agccgttatg tgtatgataa tctgattacc 540 cctgcaatga tttgtgccgg ttttctgcag ggtaatgttg atagctgtca gggtgatagt 600 ggtggtccgc tggttaccag caaaaacaat atttggtggc tgattggtga taccagctgg 660 ggtagcggtt gtgcaaaagc atatcgtccg ggtgtttatg gtaatgttat ggtttttacc 720 gattggatct atcgtcagat gcgtgcagat ggttaa 756

Literature references

Afar DE, Vivanco I, Hubert RS, Kuo J, Chen E, Saffran DC, Raitano AB, Jakobovits A. (2001) Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia. Cancer Res. 61 (4) : 1686-1692.

Antalis TM., Bugge TH., and Wu Q. (2011) Membrane-Anchored Serine Proteases in Health and Disease. Progress in Molecular Biology and Translational Science, Vol. 99. 1-50. DOI: 10.1016/S1877-1173 (11) 99001-2

Bestle D, Heindl MR, Limburg H, Van Lam van T, Pilgram 0, Moulton H, Stein DA, Hardes K, Eickmann M, Dolnik 0, Rohde C, Klenk HD, Garten W, Steinmetzer T, Bdttcher-Friebertshauser E. (2020) TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Sci Alliance. 3 ( 9 ) : e202000786.

Burnett MJ, Burnett AC. (2020) Therapeutic recombinant protein production in plants: Challenges and opportunities. Plants, People, Planet, 2:121-132. Chen YW, Lee MS, Lucht A, Chou FP, Huang W, Havighurst TC, Kim

K, Wang JK, Antalis TM, Johnson MD, Lin CY. (2010) TMPRSS2, a serine protease expressed in the prostate on the apical surface of luminal epithelial cells and released into semen in prostasomes, is misregulated in prostate cancer cells. Am J Pathol. 176 ( 6 ) : 2986-2996.

Echelard Y. (1996) Recombinant protein production in transgenic animals. Curr. Op. in Biotech. 7:536-540.

Evans JP, Liu SL (2021) Role of host factors in SARS-CoV-2 entry. J Biol Chem. 2021 Jul ; 297 ( 1 ) : 100847. doi: 10.1016/ j .j be.2021.100847.

Faller N, Gautschi I, Schild L. (2014) Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea. PLoS One. 9(4) : e94267.

Fraser BJ, Beldar S, Seitova a, Hutchinson A, Mannar D, Li Y, Kwon D, Tan R, Wilson RP, Leopold K, Subramaniam S, Halabelian

L, Arrowsmith CH, Benard F (2021) Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-1 CoV-2 activation. bioRxiv preprint doi: https : //doi .org/10.1101/2021.06.23.449282

Friis S, Godiksen S, Bornholdt J, Selzer-Plon J, Rasmussen HB, Bugge TH, Lin CY, Vogel LK. (2011) Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase. J Biol Chem. 286(7) :5793-5802.

Garenne D, Haines MC, Romantseva EF, Freemont P, Strychalski

EA, Noireaux V. (2021) Cell-free gene expression. Nature Reviews Methods Primers 1 (49) . https://doi.org/10.1038/s43586-

021-00046-x

Godiksen S, Selzer-Plon J, Pedersen ED, Abell K, Rasmussen HB, Szabo R, Bugge TH, Vogel LK. (2008) Hepatocyte growth factor activator inhibitor-1 has a complex subcellular itinerary. Biochem J. 413 (2 ) : 251-259.

Gregorio NE, Levine MZ, Oza JP . (2019) A User's Guide to Cell- Free Protein Synthesis. Methods Protoc. 2019, 2 (24) ; doi : 10.3390/mps2010024.

Heurich A, Hofmann-Winkler H, Gierer S, Liepold T, Jahn 0, Pdhlmann S. (2014) TMPRSS2 and ADAMI 7 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein. J Virol. 88 (2) :1293-1307.

Hoffmann M, Hofmann-Winkler H, Smith JC, Kruger N, Arora P, Sorensen LK, Sogaard OS, Hasselstrom JB, Winkler M, Hempel T, Raich L, Olsson S, Danov 0, Jonigk D, Yamazoe T, Yamatsuta K, Mizuno H, Ludwig S, Noe F, Kjolby M, Braun A, Sheltzer JM, Pdhlmann S. (2021) Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity. EBioMedicine . 65:103255.

Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pdhlmann S. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 181 (2 ) : 271-280.

Houdebine LM. (2000) Transgenic animal bioreactors. Transgenic

Research 9:305-320. Hunter P. (2019) The prospects for recombinant proteins from transgenic animals. EMBO Reports 20: e48757.

Kapust RB, Waugh DS. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8 (8) :1668- 1674.

Kim TS, Heinlein C, Hackman RC, Nelson PS. (2006) Phenotypic analysis of mice lacking the Tmprss2-encoded protease. Mol Cell Biol. 26 (3) : 965-975.

Ko CJ, Huang CC, Lin HY, Juan CP, Lan SW, Shyu HY, Wu SR, Hsiao PW, Huang HP, Shun CT, Lee MS. (2015) Androgen-Induced TMPRSS2 Activates Matriptase and Promotes Extracellular Matrix Degradation, Prostate Cancer Cell Invasion, Tumor Growth, and Metastasis. Cancer Res. 2015 75 ( 14 ) : 2949-2960.

Ko CJ, Hsu TW, Wu SR, Lan SW, Hsiao TF, Lin HY, Lin HH, Tu HF, Lee CF, Huang CC, Chen MM, Hsiao PW, Huang HP, Lee MS. (2020) Inhibition of TMPRSS2 by HAI-2 reduces prostate cancer cell invasion and metastasis. Oncogene. 39 ( 37 ) : 5950-5963.

Laporte M, Naesens L. (2017) Airway proteases: an emerging drug target for influenza and other respiratory virus infections. Curr Opin Virol. 2017 Jun; 24 : 16-24. doi: 10.1016/ j . coviro .2017.03.018.

Meyer D, Sielaff F, Hammami M, Bdttcher-Friebertshauser E, Garten W, Steinmetzer T. (2013) Identification of the first synthetic inhibitors of the type II transmembrane serine protease TMPRSS2 suitable for inhibition of influenza virus activation. Biochem J. 452 (2 ) : 331-343. Millet JK and Whittaker GR. (2015) Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 202:120-134.

Murray AS, Varela FA, Hyland TE, Schoenbeck AJ, White JM, Tanabe LM, Todi SV, List K. (2017) Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cellsurface localization. J Biol Chem. 292 (36) : 14867-14884.

Nakano H, Yamane T. (1998) Cell-free protein synthesis systems. Biotechnology Advances, 16(2) : 367-384.

Nonboe AW, Krigslund 0, Soendergaard C, Skovbjerg S, Friis S, Andersen MN, Ellis V, Kawaguchi M, Kataoka H, Bugge TH, Vogel LK. (2017) HAI-2 stabilizes, inhibits and regulates SEA- cleavage-dependent secretory transport of matriptase. Traffic. 18 (6) : 378-391.

Paoloni-Giacobino A, Chen H, Peitsch MC, Rossier C, Antonarakis SE . (1997) Cloning of the TMPRSS2 gene, which encodes a novel serine protease with transmembrane, LDLRA, and SRCR domains and maps to 21q22.3. Genomics. 44 (3) : 309-320.

Papa G, Mallery DL, Albecka A, Welch LG, Cattin-Ortola J, Luptak J, Paul D, McMahon HT, Goodfellow IG, Carter A, Munro S, James LC . (2021) Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion. PLoS Pathog. 17 ( 1 : el 009246.

Ragia G, Manolopoulos VG. (2020) Inhibition of SARS-CoV-2 entry through the ACE2/TMPRSS2 pathway: a promising approach for uncovering early Covid-19 drug therapies. Eur J Clin Pharmacol. 76 ( 12 ) : 1623-1630.

Ramirez Alvarez C, Kee C, Sharma AK, Thomas L, Schmidt FI, Stanifer ML, Boulant S, Herrmann C. (2021) The endogenous cellular protease inhibitor SPINT2 controls SARS-CoV-2 viral infection and is associated to disease severity. PLoS Pathog. 17 (6:el009687.

Shen LW, Mao HJ, Wu YL, Tanaka Y, Zhang W. (2017) TMPRSS2 : A potential target for treatment of influenza virus and coronavirus infections. Biochimie. 142:1-10.

Shrimp JH, Kales SC, Sanderson PE, Simeonov A, Shen M, Hall MD. (2020) An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19. ACS Pharmacol Transl Sci. 2020 Sep 7; 3 (5) : 997-1007.

Tanabe LM, List K. (2017) The role of type II transmembrane serine protease-mediated signaling in cancer. FEBS J. 284 (10) : 1421-1436.

Tomita Y, Matsuyama S, Fukuhara H, Maenaka K, Kataoka H, Hashiguchi T, Takeda M. (2021) The Physiological TMPRSS2 Inhibitor HAI-2 Alleviates SARS-CoV-2 Infection. J Virol. 95(12) : e00434-21.

Werner S, Breus 0, Symonenko Y, Marillonnet S, Gleba Y. (2011) High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector. PNAS 108 (34) : 14061-

14066. Whitworth KM, Benne JA, Spate LD, Murphy SL, Samuel MS, Murphy CN, Richt JA, Walters E, Prather RS, Wells KD. (2017) Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs. Transgenic Res. 2017 Feb;26(l) :97-107.

Claims

Claims

1 . Method for producing a fragment of a mammalian TMPRSS2 protein in a recombinant manner, characterized in that i ) said mammalian TMPRSS2 fragment comprises the mammalian TMPRSS2 ectodomain or a part thereof , comprising at least the SP domain of the mammalian TMPRSS2 protein; ii ) said mammalian TMPRSS2 fragment is expressed in a protein expression system, iii ) said protein expression system contains an inhibitor of the serine protease activity of said mammalian TMPRSS2 fragment at least during step ii ) .

2 . Method according to claim 1 , characterized in that said mammalian TMPRSS2 protein is the human TMPRSS2 protein .

3 . Method according to claim 1 or claim 2 , characterized in that said mammalian TMPRSS2 fragment has no transmembrane domain .

4 . Method according to any of claims 1 to 3 , characterized in that said mammalian TMPRSS2 fragment comprises the SP domain of said mammalian TMPRSS2 protein and the LDLRA domain and/or the SRCR domain of a mammalian TMPRSS2 protein, preferably said LDLRA domain and the SRCR domain originate from the same mammalian species where said mammalian TMPRSS2 protein originates from .

5 . Method according to any of claims 1 to 4 , characterized in that the inhibitor of said mammalian TMPRSS2 protein is a protein, preferably a protein with at least one transmembrane region, more preferably the inhibitor is selected from the HAI- 1 or HAI-2 proteins , most preferably the inhibitor is the HAI-2 protein .

78

6 . Method according to claim 5 , characterized in that said mammalian TMPRSS2 fragment and said mammalian TMPRSS2 inhibitor protein originate from the same mammalian species , preferably said mammalian TMPRSS2 fragment and said mammalian TMPRSS2 inhibitor protein originate from Homo sapi ens . i . Method according to claim 5 or claim 6 , characterized in that said mammalian TMPRSS2 fragment and said mammalian TMPRSS2 inhibitor protein are coexpressed in said protein expression system .

8 . Method according to any of claims 1 to 7 , characterized in that said protein expression system is selected from the list consisting of : cell-based expression system, eukaryotic cellbased expression system, eukaryotic cell-based expression system comprising HEK cells , eukaryotic cell-based expression system comprising HEK293F cells , cell- free expression system, transgenic animal , or transgenic plant .

9 . Method according to any of claims 1 to 8 , characterized in that either said mammalian TMPRSS2 fragment comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 1 ; or said SP domain comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO : 2 .

10 . Method according to any of claims 1 to 8 , characterized in that either said mammalian TMPRSS2 fragment consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 1 ; or

79 said SP domain consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO : 2 .

11 . Method according to any of claims 1 to 8 , characterized in that either said mammalian TMPRSS2 fragment comprises a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 1 ; or said SP domain comprises a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 2 .

12 . Method according to any of claims 1 to 8 , characterized in that either said mammalian TMPRSS2 fragment consists of a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 1 ; or said SP domain consists of a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 2 .

13 . Method according to any of claims 1 to 8 , characterized in that either said mammalian TMPRSS2 fragment comprises the polypeptide sequence according to SEQ ID NO : 1 ; or said SP domain comprises the polypeptide sequence according to SEQ ID NO : 2 .

14 . Method according to any of claims 1 to 8 , characterized in that either said mammalian TMPRSS2 fragment consists of the polypeptide sequence according to SEQ ID NO : 1 ; or said SP domain consists of the polypeptide sequence according to SEQ ID NO : 2 .

15 . Method according to any of claims 5 to 7 , characterized in that said mammalian TMPRSS2 inhibitor protein comprises a

80 polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 3 .

16 . Method according to any of claims 5 to 7 , characterized in that said mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98 % similarity with SEQ ID NO : 3 .

17 . Method according to any of claims 5 to 7 , characterized in that said mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 3 .

18 . Method according to any of claims 5 to 7 , characterized in that said mammalian TMPRSS2 inhibitor protein consists of a polypeptide sequence having at least 95% identity, more preferably at least 98 % identity with SEQ ID NO : 3 .

19 . Method according to any of claims 5 to 7 , characterized in that said mammalian TMPRSS2 inhibitor protein comprises a polypeptide sequence according to SEQ ID NO : 3 .

20 . Method according to any of claims 5 to 7 , characterized in that said mammalian TMPRSS2 inhibitor protein consist of a polypeptide sequence according to SEQ ID NO : 3 .

21 . Method according to any of claims 1 to 20 , characterized in that it comprises the steps of a ) providing a first DNA construct suitable for the expression of the protein encoded by the first DNA construct in said protein expression system, where the first DNA construct is encoding said mammalian TMPRSS2 fragment ; b ) providing the inhibitor of said mammalian TMPRSS2 protein of step iii ) ;

81 c ) introducing said first DNA construct and said inhibitor into said protein expression system; d) producing said mammalian TMPRSS2 fragment by said protein expression system .

22 . Method according to claim 21 , characterized in that in step b ) a second DNA construct suitable for the expression of the protein encoded by the second DNA construct in said protein expression system is provided, where the second DNA construct is encoding a mammalian TMPRSS2 inhibitor protein according to any of claims 5 and claims 15 to 20 ; and step c ) comprises the introduction of said second DNA construct into said protein expression system .

23 . Method according to claim 21 or claim 22 , characterized in that said mammalian TMPRSS2 fragment is the mammalian TMPRSS2 fragment according to any of claims 9 to 14 .

24 . Method according to any of claims 21 to 23 , characterized in that said protein expression system is a cell-based expression system, preferably a eukaryotic cell-based expression system, more preferably a eukaryotic cell-based expression system comprising HEK cells , most preferably a eukaryotic cell-based expression system comprising HEK293F cells ; and step c ) involves a trans fection with said first DNA construct .

25 . Method according to claim 22 or claim 23 , characterized in that said protein expression system is a cell-based expression system, preferably a eukaryotic cell-based expression system, more preferably a eukaryotic cell-based expression system comprising HEK cells , most preferably a cell-based expression system comprising HEK293F cells ; and step c ) involves a trans fection with said first DNA construct and said second DNA

82 construct, and the transfections with said first and second DNA constructs are carried out simultaneously.

26. Method according to any of claims 21 to 25, characterized in that said first DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 9, most preferably it comprises the nucleic acid sequence of SEQ ID NO: 9.

27. Method according to any of claims 21 to 24, characterized in that said first DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 4, even more preferably it comprises the nucleic acid sequence of SEQ ID NO: 4, most preferably it consists of SEQ ID NO: 4.

28. Method according to any of claims 22 to 25, characterized in that said second DNA construct comprises a nucleic acid sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 5, even more preferably it comprises the nucleic acid sequence of SEQ ID NO: 5, most preferably it consists of SEQ ID NO: 5.

29. A mammalian TMPRSS2 fragment of a mammalian TMPRSS2 protein, characterized in that the mammalian TMPRSS2 fragment is the mammalian TMPRSS2 ectodomain or a part thereof, comprising at least the SP domain of the mammalian TMPRSS2 protein .

30. The mammalian TMPRSS2 fragment according to claim 29, characterized in that said mammalian TMPRSS2 ectodomain is the human TMPRSS2 ectodomain.

83

31. The mammalian TMPRSS2 fragment according to claim 29 or claim 30, characterized in that either said mammalian TMPRSS2 ectodomain comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:1; or said SP domain comprises a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:2.

32. The mammalian TMPRSS2 fragment according to claim 29 or claim 30, characterized in that either said mammalian TMPRSS2 ectodomain consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:1; or said SP domain consists of a polypeptide sequence having at least 95% similarity, more preferably at least 98% similarity with SEQ ID NO:2.

33. The mammalian TMPRSS2 fragment according to claim 29 or claim 30, characterized in that either said mammalian TMPRSS2 ectodomain comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO:1; or said SP domain comprises a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 2.

34. The mammalian TMPRSS2 fragment according to claim 29 or claim 30, characterized in that either said mammalian TMPRSS2 ectodomain consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO:1; or said SP domain consists of a polypeptide sequence having at least 95% identity, more preferably at least 98% identity with SEQ ID NO: 2.

35. The mammalian TMPRSS2 fragment according to claim 29 or claim 30, characterized in that either said mammalian TMPRSS2

84 ectodomain comprises the polypeptide sequence according to SEQ ID NO:1; or said SP domain comprises the polypeptide sequence according to SEQ ID NO: 2.

36. The mammalian TMPRSS2 fragment according to claim 29 or claim 30, characterized in that either said mammalian TMPRSS2 ectodomain consists of the polypeptide sequence according to SEQ ID NO:1; or said SP domain consists of the polypeptide sequence according to SEQ ID NO: 2.

37. The mammalian TMPRSS2 fragment according to any of claims 29 to 36, characterized in that said mammalian TMPRSS2 ectodomain is produced according to the method of any of claims 1 to 28.

38. Transfection vector comprising a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 4, even more preferably it comprises the nucleic acid sequence of SEQ ID NO: 4, most preferably it consists of the nucleic acid sequence of SEQ ID NO: 4.

39. Transfection vector comprising a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 5, even more preferably it comprises the nucleic acid sequence of SEQ ID NO: 5, most preferably it consists of the nucleic acid sequence of SEQ ID NO: 5.

40. Combination of two transfection vectors, characterized in that one of the transfection vectors comprises a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 4, more preferably it comprises the nucleic acid sequence of SEQ ID NO: 4, most preferably it consists of the nucleic acid sequence of SEQ ID NO: 4; and where the other one of the transfection vectors comprises a nucleic acid sequence having at least 95% sequence identity, preferably 98% sequence identity with SEQ ID NO: 5, more preferably it comprises the nucleic acid sequence of SEQ ID NO: 5, most preferably it consists of the nucleic acid sequence of SEQ ID NO: 5.

41. Cell line, characterized in that the cell line comprises eukaryotic cells, and the cells of the cell line are transfected with the combination of two transfection vectors according to claim 40.

42. Cell line according to claim 41, characterized in that said eukaryotic cells are preferably HEK cells, more preferably HEK293F cells.

43. Cell line according to claim 41 or claim 42, characterized in that the cell line is a permanent cell line.

44. Method for determining whether a compound is an inhibitor of a mammalian TMPRSS2 protein, characterized in that it comprises the following steps: a) providing the mammalian TMPRSS2 fragment according to any of claims from 29 to 37, originating from the same species as said mammalian TMPRSS2 protein; b) contacting said mammalian TMPRSS2 fragment with said compound; c) identifying whether said compound acts as an inhibitor of said mammalian TMPRSS2 protein.

45. Method according to claim 39, characterized in that said mammalian TMPRSS2 protein is the human TMPRSS2 protein.

46. Use of the mammalian TMPRSS2 fragment according to any of claims from 29 to 37 in screening methods, where said screening method is suitable for identi fying inhibitors of a mammalian TMPRSS2 protein, preferably said mammalian TMPRSS2 fragment is originating from the same species as said mammalian TMPRSS2 protein .

47 . Use according to claim 46 , characterized in that said mammalian TMPRSS2 fragment is the human TMPRSS2 fragment and said mammalian TMPRSS2 protein is the human TMPRSS2 protein . 48 . Use of the combination of trans fection vectors according to claim 40 in trans fecting cells , wherein said cells after trans fection are suitable to produce the mammalian TMPRSS2 fragment according to any of claims from 29 to 37 .

87