SUBTILISIN INHIBITOR OF STREPTOMYCES VENEZUELAE, AND USE OF THE GENE SEQUENCES FOR EXPRESSION AND/OR SECRETION OF HETEROLOGOUS PROTEINS IN STREPTOMYCES

Abstract: The present invention relates to a polynucleic acid coding for a protein of Streptomyces venezuelae with subtilisin inhibitor activity. The gene regulating sequences (promoter, secretion signal sequence etc.) can be used in a vector for the expression and secretion of heterologous proteins in Streptomyces.

Description: SUBTILISIN INHIBITOR OF STREPTOMYCES VENEZUEUE, AND USE OF THE GENE SEQUENCES FOR EXPRESSION AND/OR SECRETION OF HETEROLOGOUS PROTEINS IN STREPTOMYCES The present invention relates to a novel subtilisin inhibitor protein ot Streptomyces \ enezuelae and uses thereof, polynucleic acids encoding this protein, probes and primers derived from the same, the use of these polynucleic acid sequences in the expression and secretion of heterologous proteins and processes tor production ot heterologous proteins based thereon. Protein protease inhibitors are widespread in nature They occur in animal and plant cells as well as in microorganisms. They can be classified into at least 10 families on the basis of their topologicai structure (Laskowski & Kato, 1980) Recently, it has been shown that proteinase inhibitors are also widely distributed among Streptomyces species (Taguchi el al , 1993) and they secrete these compounds into the medium Most ot these are inhibitors ot serine proteases with a strong inhibitor activity towards subtilisin BPN' , but some also interact with trypsin or chymotrypsin. In addition, an inhibitor ot metal lo-proteases has been described in Streptomyces (Kumazaki et al., 1993) To date, the complete amino acid sequence ot several Streptomyces subtilisin inhibitors (SSI) has been determined These comprise plasminostreptin trom Streptomyces antήibrinolyticus (Sugmo et al., 1978) (PSN), alkaline protease inhibitor (API-2C) trom Streptomyces griseoincarnatus (Suzuki et al. , 1981 ), SSI trom Streptonnces albogri eoliis S- 3253 (Obata et al., 1989), SLP1 (synonymous tor STH or LEP 10) and LT1 (synonymous tor STI2) from Streptomyces lividans and Streploim'ces longispoms respectively (Stπckler el ai , 1992; WO 88/01278), SIL2, SIL3 and SIL4 trom Streptomyces parvuius, Streptomyces toelicolor and Streptomyces lαvendulαe, respectively (Taguchi et αl . 1994) According to the primary sequence similarity, these eight inhibitors can be divided into three groups (Taguchi el αl. , 1994). In addition a number of other Streptomyces proteinase inhibitors were characterized, namely SIL1 from Streptomyces cαcαoi (Kojima et αl 1994), SIL8 trom Streptomyces virginαe (Terabe et αl. , 1994a), SIL10, SIL 13 and SIL14 produced by Streptomyces thermotolerans, Streptomyces galbus. and Streptomyces azureus respectively (Terabe et al. , 1994b) and SIL15 from Streptomyces bikiniensis (Terabe et αl , 1995) The total length of these different mature protease inhibitors trom Streptomyces varies between 107 and 116 amino acids and a molecular weight ot 10 9 and 1 1 8 kDa They form stable dimers (I 2 ) composed of two identical subunits and tor subtilisin BPN' (E) it has been shown that they form an E,I 2 complex (Takeuchi et al 1991 ) Structure function relationship ot SSI has been investigated by crystallographic studies (Mitsui et al 1979 Takeuchi et al 1992) and by site-directed mutagenesis (Kojima et al 1990) Streptomyces has already been extensively studied as a host tor heterologous expression (for a review, see Brawnei et al ( 1991 ) and Anne and Van Mellaert ( 1993)) As a gram-positive bacterium, it offers the advantage ot possible protein secretion directly in the culture medium Moreover, extensive fermentation experience is available with this organism tor antibiotic production However, as compared to E coli, the development ot Streptomyces as a host for toreign expression is still in its very early stages Although there are many examples of heterologous expression in Streptomyces, the expression levels tor all but a tew proteins have been too low for most industrial applications There is still a need tor no el expression and/or secretion signals which may lead to higher expression levels and/or more efficient secretion of heterologous proteins. Because of the efficient expression and secretion ot subtilisin inhibitors, their promoters and signal peptides have been investigated tor the secretion ot heterologous proteins in Streptomyces including apidaecin lb (Taguchi et al 1992) and soluble torms of human T-cell receptor CD4 (Fornwald et al 1993) It is the aim of the present invention to provide tor the polynucleic acids encoding a novel subtilisin inhibitor protein of Streptomyces \ enezuelae and the use ot these new polynucleic acid sequences or fragments thereof tor the heterologous expression ot proteins in host cells, more particularly in Streptomyces It is more specifically an aim of the present invention to pro\ ide tor new polynucleic acid sequences which can be used to direct expression and/or secretion ot heterologous proteins in host cells, more particulaily in Streptomyces It is more specifically an aim of the present invention to provide tor new regulatory sequences which can be used to direct expression and/or secretion ot heterologous proteins in host cells, more particularly in Streptomyces It is in particular an aim of the present invention to prov ide tor a new promoter sequence which can be used to express heterologous proteins in host cells more particularly in Streptomyces. It is in particular another aim of the present invention to provide tor a new ribosome binding site sequence which can be used to express heterologous proteins in host cells, more particularly in Streptomyces. It is in particular another aim of the present invention to provide tor new sequences encoding a secretion signal, capable of directing and/or optimalizing the secretion of heterologous proteins in host cells, more particularly in Streptomyces It is more particularly an aim of the present invention to provide tor specifically mutated (secretion signal encodιng)-sequences, resulting in varying efficiencies of secretion ot heterologous proteins in host cells, more particularly in Streptonwces . Said signal sequences may be used to optimize the secretion efficiency of heterologous proteins in Streptomyces It is also an aim of the present invention to provide tor recombmant DNA molecules, more particularly recombinant vectors, for use in heterologous expression ot proteins in host cells, more particularly in Streptomyces. It is moreover an aim of the present invention to provide tor a process tor producing heterologous proteins in host cells, more particularly in Streptomyces It is in addition an aim of the present invention to provide tor probes hybridizing specifically to the above-mentioned new polynucleic acid sequences It is in addition an aim of the present invention to provide tor primers capable ot specific amplification ot the above-mentioned new polynucleic acid sequences. It is also an aim of the present invention to provide tor a novel subtilisin inhibitor protein isolated from Streptomyces xenezaelae, functional analogues and fragments thereof, and the use of these proteins or protein fragments in the medical and/or non-medical field All of the above-mentioned aims have been achieved bv the following embodiments ot the invention As described in more detail in the Examples section, the current invention identities a novel subtilisin inhibitor protein (VSI-protein) with a primary structure that is rather different from other known Streptomyces subtilisin inhibitors It is secreted in high amount bv Streptomyces vene∑uelae CBS 762.70 The isolation ot the gene and its sequence analysis is disclosed. The deduced primary amino acid sequence is compared to the already known sequences of other known Streptomyces subtilisin inhibitor proteins In addition, the current invention shows that the promoter and signal sequence ot the \ -gene can be used tor the highly efficient secretion of cytokines, such as mouse tumor necrosis factor (mTNF-α), human IL-2, human IL-10, and human GM-CSF by Streptomyces I ans The invention describes a polynucleic acid in substantially isolated form comprising a sequence of at least 10, more prefei ably at least 15 most preferably at least 20 contiguous nucleotides selected from: (a) the polynucleic acid sequence as shown in Fig 1 (SEQ ID NO 1 ). or (b) the polynucleic acid sequences which are degenerate as a result ot the genetic code to the polynucleic acid sequence as defined in (a) and which code tor a polypeptide showing a subtilisin inhibitory activity, or (c) the polynucleic acid sequences which hybridize to the polynucleic acid sequences as defined in (a) or (b). Fig. 1 represents the sequence of the i -gene ot this invention, isolated trom Streptomyces venezuelae CBS 762.70, encoding a novel subtilisin inhibitor protein. The term "polynucleic acid" corresponds to either double-stranded or single-stranded cDNA or genomic DNA or RNA, containing at least 10. 20, 30 40 or 50 contiguous nucleotides. A polynucleic acid which is smaller than 100 nucleotides in length is often also referred to as an oligonucleotide. Single stranded polynucleic acid sequences are always represented in the current invention from the 5' end to the 3 ' end The term "hybridize to" refers to preferably stringent hybridization conditions, allowing hybridization between sequences showing at least 70 % 80% . 90% , 95 % or more homology with each other. The terms "homologous" and "homology" are used in the current application as svnonyms for "identical" and "identity": this means that polynucleic acid (or amino acid) sequences which are said to be 80% homologous show 80% identical base pairs (or amino acids) in the same position upon alignment ot the sequences The Streptomyces polynucleic acids as disclosed abov e are preferably more than 70% , more preferably more than 80% , most preferably more than 90% homologous to the polynucleic acid sequence as shown in SEQ ID NO 1 The term "degenerate" refers to possible variations in the polvnucleic acid sequence encoding the same protein, due to the possible occurrence ot different codons tor the same ammo acid. The expression "in substantially isolated form" refers to the tact that the polynucleic acid has been isolated from its natural environment, e g by purification techniques, or by recombinant DNA techniques, or by techniques ot chemical synthesis ot DNA It implies that said polynucleic acid is 90%, more preferably 95 % , most preferably 98% pure as measured by its weight versus the weight of possible contaminants The expression "subtilisin inhibitory acitivity" refers to an inhibitory action ot the protein of the invention towards the activity ot subtilisin. a serine protease. It is to be understood however that said inhibitory activity is not limited to subtilisin. and that it is also applicable to other serine proteases, and possibly also to trypsin and/or α-chymotrypsin. In a more specific embodiment, the invention provides tor a polynucleic acid in substantially isolated form comprising or consisting ot at least part ot the sequence as shown In another embodiment the invention provides tor a recombinant DNA molecule comprising any of the polynucleic acid sequences as described above or at least part of them The term "recombinant DNA molecule" refers to any DNA molecule which has been made in vitro by DNA cloning techniques, and which mav contain DNA originating from different sources ligated to each other. Another embodiment of the invention provides tor a recombinant DNA molecule comprising the gene expression unit of any of the polynucleic acid sequences as described above, or at least part of said gene expression unit The expression "gene expression unit" refers to those sequences involved in the expression of the gene to protein, i.e. its transcription and/or translation and/or the regulation thereof The gene expression unit may thus comprise at least the promoter sequence, including possible promoter regulation sequences, the ribosome binding site, the sequence coding for the secretion signal, the sequence coding tor the mature protein, and the termination sequence. Another specific embodiment of the invention provides tor a recombinant DNA molecule comprising the gene expression unit of the polynucleic acid sequence as represented by SEQ ID NO 1 , or part(s) of it, said gene expression unit comprising: - the promoter sequence located in the region extending trom pos (-686) to pos (-38) in Fig. I (SEQ ID NO 1), more specifically located in the region extending trom position (-81 ) to pos (-38), - the ribosome binding site (RBS) located in the region extending from pos (-37) to pos (-1) in Fig. 1 (SEQ ID NO 1 ), more specifically located in the region extending trom pos (-15) to pos (-1) in Fig. 1 , - the sequence coding for the secretion signal, extending from pos ( + 1 ) to pos ( + 84) in Fig. 1 (SEQ ID NO 1), - the sequence coding for the mature protein, extending from pos ( + 85) to pos ( +439) in Fig. 1 (SEQ ID NO 1), and/or - the termination sequence located in the region extending from pos ( +442) to pos ( + 976), more specifically located in the region extending from pos ( + 518) to pos ( +558) in Fig. 1 (SEQ ID NO 1). It is to be understood that some elements of the gene expression unit (promoter, RBS, termination sequence) are localized in a sequence region, determined by way of sequence comparison with other gene sequences. The numbers delineating these regions therefore do not have to be taken exactly, but they can differ by one or several units. Another specific embodiment of the invention provides for a recombinant DNA molecule as described above, compnsing the regulatory sequence of SEQ ID NO 1 , or part of it, operably linked to a heterologous coding sequence. The expression "regulatory sequence" can encompass the promoter sequence, and/or the ribosome binding site and/or the nucleotide sequence encoding the secretion signal. Another specific embodiment provides for the use of any of the regulatory sequences of this invention in a method to direct and/or optimalize the expression and/or secretion of heterologous proteins in recombinant host cells. The expression "operably linked to" refers to a juxtaposition where the components are configured so as to perform their usual function. Thus, regulatory sequences operably linked to a coding sequence are capable of directing the expression and eventually secretion of the coding gene. The term "heterologous" means originating from another source organism than the host cell in which the protein is expiessed. It the context o the present invention, where the host cell is preferably belonging to the genus Strepton es. heterologous may refer to any organism other than Streptomyces, o * to a Streptomyces strain which is different from the host strain which is used for expression. Another specific embodiment of the invention provides tor a recombinant DNA molecule as described above, comprising the promoter sequence ot SEQ ID NO 1 . or part of it, operably linked to a heterologous coding sequence. The term "promoter sequence" refers to the nucleotide sequence in the DNA to which RNA polymerase binds to start transcription. In the context ot the present invention, it may also include upstream sequences which may be involved in the regulation ot said binding process. Still another specific embodiment provides tor a recombinant DNA molecule as described above, comprising the ribosome binding site ot SEQ ID NO 1. or part ot it, operably linked to a promoter sequence and to a second nucleotide sequence encoding a heterologous protein. The term "ribosome binding site" refers to the sequence upstream ot the initiation codon determining the site where the ribosomes bind to the mRNA to initiate protein translation. Said ribosome binding site is complementary to the 3 'end ot the 16S rRNA of the same organism. Still another specific embodiment provides tor a recombinant DNA molecule as described above, comprising the sequence encoding the secretion signal ot SEQ ID NO 1 , or part of it, operably linked to a promoter sequence and to a second nucleotide sequence encoding a heterologous protein. The term "secretion signal" or "signal peptide" refers to an oligopeptide, in most cases localised at the N-terminus of a polypeptide. said oligopeptide governing the secretion ot said polypeptide, upon which cleavage of the secretion signal follows The resulting cleaved protein, which will in most cases be secreted, is also referred to as the "mature protein". Still another specific embodiment provides tor a recombinant DNA molecule as described above, comprising the regulatory sequence ot SEQ ID NO I . or part ot it, operably linked to the coding sequence of SEQ ID NO 1. or part ot it wherem said coding sequence is fused in frame with a second heterologous coding sequence. This recombinant DNA molecule aims at the production of a fusion protein The term "fusion protein" refers to a hybrid protein consisting ot a sequence originating trom the VSI-protein, or part ot it. linked to the sequence ot a heterologous protein. In another embodiment, the invention provides tor a recombmant DNA molecule as described above, wherein the sequence encoding the signal peptide ot SEQ ID NO 1. i.e. the sequence extending from position (+ 1) to position ( + 84) in the polynucleotide sequence as represented in figure 1 , is specifically mutated resulting in one at least ot the following ammo acid substitutions: R substituted by Q at amino acid position 2 of Figure 1 , and/or R substituted by Q at am o acid position 3 of Figure 1 , and/or T substituted by R at amino acid position 4 of Figure 1 , and/or L substituted by R at amino acid position 5 of Figure 1. and/or K substituted by Q at amino acid position 6 of Figure 1. As will be exemplified further on in the Examples section, the above-specified mutations in the signal sequence lead to varying efficiencies ot secretion of heterologous proteins in Streptomyces. Another embodiment of the invention provides tor a recombinant vector capable of transforming a host cell, and capable of replicating and/or directing expression in said host cell , wherein said recombinant vector compπses a recombinant DNA molecule as described above. The term "vector" refers to any DNA molecule that replicates on its own in a host cell and can be used as a vehicle for replicating and/or directing the expression ot another type ot DNA (insert), and may include a plasmid, a phage, a cosmid or a virus. Preferably said vector is a plasmid. The term "recombinant vector" refers to a vector containing DNA trom different sources ligated together using DNA cloning-techniques. The invention more particularly provides tor a recombinant \ ector as described above wheπn said host cells can be procaryotic or eucaryotic, but preferably belong to the genus Streptomyces, and more preferably to the species Streptonn es Imdans In the latter case, the vector is most preferably chosen from the plJ-type plasmids e g p[J702, pIJ486/7 (Hopwood et al. , 1987). The invention also provides for a host cell transformed by a recombinant vector as described above, wherein said host cell can be procaryotic, e.g a bacterium like Eschericia coli or Streptomyces , or eucaryotic, such as plant cells, animal cells , or yeast cells. In a specific embodiment the invention provides tor a transformed host cell belonging to the genus Streptomyces, and being preferably Streptomyces Imdans In another embodiment the invention provides tor a process tor producing a heterologous protein from a host cell, comprising the steps ot - growing a culture of a host cell transformed by a recombinant DNA vector as described above under conditions which enable the expression and possibly secretion of the heterologous protein, - recovering said heterologous protein from said culture In a specific embodiment the invention provides tor a process as described above, with said host cell belonging to the genus Streptomyces, and being most preferably Streptomyces Imdans. In another embodiment, the invention provides tor a recombinant polypeptide being the product of a process as described above. In a subsequent embodiment the invention provides tor a polypeptide in substantially pure form comprising an amino acid sequence, or part ot it encoded by any ot the polynucleic acid sequences as defined above. More specifically, the invention provides tor a polypeptide in substantially pure form, comprising a sequence of at least 9 contiguous ammo acids selected from (a) an amino acid sequence encoded by any of the polynucleic acid sequences as defined above, or (b) the sequence of 146 amino acid residues as represented in Fig 1 (SEQ ID NO 2). or (c) polypeptide sequences resulting from the modification ot one or several amino acids in the polypeptide sequence as represented by SEQ ID NO 2, said modifications comprising substitution and/or deletion and/or addition ot one or several amino acids, provided that said modficiation do not abolish the subtilisin inhibitory activity of the polypeptide encoded by SEQ ID NO 2. The sequence as represented in SEQ ID NO 2 refers to a novel subtilisin inhibitor protein isolated from Streptomyces venezue/ae CBS 762 70 (VSI-protein) as described in the examples section. It is characterized by an inhibitory activity towards proteases like subtilisin. The subtilisin inhibitor activity can be determined by any method know n by the man skilled in the art, and more specifically by a method as described in the examples section, or as described by Kojima et al. (1990). The term "polypeptide" designates a linear series ot amino acids connected one to the other by peptide bonds between the alpha-ammo and carboxy-groups ot adiacent ammo acids Polypeptides can be in a variety of lengths, either in their natural (uncharged) torms or in torms which are salts, and either free ot modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications It is well understood in the art that amino acid sequences contain acidic and basic groups, and that the particular lonization state exhibited by the polypeptide is dependent on the pH ot the surrounding medium when the protein is in solution, or that ot the medium trom which it was obtained it the protein is in solid form. Also included in the definition are proteins modified by additional substituents attached to the amino acids side chains, such as glycosyl units, lipids. or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains, such as oxidation of sulphydryl groups Thus, "polypeptide" or its equivalent terms is intended to include the appropriate amino acid sequence referenced. sub|ect to those of the foregoing modifications which do not destroy its functionality. The polypeptides of the invention can be prepared by classical chemical synthesis. The synthesis can be carried out in homogeneous solution or on solid phase. For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book entitled "Methode der organischen chemie ' (Method ot organic chemistry) edited by E. Wunsh, vol. 15-1 et II. THIEME. Stuttgart 1974. The polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989). The polypeptides according to this invention can also be prepared by means of recombinant DNA techniques as described by Sambrook el al . (Molecular Cloning: A Laboratory Manual, 2nd Edition, New York, Cold Spring Harbor Laboratory Press, 1989). The expression "in substantially pure form " signifies that the polypeptide has been purified from its natural environment, e.g. by protein purification techniques, or by production in vitro via recombinant DNA techniques, or by chemical protein synthesis techniques. It implies that said polypeptide has a degree ot purity ot at least 90% , preferably 95 % and more preferably of 98% o the protein expressed in its weight versus the weight ot possible contaminants, as determined by one or two dimensional PAGE (polyacrylamide gel electrophoresis). The present invention also relates to proteins characterized bv an ammo acid sequence showing a homology of at least 60% , more preferably at least 70% more preferably at least 80% and most preferably at least 90% to the amino acid sequence as described in SEQ ID NO 2. Said "related" proteins may also be referred to as "muteins" ot the protein shown in SEQ ID NO 2. The term "muteins" (or "functional analogues") may be defined as proteins containing substitutions and/or deletions and/or additions ot one or several amino acids, provided that said muteins have retained the subtilisin inhibitor activity characteristic for the Streptomyces protein of the invention. It should be evident that such muteins might have a slightly different molecular weight from the protein described by SEQ ID NO 2 as determined by SDS-PAGE. An overview of the amino acid substitutions which could form the basis for the above-mentioned muteins is shown in Table 1. It is known from the prior art (e.g. Kojima el al. 1990) that specific mutations in the active site of subtilisin inhibitor proteins may cause alterations ot the substrate specificity. It is therefor to be understood that the above-mentioned muteins may also show a different substrate specificity than the VSI-protein as isolated trom Sirepionn ces venezuelae CBS 762.70. In a very specific embodiment the invention provides tor the novel VSI-protein of Streptomyces venezuelae CBS 762.70, as shown in Fig. 1 (SEQ ID NO 2). This invention thus provides for a polypeptide in substantially pure form comprising or consisting of at least part of the amino acid sequence as represented by SEQ ID NO 2. In addition, the invention also provides fragments ot said polypeptides, with said fragments still showing subtilisin inhibitory activity and/or with said fragments being able to elicit antibodies binding specifically to the above-mentioned polypeptides. Most preferably, said fragments are located within the region ot the polypeptide sequence extending from amino acid pos. 55 to amino acid pos. 1 18 as shown in Fig. 1 (SEQ ID NO 2), and most preferably from amino acid pos. 55 to amino acid pos. 80. The invention also provides for a fusion protein comprising a polypeptide of the invention, or a fragment thereof, fused to a heterologous protein. Another embodiment of the invention provides tor the above-mentioned polypeptides or polynucleic acids for use as a medicament, or for use as an additive in the non-medical field, for instance food/feed processing. The polypeptides of the invention can be used in applications ot different kind. In the medical and/or pharmaceutical field, the subtilisin inhibitory activ ity ot the above-mentioned polypeptides may be applied to treat medical distresses e.g in the modulation ot blood coagulation (inhibition of plasminogen activator), in the do nregulation ot inflammatory reactions, in the treatment of invading tumors (inhibition ot urokinase) Moreover, in the non- medical field (e.g. food and fermentation industry) the above-mentioned polypeptides may be used to prevent proteolytic degradation (e.g. as additives) Another embodiment of the invention provides tor the use ot the polypeptides or the polynucleic acids of the invention for the manufacture ot a medicament tor therapeutic use as specified above. According to another embodiment, the present invention also relates to an oligonucleotide comprising at least 10 contiguous nucleotides selected trom any ot the polynucleic acid sequences as described above, tor use as a specific hybridization probe detecting the polynucleic acids of the invention. The term "probe" refers to single stranded sequence-specific oligonucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected. Preferably, these probes are about 5 to 50 nucleotides long more preferably from about 10 to 25 nucleotides. According to the hybridization solution (SSC, SSPE. etc ) these probes should be stringently hybridized at their appropriate temperature in order to attain sufficient specificity. However, by slightly modifying the DNA probes, either by adding or deleting one or a tew nucleotides at their extremities (either 3' or 5'), or substituting some non-essential nucleotides ( l e nucleotides not essential to discriminate between types) b\ others (including modified nucleotides or inosine) these probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions (i.e the same temperature and the same hybridization solution). Also changing the amount (concentration) ot probe used mav be beneficial to obtain more specific hybridization results. It should be noted in this context that probes ot the same length, regardless of their GC content, will hybridize specifically at approximately the same temperature in TMAC1 solutions (Jacobs et al , 1988). The latter implies that variant probes contemplated within this aspect ot the present invention can be defined as probes hybridizing with the same speciticitv as the probe they are derived trom under different, but stringent, hybridization and wash conditions (different solutions, different concentrations of butter, different concentrations ot probe, and/or different temperatures). The oligonucleotides used as primers or probes may also contain or consist of nucleotide analoges such as phosphorothioat.es (Matsukura et al 1987), alkylphosphoro- thiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al . 1991 Nielsen et al.. 1993) or may contain intercalating agents (Asseline et al.. 1984). As most other variations or modifications introduced into the original DNA sequences of the invention, these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However, the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides. The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc. In a very specific embodiment the oligonucleotide probe comprises a sequence chosen from among the following group of sequences: SEQ ID NO 3: CGCTCTACGCCCCCTC, or SEQ ID NO 4: CTTCATSGTGCACTGGTTG. According to another embodiment, the present invention also relates to an oligonucleotide comprising at least 10 contiguous nucleotides selected from any of the polynucleic acid sequences as described above, for use as a primer tor specific amplification of any of the polynucleic acids of the invention. The term "primer" refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis ot extension products. Preferably the primer is about 5-50 nucleotides long, more preferably trom about 10 to 30 nucleotides long. Specific length and sequence of the primer will depend on the complexity of the required DNA or RNA targets, as well as on the conditions ot primer use such as temperature and ionic strength. The fact that amplification-primers do not have to match exactly with the corresponding template sequence to warrant proper amplification, is amply documented in the literature (Kwok el al. , 1990). In a specific embodiment, the oligonucleotide primer comprises a sequence chosen trom among the following group of sequences: SEQ ID NO 7: TTCGCCAACCAGTGCACGAT or SEQ ID NO 8: CTTGAGGGTGCGACGCATGG or SEQ ID NO 9: ACGAAAGGTCACCGGGACAT or SEQ ID NO 10: CATTTCAGATCTGCACCC. In a specific embodiment, the present invention also relates to the optimalisation of the secretion of heterologous proteins in host cells by modifying the amino acid sequence of said secretion signal through site-specific mutagenesis. As further illustrated in the examples section, said site-specific mutagenesis may be achieved using at least one of the following oligonucleotide primers: SEQ ID NO 16: GCTCCCACGGCCTGCAGGGTCTGCTGCATGGTGAACTCTCC SEQ ID NO 17: GCTCCCACGGCCTGCAGGGTCTGACGCATGGTGAAC SEQ ID NO 18: GCTCCCACGGCCTGCAGGGTGCGACGC SEQ ID NO 19: CCACGGCCTTGAGACGTCGACGCATGGTGAAC SEQ ID NO 20: CCCACGGCCTTGCGACGTCGACGCATGGTGAAC resulting respectively in modification of the following amino acids in the signal sequence: R substituted by Q at position 2 of Figure 1 , R substituted by Q at position 3 of Figure 1 , T substituted by R at position 4 of Figure 1 , L substituted by R at position 5 of Figure 1 , K substituted by Q at position 6 of Figure 1. Table Legends Table 1 : Amino acid substitutions which may form the basis ot the muteins according to the present invention. Table 2: E. coli strains used and their genotype. Table 3: 3a: E. coli plasmids used in the current invention. 3b: Streptomyces plasmids used in the current invention. Table 4: Primers used in the DNA sequencing reactions. Table 5: Comparison of mTNF yield detected in supernatants of transformed S. Imdans cultures. Table 6: Oligonucleotides used for in vitro mutagenesis. Figure legends: LXgA: Nucleotide sequence of the cloned 1.6 kb fragment and the deduced ammo acid sequence of the Svenezuelae subtilisin inhibitor. The ammo acid residues identified by N-termmal amino acid sequence analysis are underlined. Direct and inverted repeats are indicated by specific arrows above the lines and the -35 and -10 region of the r.sv putative promoter are underlined, is the transcription initiation site and "Ψ" is the signal peptidase cleavage site. £lfi_2: (a) SDS-PAGE of the culture filtrate of S. venezuelae CBS 762.70. standard: low molecular weight marker (3 μg/protein: Pharmacia) lane 1 : about 2 μg total protein lane 2: about 10 μg total protein (b) SDS-PAGE of fractions 47, 49, 51 , 53 and 55 following elution from the Sephadex G50 column. (a) Restriction map of the 5kb Pvuϊl fragment ot S venezuelae CBS 762 70 chromosomal DNA hybridizing with the deoxyoligonucleotides 5'-CGCTCTACGCCCCCTC-3' (SEQ ID NO 3) and 5'-CTTCATSGTGCACTGGTTG-3' (SEQ ID NO 4) complementary to the DNA sequence of the N- and C-termmal part of VSL (b) Sequencing strategy of the 1.6 kb subfragment ot the 5.0 kb IXuW fragment containing the vs/ gene. (c) FRAME analysis (Bibb et al. , 1984) of the 1 .6 kb nucleotide sequence with potential start ( | at the bottom line) and stop ( | in the middle) codons. Fig 4: Comparison of the signal peptides of subtilisin inhibitors from S ve ezuelae (VSI), S Imdans (SLP1), S.longisporus (STI2) and S albogriseo/us (SSI). Italic sequences represent possible pro-protein sequences which are possibly cleaved off during maturation ot the protein. Fi£_5_: Comparison of the different promoters of Slreptonn tes subtilisin inhibitors ot S albogriseolus ( ss/77 and ssιP2), S Imdans 1362-K12 (stiPI) and S venezuelae \ sιlX Short direct repeats are indicated by arrows. Fig 6: Alignment of amino acid sequences ot the mature forms ot 18 protease inhibitors of Streptomyces spp. of which the complete amino acid sequence is known β-strands and Ot- helices known from crystallographic studies (Takeuchi et al 1992 ) are indicated. The proteinase inhibitors are trom S venezuelae CBS 762 70 (VSI) S albogrιset>lus (SSI), S gi'iseomcarnatus (API-2c'), S Imdans (STH ), S coe/ico/or (S\L3). S ambofaciens (S1L ~ ), S pan-ulus (S1L2), S antifibrmolyticus (PSN), Sgalbus (SILO) S (STI2), S kiΛ'endulae (SIL4), S thermotokrans (SIL10) S azureus (SIL 14) S fraώae (SIL5), S hygroscopicus (SIL12), S virginae (SIL8), S bikuvensis (SI L 15 ) and S acaoi (SIL 1 ) Bold characters: residues common to eight or more proteinase inhibitors among the 18 alhgned; "*": perfectly conserved residues, " " reactive site ot inhibitors," ' well conserved residues FJS-2 N-terminal amino acid sequence analysis ot the abundantly secreted proteins in 48 h cultures ot S lividans TK24[pCBS(2)mTNF]. Els t SDS-PAGE [12.5 %(w/v)gels] (A) and immunoblot analysis (B) ot culture filtrate of Slividans TK24 [pCBS(2)mTNF] grown for 48h in NM-medium. Samples in panel A are from cultures' 1 ot Slividans TK24 [pIJ486| (lane 1 ). (250μl), and S lividans [pCBS(2)mTNFl (lane 2-4) (250μl, 125μl. 62μl. resp ) Lane 5 is rmTNF ( lμg) and lane 6 is Gibco-BRL low molecular weight protein standard Panel B represents 50ng rmTNF (50ng) and 10μl of Slividans TK24[pCBS(2)mTNF| All culture filtrates were precipitated with 20% tπchloroacetic acid and dissolved in gel loading butter, 'protein content of culture filtrate is 60-80μg/ml. Phylogenetic tree of the subtilisin inhibitor proteins ot the 18 Streptoi ces spp tor which the ammo acid sequence of the complete mature protein was available Abbreviations see legend to Fig. 6. Immunoblot analysis ot culture filtrate of S Imdans TK24 |pCBS2hlL 10| grown tor 30h in NM medium. Lane 1. Gibco-BRL low molecular weight protein standard, lane 2 1 ml ot S Imdans TK24 fpCBS2hIL10] culture filtrate (protein content 80 μg/ml )) precipitated with 20% tπchloroacetic acid and dissolved in gel loading butter No immunoreactive bands were visible in the S Imdans TK24[pIJ486] culture filtrate (not show n) Figure 1 1. Schematic representation of A the construction of the vsi-mTNF secretion cassette from the plasmid pCBS2mTNF and B. the mutations introduced in the original VSI signal peptide (vsi-P l : promoter of the vsi- gene, SSvsi: signal sequence of the vsi-gene, SD: Shine Dalgarno sequence, SPase: signal peptidase cleavage site, N: hydrophilic region of the signal peptide. H : hydrophobic region of the signal peptide, C: C-terminal region of the signal peptide). Figure 12. A. Intracellular. extracellular and total amounts of mTNF produced by S. imdans under the influence of the different signal peptide mutants as obtained by biological activity measurements. B. Coomassie staining of secreted proteins of Imdans cultures, harbouring the different signal peptide mutants, after TCA precipitation and SDS-PAGE. Equivalents of 50 μl culture fluid were applied on gel. C. Graphical representation of secretion efficiencies calculated as secreted mTNF/(secreted mTNF + intracellular mTNF) x 100. Figure 13. Immunoblot of secreted (a) and intracellular mTNF (b). For each mutant, an equivalent of 20 μL culture fluid ar.d a corresponding amount of cell lysate were used for SDS-PAGE and subsequent blotting.-: Slividans [plJ486/ Figure 14. Slot blot of total RNA isolated from S. lividans strains, containing the different signal peptide mutants, hybridised with A: a mTNF cDNA specific probe and B: a chromosomal sti l -probe. Next to the blots the average OD. obtained by scanning, is indicated. Table 1 : Amino acid substitutions which may form the basis of the muteins accenting to the present invention. Amino acids Synonymous gioups Seτ (S) Ser, Thr. Gly. Asn Arg (R) Arg, His. Lv s. Glu. Gin Leu (L) Leu. He. Met. Phe. Val. Tyr Pro (P) Pro. Ala. Tin. Gly Thr (T) Thr. Pro. Ser. Ala Gly. His. Gin Ala (A) Ala Pro, Gly. Tin Val (N) Val, Met. He. Tyr. Phe. Leu Gly (G) Gly, Ala Thr. Pro. Ser H (I) He. Met. Leu. Phe. Val. Tyr Phe (F) Phe. Met. He. LeiL Tip. Val Tyτ (Y) Tyr. Phe. Tip. Met. He. Val. Leu Cys (C) Cys, Ser. Thr. Met His (H) His. Gin. Arg. Lys. Glu. Thr Gln (Q) Gin. Glu. His. Lys. Asn. Thr. Arg Asn (Ν) .Asn. Asp. Ser. Gin Lys (K) Lys, Arg. Glu. Gin. His Asp (D) Asp. Asn. Glu. Gin Glu (E) Glu, Gin. .Asp. Lys. .Asn. His. Arg Met (M) Met. He. Leu. Phe. Val Table 2. Ecoli strains used and their genotype Strain Genotype HBI01 F Δ(gp1-p>vA)62.leuB6, supi.44. antl 4. alK2, lacYl, proΛ2, rspl.20. wl-5.mil- 1, rec A IS. ΔfmcrC, mn)', (stι) SURE ■ TM el 4- (mcrλ), ΔmcK ' B-hsdSMR-mπiri. sbc( recB, recJ. umuCXnJ (kcoi).uvιi ' . supE-14, in lac, gvrA 6, ivIAl. ilu-l. eiu/A //F /vvAB. laPZΔM15, TnW.(tei')] XL 1 -Blue MRF Λ(mcrA)lS3, ΔfmcK endAl. supE-(4, thi-1, ivcA, χrAl>6, iv/Al. lac; ):[¥' proAB, IOCFZΔMID. TnW fief)] 15 DH5αF F, endAl. hsdRX ιhι-1. recAl, gyrAXi re/Al. Nthl, luXZ\\I Δ(lacZYAargF)U169 JM109 endAl, recAl, μ A'X ihi. h.sdR X (ι . ///,, ). ivIAl. supE44. el4 ' (mcτA ' )X.Mac-pιvAB), // ■' . i ml XX. pro A B " >0 lacWMlL . (NaD BMH 71-18mutS thi, supE iac-piv B). [iiiuiS TnIO]. [/ •" . proA B , laci'OZMlL TGI supE, iχhsdXl-mcrB)5(r U^nicrBl. Xkv-pmA B). thi. [E. tntlXό, proA B lacr'λZM415/ Table 3a Ecoli plasmids used in the cuirent invention Name Description Reference pACYC184 Tc R , Cm R Chang & Cohen, 1978 pUC19 MCS, Ap R , LacZ Norrander el ai. 1983 pBluescriptπ KS(+) MCS, Cm R , LacZa, Stratagene n(+)ori pBSDK pBluescript KS(+) missing this work Droll pIG2 mTNF Tc R , P L promoter. mTNF Innotienerics pIG2 mTNF-m7 pIG2 mTNF with a newly this work formed Snά l site at the lst codon of mTNF pBSDK-CBSS pBSDK containing a 574bp this work Smal fragment with part of vsi gene pBS-CBSN pBluescript II KS(+) with vsi this work signal sequence followed by N.si site and MCS pCBS2 mTNFEc pBSDK-CBSS with mTNF gene this work fused to vsi 2 codons downstream the vsi signal sequence 11 pCBS TNFEc pBS-CBSN with mTNF gene this work fused directly to vsi signal sequence pCBS2hIL10Ec pBSDK-CBSN with hIL 10 gene thi s work fused to vsi, 2 codons downstream the vsi signal sequence pIGFH20hTL10 Tc R , P L promoter, hILlO Innogenetics pUC18hTL2 Ap R , hIL2 (cDNA) Innogenetics pIGH2hGM-CSF Tc R , P L promoter, hGM-CSF (cDNA) Innogenetics pIGH2hGM-CSF-m5 pIGH2hGM-CSF with a newh this woik formed Espl site at the first codon of hGM-CSF (cDNA) (oligo CSFBL) pEPsBLA Ap κ , synthetic M/ gene this work pBSDK-CBSS pBSDK containing a 574 bp this k Smal fragment with part of pCBS2hIL10Ec pBS-CBSN with hILl O gene this oik fused to v.sv 2 codons downstream v.sv signal sequence pCBS2hIL2Ec pBSDK-CBSS with hIL2 gene this k fused to v.sv 7. codons downstream v.sv signal sequence pCBShlL-lEc pBS-CBSN with hIL2 fused this work directly to v.sv signal sequence pCBS2hGM-CSFEc pBSDK-CBSS with hGM-CSF fused this work to v.sv 2 codons downstream vs/ signal sequence pCBS2sBLAEc pBSDK-CBSS with synthetic bla-fυsed this work to VΛV 2 codons downstream vs/ signal sequence pCBSsBLAEc pBS-CBSN with synthetic Na fused this work directly to v.S7-sιgnal sequence TaUe 3b: Streptomyces plasmids used in the piesent invention Name Description Reference pIJ486 promoter probe vector, Ward el ul 1986 Tsr 1 *, promoterless Neo R pCBS2mTNF pIJ486 containing the module this work of v.sv signal sequence, 2 codons of mature vsi and mTNF cDNA pCBSmTNF pIJ486 conta.ning the module this work of vsi signal sequence directly fused to mTT cDNA pCBS2hTL10 pIJ486 containing the module this work of v.sv signal sequence, 2 codons of mature vsi and hILl O cDNA pCBS2hIL10 pIJ486 containing the module of v.sv this work signal sequence, 2 codons of mature v.sv and hILl O cDNA pCBS2hIL2 pIJ486 containing the module of v.s this work signal sequence, 2 codons of mature v.sv and hTL2 cDNA pCBShIL2 pIJ486 containing the module of v.sv this work signal sequence directly fused to hIL2 cDNA pCBS2hGM-CSF pIJ486 containing the module of vsi this work signal sequence, 2 codons of mature pCBS2sBLA pU486 containing the module of vsi this work signal sequence, 2 codons of mature vsi and synthetic bla pCBSsBLA pIJ486 containing the module of vsi this work signal sequence directly fused to synthetic bla Abbreviations: MCS = multiple cloning site, Tc R = tetracycline resistance, Ap R = ampicillin resistance, Cm R = chloramphenicol resistance, Neo R = Neomycin resistance, Tsr R = thiostrepton resistance, sBLA= synthetic Bovine alpha-LActalbumine Table 4. Primeis used in the DNA sequencing reactions Oligonucleotide sequence Name GTAAAACGACGGCCAGT M I3 mm ersal primer AAC AGCTATGACCATG 1 reverse pri er ATTAACCCTCACΓAAAG T3 primer AATACGACTCACTATAG T7 primer ACCAGTTTTGATTTAAACGTG (SEQ ID NO ) 93F 17 GACGGCATGATGAACCTGAAT (SEQ ID NO 6) 93F 18 TTCGCCAACCAGTGCACGAT (SEQ ID NO 7) 93G234 C TTGAGGGTGCGACGCATGG (SEQ ID NO 8 ) 93G 14 ACGAAAGGTCACCGGGACAT (SEQ ID NO 9) 93G315 CATTTCAGATCTGCACCC (SEQ ID NO 10) 93k 145 CGAGAACGGGTGCGC ATAGAA ATTGC I G 1565 (SEQ ID NO 11) CTCGGCACCGTCACCCTGGA ( SEQ I D NO 12 ) I G 1566 Table 5. Comparison of mTNF yield detected in supernatants of 48 h cultures of SAividans with mTNF under the control of (a) S.venezuehιe α-amylase (Van Mellaeit et id., 1994) or ( b) vsi regulatoiy sequences and signal peptide. In both cases (a) and (b) below. N-terminal sequence analysis was canied out on 2 distinct immunoreactive bands. The higher band contained the correctly cleaved fusion protein (+1) or a truncated form (+4): the lower band consisted of tnincated fonns of mTNF. the numbering of which starts at the first amino acid of mTNF. (a) (b) Table 6. Oligonucleotides used for in vitro mutagenesis EXAMPLES Example 1: Materials and Methods Strains and plasmids Streptomyces venezuelae CBS 762.70 (= ATCC 14583) was used as source for the isolation ofthe subtilisin inhibitor gene. S.lividans TK24 (Hopwood et ai. 1985) was the host strain for maintenance and isolation ofthe Streptomyces plasmids pl.1486 (Ward el ai. 1986) and the newly constructed secretion vectors (table 3). For cloning, several Escherichia coli strains have been used (Table 2). Clones and subclones were constructed with the Ecoli vectors summarized in Table 3. Media and Buffers S. venezuelae and S.lividans were grown at 27°C. 300rpm in NM medium, i.e. Nutrient Broth N°2 (Lab B Bury, U.K.) at pH 7.0 with 0.05M MOPS (= 3-[-N-motpholino]-propane sulfonic acid). S. lividans tiansformants were obtained and grown on Rl I -medium as described by Hopwood et al. (1985). Ecoli was cultured at 37°C in Luria-Bertani (LB) medium (Miller, 1972). where necessary supplemented with ampicillin ( 1 OOμg'ml ). chloramphenicol ( 1 Oμg/ml). or tetracycline (15μg/ml) for selection, and containing 15g agai/L. when applicable. Purification of extracellular proteins The proteins in the supernatant of a 48h culture of \ venezuelae. obtained by centrifugation (3000xg, lOmin, 4°C), were first precipitated by the addition of solid (NH_,) : SO^ to 80% saturation. The precipitate was collected by centrifugation ( 1 Omin. 4 C C) and subsequently dissolved in 7ml of water. Next, the solution was applied on a Sephadex superfine G50 column (2.4x280cm) and the proteins were eluted with 50mM NH 4 HCO, in the presence of 0.2% NaN at a flow rate of 20mL/h. Tlie effluent was collected in 2ml fractions and detection of the protein content was at A 2MI and A ; „,. Fractions containing proteins were controlled by SDS-PAGE ( 12.5%) (Laemmli. 1970) to identify the protein content of the tractions. Endoproteinase Lys-C digest of subtilisin inhibitor was separated by HPLC on an Aquapore C8RP column (2.1x220mm) (Applied Biosystems). equilibrated with a solution of 0. 1 % trifluoro- acetic acid in water. The column was eluted with a linear uradient from 0 to 100% with solvent (0.1% trifluoro-acetic acid and 80% acetoniuile in water) at a rate of 400μl/min and 400μl fractions were collected. Amino acid sequence analysis The N-terminal amino acid sequence of the relevant proteins were detennined by Edman degradation on an automatic 477A-120A protein sequencing system (Applied Biosystems). To determine internal amino acid sequences, the purified subtilisin inhibitor protein was digested in a total volume of 1ml using 1 μg of endoproteinase Lys-C (sequencing giade, Boehringer Mannheim) for 18 h at 37°C using a protocol as described by the manufacturer. After digestion the peptides were sepaiated on HPLC as described above, and particular fractions were applied to the automatic protein sequencer DNA manipulations Chromosomal DNA of S.venezuelae was extracted fiom cells by a modified method of Womble et al. (1977). Briefly, S.venezuelae was grown in S medium supplemented with 0.5% glycine inoculated with a seed culture (0.1%). After growth (27°C. 350rpm) for 24h. 40ml cultures were collected by centrifugation (3000xg. lOmin) and washed twice with buffer A (0. 15M NaCl 0.1M EDTA. lOmM HEPES. pH8.0) prior tυ incubation with lysozyme ( 500μιg/ml) in 5ml of buffer A. After lOmin at 37 C. the cells were K sed by the addition of 50μl of 25% (w/v) SDS. Subsequently, 0.5 ml pronase solution (20mg pronase/ml H 2 0 and placed at 30°C for 10 min for autodigestion) was added and the suspension was further incubated for 2h (37°C) followed by 30min at 65 C C and shearing by repeated suckening in a syringe. Chromosomal DNA was purified by EtBr-CsCl gradient centrifugation ( Ig CsCl/ml) (45h. lOO.OOOxg). Purified chromosomal DNA was digested with several restriction endonucleases and separated on a 1% (w/v) agarose gel. Tlie DNA digest was blotted onto Hybond-N (Amersham) using a Vacugene blotting system ( Phaimacia). Restriction endonucleases and DNA modifying enzymes were fiom Boehringer Mannheim. Biolabs and Gibco-BRL and tiiey were used according to the manufacturers ' protocol. The DNA fragment containing the subtilisin inhibitor gene was isolated by hybridization experiments with synthetic deoxy-oligonucleotides as probe. These deoxy-oligonucleotides consisted of 5'-CGCTCTACGCCCCC C-3' (SEQ ID NO 3 ) (sense strand) (T ιn - 58°C) and 5'-CTTCATSGGTGCACTGGTTG-3' (SEQ ID NO 4) (antisense strand) (T m = 56°C), conesponding to the N-tenτιinal sequence of the \- and C-temiinal pait of the protein, respectively. They were labeled with γ-[ ",2 P] ATP (3000Ci/mmol ) ( Amersham) using T4- polynucleotide kinase (Boehringer- Mannheim). Hybridization of these probes with chromosomal DNA cross-linked to Hybond-N was canned out at 48 ' C overnight in the hybridization solution as described in the Amersham protocol. After hybridization, the membranes were washed in 2xSSC, 0.1% SDS twice at room temperature, and subsequently, at the T n -value of the deoxyoligonucleotide. DNA sequence analysis DNA sequencing was carried out using the chain terminating method developed by Sanger et al. (1977) either with [ 35 S]-labeled fragments or using fluoro-labeled primers, when using the automatic ABI DNA sequencing system (Applied Biosystems Inc.). Fragments were separated on a 6% polyacrylamide gel [acrylamide:bis-acι lamide ( 1 . 1 )) with 7.0M urea in TBE buffer (60mM Tris, 50mM boric acid 1.5mM EDTA) at 15U0Y (35 W. 35mA). Prior to sequencing, several restriction fragments, obtained by digesting the original approx. 5kb IXull fragment oi S. venezuelae chromosomal DNA. containing the subtilisin inhibitor gene with several restriction enzymes, were subcloned in pBluescript KS ( -+- ). p AC ' C 1 4 or pUC 19. All DNAs for sequencing were purified by CsCl-EtBr gradient centrifugation. Sequencing primers were the Ml 3 universal and reverse primer for subclones in pUC I9. T3 and T7 primers for those in pBluescript and primers 1565 (SEQ ID NO 1 1 ). 1566 (SEQ ID NO 12). 93F17 (SEQ ID NO 5) and 93F18 (SEQ ID NO 6) (Table 4) for subclones in p.ACYC 184. In addition, a number of subtilisin inhibitor specific primers were used: 93G234 (SEQ ID NO 7). 93G314 (SEQ ID NO 8). 93G31 (SEQ ID NO 9). 93K145 (SEQ ID NO 10) (Table 4). Computer aided analysis The sequencing data were processed using the Gel Assembler system of PC Gene (Genofit. IntelliGenetics. Inc.) and the protein coding sequences were identified by virtue of the similarity of frieir codon usage to a codon frequency table by FRAME analysis (Bibb et ai. 1984). A phylogenetic tree was constructed using the CLUSTAL program of PC Gene. Primer extension studies Total RNA was extracted as dsscribed by Hopwood ei ai ( 1 c >85 ) from 24h cultures of S. venezuelae CBS 762.70 grown in NNi medium. Seventy μg of RN in a total volume of 30μl was hybridized for 2h at 60°C with a [ P]-y-ATP labeled (6.6 l 0^cpm) antisense oligonucleotide 93G314 5'-CTTGAC 3GTGCGACGCATGG-3' ( SEQ 1 D NO 8) (T m - 66°C) complementary to the sequence of the 5' end of the subtilisin inhibitor gene. .After hybridization, the solution was unmediately cooled to 0°C. Next, primer extension was canied out by the addition of 30 u AMV-reverse transcriptase (AMY-RT) ( Boehringer Mannheim) in 70μl AMV-RT reaction buffer (Yόgtli and Hiitter. 1987 ) and incubation at 42°C for lh. Tlie piimer extension product was purified by consecutive tieannent with phenol: chlorofonn ( 1 : 1 ) and precipitation with ethanol. Further purification was by elution fiom a NAP- 10 column (Phamiacia). By comparison ofthe labeled primer extension pioduct with a sequence ladder on a 6% polyacrylamide gel containing 7.0M urea in TBE-buffer. the nucleotide conesponding to the mRNA start could be detennined. Protease inhibitor test Inhibitory activity against subtilisin BPN (7.81 ' mg) ( Sigma. Protease iI), trypsin (10.600U/mg) (Sigma) and α-chymotrypsin (44U, mg) ( Sigma ) were tested using Azocoll (Sigma) as a substrate for protease activity. Therefore. I 50μl supernatant of ,S " . venezuelae cultures grown for 33h in NM-medium were incubated with 0.04 to 10 units of protease in lOmM sodium maleate buffer (pH 6.2 ) in a total \ olume of 500μl. After lOmin incubation at room temperature, 5ml of buffer solution containing Azocoll ( 5mg/ml ) was added and slowly shaken at 27°C. At regular time intervals the suspension was centrifugated and against subtilisin BN ' P' was also tested using N-succinyl-L-Ala-L-Ala-L-PiO-L-Phe-p-nifioanilide as described by Kojima el ai ( 1990). Construction of the pCBS2mTNF and pCBSmTNF secretion \ ectors For the construction ofthe pCBS(2)mTNF secretion vector, a 574 bp (nucl. pos. 319- 893) Smal -fragment (see Fig. 3a) containing part ofthe subtilisin inhibitor gene including the promoter. RBS. signal sequence and the coding region of the Vterminal end of the mature protein was cloned in the Smal site of pBSDK. resulting in pBSDK-CBSS Tlie 5 , -piOtruding ends of the røll digested (unique site) pBSDK-CBSS ere filled in using a Klenow polymerase fill-in reaction (by which manipulation a blunt end site was fonned 2 ttiplets downstream the vsi signal sequence). Subsequently. pBSDK-CBSS was digested with EcoRI. A 0.7kb SnaBl/EcoRl fragment of pIG2mTNF-m7 containing the coding region of recombinant mTNF (in pIG2mTNF the first amino acid of mTNF was changed from L to Y) could thus be fused in frame in such a manner that the second amino acid of the subtilisin inhibitor gene was followed by the first amino acid (Y) of mTNF. A direct fusion between the vsi signal sequence and the mTNF cDNA was also achieved by introducing a unique N.sv ' I site at the 3' end ofthe v.sv signal sequence using PCR mutagenesis. Tlie resulting pBS-CBSΝ vector was digested with Nsil. Subsequently, the 3'-protruding ends were removed in a T4 DΝA polymerase reaction. After £coRI digestion of the vector, the above-mentioned 0.7 kb Sn B EcoRI fragment of pIG2mTΝF-m7 was cloned in the vector Tlie coπectness of both constructs, i.e. pCBS2mTNFEc and pCBSmTNFEc. was verified by DNA sequence analysis. From both plasmids the module containing the subtilisin inhibitor transcription and translation start signals and the mTNF gene fused, either directly or indirectly, in flame with the subtilisin inhibitor signal sequence was ligated in pl.1486 after proper digestion with BamHI and EcoRI. Construction of the pCBS2hIL10 secretion vector For the construction of pCBS2hlL10, the pBSDK-CBSS v ector was digested with DraW. The 5'-recessed ends were subsequently filled in using a Klenow poKmerase fill-in reaction. In this vector the 530 bp Nsil (blunted in a T4 DNA poKmerase reaction ) HiniΛ II fragment of pIGFH20hIL10 containing the WLIO cDNA could be cloned In pCBS2hIL10Ec plasmid the second codon of mature vsi was followed by the first hILlO cDNA codon. The conectness of the fusion was checked restriction analysis of the plasmid since an in-frame fusion resulted in a unique .S7//I site Tlie module fiom pCBS2hIL10. containing the v.sv ' regulatory sequences and the hIL l gene fused in frame with the r.s7 signal sequence, was cloned in a BamHI digested pl .I486 \ ectoi Construction of the pCBS2hIL2 ana pCBShIL2 secretion v ectors In order to obtain the pCBS21τlL2 secretion \ ector. pBSDK-CBSS was digested with Droll. After filling in the/ rall 5'-protruding ends using a Klenow poKmerase fill-in reaction, the vector was digested with EcoRI. Subsequently, the 412bp BspWl (with filled in 5 ' - protruding ends)/£coRI fragment from pUC 18hlL2 containing the hIL2 cDNA was ligated to the vector resulting in pCBS2hIL2 ]Ξc in which the hJL2 cD A was fused downsfream the first 2 codons of mature v.v/. For the construction of the pCBShIL2 secretion vector, the mentioned BspMU EcoRI tiagment was ligated to pBS-CBSN digested with A'.s/I (3 " -protruding ends blunted in a T4 DNA polymerase reaction) and EcoRI. These manipulations resulted in the plasmid pCBShIL2Ec in which the hIL2 cDNA was directly fused to the vsi signal sequence. The fusions in pCBS2hIL2Ec and pCBShIL2Ec were controlled by DNA sequence analysis. From each plasmid the module containing the vsi regulatoiy sequences and the hIL2 cDNA fused to the vsi coding sequence was cloned in the BamHI site of pl.1486 Construction of the pCBS2hGM-CSF secretion vector For the construction of pCBS2hGM-CSF. the 525bp hspl Hindlll tiagment of plGH2hGM-CSF-m5 containing tlie hGM-CSF cD A was ligated to pBSDK-CBSS. Tlierefore. this vector was digested with DiτA and the 5 ' -protruding ends blunted using a Klenow polymerase fill-in reaction. Subsequently, the vector was digested with Hindlll. The ligation reaction resulted in a fusion of the hGM-CSF cDN \ 2 n iplets down-stream the vsi signal sequence. The conectness of the fusion was v erified sequence analysis The module of the plasmid designated pCBS2hGM-CSF Ec. containing the vsi regulatory sequences and the hGM-CSF cDNA fused in-frame downstream the first 2 codons of the mature vsi sequence was cloned in a Scxil Hi dlll digested pl.1486 v ector Construction of the pCBS2sBLA and pCBSsBLA secretion ectoi As in the other examples, a direct and indirect fusion with the vsi signal sequence was established. The sbla gene located on a 399bp Mn /JXoR riagment of pEPsBLA was. therefore, ligated either to pBSDK-CBSS digested with / ) ra\l ( blunted using a till-in Klenow polymerase reaction and EcoRI or to pBS-CBSN digested with A s/ 1 ( treated with T4 DNA poKmerase) and £lcoRI. In this way, the plasmids pCBS2sBL.A£c and pCBSsBLAEc were constructed with the sb/a gene fused 2 triplets downstream the vsi signal sequence or directly to the vsi sequence, respectively. The co ectness of these fusions was verified by DNA sequence analysis. From both plasmids the module containing the v i regulatoiy sequences and the sbla gene fused directly or indirectly in-frame with the vsi signal sequence was inserted in pl.1486 digested with BamHI and £cσRI. Construction of the pCBS2hIL10Ec secretion vector For the construction of pCBS2hIL10. the pBSDK-CBSS vector was digested with Dral . Tlie 5'-recessed ends were subsequently filled in using a Klenow poKmerase fill-in reaction. In this vector the 530 bp Nsil (blunted in a T4 DNA polymerase reactionVH/ κ/TII tiagment of pIGFH20ML10 containing the hILlO cDNA could be cloned. In pCBS2hIL10Ec plasmid the second codon of mature vsi was followed by the fiist hlLIO cDNA codon. The correctoess ofthe fusion was checked by restriction analysis of the plasmid since an in-frame fusion resulted in a unique .S7//1 site. The module from pCBS2hIL10Ec, containing the vsi regulatoiy sequences and the hlL IO gene fused in frame with the v.s7 signal sequence, was cloned in a BamHI digested pl.1486 vector. Biological activity determination, immunoblot assay and N-teiminal amino acid acid sequence analysis of mTNF Methods for biological activity determination and immunoblot analysis have been previously described (Van Mellaeit et ai. 1994). N-tenninal amino acid sequence analysis has been carried out on proteins in culture filtrates sepaiated on 1 .5.% SDS-PAGE gels stained with Coomassie blue and blotted on PVDF membranes ( BioRad) Biological activity determination and immunoblot assay of hlL I Biological activity detennination of hlLIO is canied out using a I L- 10 sensitive mouse cell line MC9. which proliferates in the presence of IL- l 0. as described by Thompson-Snipes el ai, ( 1991). λ H-Thymidine is added after 24 h of culture and counting of cell proliferation is measured in a γ-counter after another 18 h of incubation. One unit (ϋ) of biological activity is defined as the amount of hlLIO required to mediate half maximal proliferation of MC9 cells. The immunoblot analysis was αanied out in the same as described for mTNF using rabbit hlLIO antibodies (Innogenetics) as first antibody Immunoblot assay of hIL2 and hGM-CSF Immunoblot analysis of hlL2 and hGM-CSF was canied out as described for mTNF using rabbit antibodies (Innogenetics] against hlL2 and hGM-CSF. respectiv ely. Detection of sBLA The detection of sBLA occurred by a sandwich immunoradiometric assay (iRMA. Yiaene et al., 1991). Therefore, the wells of a microtiter plate were coated with polyclonal anti-BLA antibodies. After contact with the samples. ' , I-labeled anti-BLA antibodies were added. Binding of those antibodies - and thus the presence of sBLA - was detected by measurement of the radioactivity. Example 2: Cloning and sequence analysis of the vAV-gene of Streptomyces venezuelae CBS 762.70 I N-terminal determination of an abundantly secreted ! 3kPa protein During a search for Streptomyces spp. secreting high lev els of a protein, we have identified following SDS-PAGE and Coomassie staining, λ venezuelae ( " BS 762.70 as a strain producing high amounts of a protein appealing as a double band of approximately 13kDa in size. Besides this 13kDa protein, only a few other proteins, larger in size and at much lower quantities were observed (Fig. 2a). Quantitative estimations indicated that this 13kDa protein was secreted using shake flask cultures in amounts of up to 300mg'L sepaiation on a Sephadex G50 column, the proteins in the double band eluted in separate fractions (tract. 47 and 55) (Fig. 2b). N-tenninal amino acid sequence analysis of the proteins in fraction 47 and 55 indicated that the proteins in both fractions were identical at their N-temiinal end except that in fraction 47 the protein was truncated (+7 to + 1 ). with the -"9 foπn being predominant, comprising 45 pet. of the total amount. More extensiv e detennination of the 13kDa protein was in a first instance by N-tenninEil sequencing of sev eral fragments obtained following endoproteinase Lys-C digestion and separation o er HPLC ( see in Fig. I ). A comparison in the SW1SS-PROT protein database ofthe amino acid sequences obtained with those of other proteins revealed that the 13kDa protein belongs to the family of subtilisin inhibitors. The identification ofthe 13kDa protein secreted by λ. venezuelae as a subtilisin inhibitor (VSI) was 5 also proven by protease inhibitor tests, which showed that YSI had inhibitory activity towards subtilisin BPN and trypsin, but not for α-chymotrypsin (data not shown). Furthennore, with respect to the heterogeneous N-teπriinal end of VSI. similai heterogeneous N-tennini were observed for STI2. when produced in S.lividans (Strickler et ai. 1 92). They were supposed to be the result of a protease activity. It could howev er, also be the result of an incorrect 10 processing ofthe signal peptidase, since pre-SSI S-3253 was shown to have different signal peptidase processing sites when cloned in S.lividans 66 (Obata et ai. 1989) or Ecoli (Taguchi et ai. 1990). On the other hand it might be that the λ. venezuelae subtilisin inhibitor is secreted as a pro-protein. I 5 2. Isolation and sequence analysis of the v.v/-gene We used the infonriation of the N-teiminal amino acid sequence analysis of the abundantly secreted 13kDa protein and of one of its internal fragments to develop 2 oligonucleotides. in order to isolate the r.v/-gene fiom chromosomal DNA of S.venezuelae. Tliese oligodeoxvnucleotides were designed taking into account the codon bias of 0 Sireptomyces (Wright & Bibb, 1992). They were radioactively labeled and probed to the .V. venezuelae chromosomal DNA digested with Apa . BssHλl. iψ . Kpnl. Noel. Narl, Ncol. Noll, NIΉI. PstI, Sail, Smal or StuI, and separated on an agarose gel. A /V//II restriction fragment of 5kb hybridized with both probes and was. therefore, supposed to contain the complete v.s/ ' gene. This fragment was subcloned into the IXoRN site of 5 pACYC184 and multiplied in Ecoli XLl-Blue MRF. In case the high copy number pBluescriptll KS (+) was used to clone the 5kb D ull fragment and with Ecoli HB101 as host, no transfonnants could however, be detected. A restriction map of the l ull insert was constructed for Apal, Ncol. Noll, Nru . D'ul and Smal ( Fig. 3a) and fragments were subcloned in pBluescript II KS (+), pUC19 or pACYC 184 for sequencing. Fig. 1 shows the 0 nucleotide sequence of the 1.662bp fragment, obtained following sequence analysis on both strands using a sequencing strategy outlined in Fig. 3b. Potential protein-coding sequences were predicted by the computer-aided "FRAME" analysis dev eloped by Bibb el ai ( 1984). based on the extremely biased codon usage for a G or C in the third and most degenerate position of the codon (Wright & Bibb, 1992). The analysis rev ealed 1 complete ORF (from 687 to 1 124) and 2 incomplete ORFs (from nt 1 to 324 and fiom 1282 to 1662) (Fig. 3c). Translation ofthe nucleotide sequence in ORF2 revealed a complete subtilisin inhibitor gene. 5 It is assumed that the ATG at nt687-689. which was situated 8nt downstream a consensus RBS-sequence ('5-AAGGAG-). is the translational start codon. Tlie most probable signal sequence is fonned from 28 amino acids containing 3 positiv e charges (Arg at position +2 and +3 and Lys at +6) and with Ala-Gb-Ala at the end of the signal sequence (Fig. 4). This is in agreement with the general role that signal peptidases recognize a (-3. - 1 ) pattern with 1 0 small uncharged residues, usually alanine. in position -3 and - I . relative to the signal peptidase cleavage site (von Heijne & Abrahamsen. 1989) A comparison of the VSI signal peptide with those ofthe other Streptomyces protease inhibitors shows that the foimer is quite different from the subtilisin inhibitor SSI of S.alhoχriseo/us and the tiypsin inhibitors SLP1 (STH ) and ST12 of S.lividans 1326 or 66 and S.longisporus. respectiv ely (Fig. 4). The VSI I 5 signal peptide contains 3 positive charges compared to +2 for the others. In addition, the hydrophobic region of VSI signal peptide has a very high number of Ala residues. The precursor thus consists of 146 amino acids and the mature Ibim is composed of 1 18 amino acids. The size of VSI is 12.03kDa. closely to the size estimated by SDS-PAGE and its isoelectric point is 7.02. Downstream fiom the stop codon is a 15bp inverted repeat 0 followed by a A/T-rich sequence which may serve as a transcriptional teπninator ( Fig. 1 ). 3 Primer extension studies and promoter identification To localize the promoter sequence preceding the vsi ORF. the start point of the vsi transcript was detennined by primer extension experiments using mRNA extracted fiom 23h- 25 old cultures of S.venezuelae and the 2 P-labeled antisense oligonucleotide 93G3 14. The size of the extended product was determined by perfoπning DNA sequence analysis using the same primer. The results indicated the presence of 2 transcripts starting at nucleotide -37 and -36 upstream the initiation codon. Upstream the mRNA transcripti n start site, the DNA sequence showed at the -10 but not at the -35 region a high similarity with the sequence of 3 ( > the ssiP2 promoter ofthe S. a/bogriseo/us s.si gene (Taguchi et al.. 1989) ( Fig. 5). Furthermore, the putative v.s promoter did not show any obvious similarity with the SEP-like promoters ( Strohl. 1992) which is consistent with the observation that the vsi promoter failed to function in Ecoli. Direct repeats in the neighbourhood of the promoter sequence, a common phenomenon was seen in regulated promoters (Delic et ai. 1992). also observed for vsi (Fig. I ). However, so far there are no indications that the v i promoter is subjected to regulation. The piimer extension studies did not reveal any other transcript, indicating that the vsi ORF was preceded by a single promoter sequence in contrast to e.g S.albogiiseolus s.si. Example 3: Comparison of the VSl-sequence to oύvtr Streptomyces subtilisin inhibitor protein sequences The secondary structure of Streptomyces subtilisin inhibitors ( Fig. 6) shows 5β-strands and 2α-helices. with the reactive site located on a flexible loop between the αl -helix and the f. -strand as elucidated from crystallographic studies (Takeuchi ei ai. 1992). The most conserved region resides in the core of the β-sheet. the β 1. B2 and IM-strand and to some extent in B3. which all are of structural importance. Tlie B4- strand contains most of the residues in contact with the proteolytic enzyme. Replacement of amino acids in these regions may result in less stable proteinase inhibitors, which might be subjected to degradation by various proteases. The flexible loop contains the reactive site. It shows surprisingly high variability between the different inhibitors. As a consequence, the amino acid sequence seems not to be important, as long as flexibility is prov ided. However, the amino acid residue at Pl detennines the specificity of the inhibitory activity A mutation of this amino acid residue results in an alteration of the specificity ( Kojima el al . 1990). When comparing the VSI amino acid sequence with those of other inhibitors (Fig. 6). the alignment indicates that VSI has an amino acid identity with other proteinase inhibitors from 48.7% (for STT2) to 54.0% (for SIL2). Based on the similarity, the reactive site of VSI is supposed to be Lys-Glu. This is consistent with its trypsin inhibitory activity since other Streptomyces subtilisin inhibitors with trypsin inhibitory activ ity contain at the Pl site Lys or .Arg (Kojima et ai, 1990). YSI shows a relatively high identity of amino acids in the conserved domains, including the presence of 4 Cys. which are considered to foim two intrachain disulfide bridges. On die other hand VSI shows also some striking differences. Several amino acid residues that are completely conserved in the already published protease inhibitors are changed to amino acids of low or no similarity, e.g.. YaK,. Sei> or Argμ instead of Pro. Ala or Asp, respectively. These amino acids aie situated outside the β-strands and αl -helix. Therefore, of more fundamental importance might be the difference of amino acids in the B5-strand for which the conserved sequence Ser-T\τ-Glu-Arg is replaced by the non-similar amino acids Tlie substitution of these highly conserved 5 amino acid residues might suggest diat VSI belongs to a class of Siivptomyce.s subtilisin inhibitors, different from the 3 classes mentioned by Taguchi et al. ( 1 94) as also suggested by phylogenehc tree analysis (Fig. 9). For this phylogenetic analvsis. a 1 10 amino acid alignment was made, starting with the conserved Tyr at the beginning of the βl strand and ending with the carboxyterniinal Phe. t o Example 4: Secretion of mTNF in S.lividans mediated by the regulatory sequences and signal sequence of vsi S.lividans TK24[pCBS2mTNF] transfonnants were cultured in NM medium (Van Mellaert et al., 1994) for 32 and 48 h. Culture filfrates were assayed for mTNF production by SDS-PAGE, immunoblot analysis and bioassay. On Coomassie stained SDS-PAGE gels 3 abundantly secreted proteins were detected in 48 h cultures t o of which were about 17kDa and a third of 13kDa (Fig. 8). N-temiinal sequence analysis of the I ' SI proteins in band 1 showed that the I S/-mTNF fusion protein was precisely processed by the S.lividans signal peptidase. However. mTNF with heterogeneous N-tennini (+3. +4. +5 foims) were detected in band 2 (Fig. 7). This heterogeneity was the result of aminopeptidase activity of S.lividans, as already mentioned previously (Van Mellaert et ai. 1994). N-teiminal analysis of the proteins in band 3 indicated that this band consisted of the S.lividans subtilisin inhibitor. Immunoblot analysis of S.lividans TK24[pCBS2mTNF] showed that the protein bands 1 and 2 were immunoreactive, but also band 3 showed some cross-reactivity. Biological assays gave values of 2.5 - 8 x IO 10 U mTNF/L culture corresponding to 1 10 - 300 mg mTNF/1. Upon culturing TK24 [pCBSmTNF] transfonnants in NM medium, biological mTNF activities up to 2.5xl0 lϋ U/l were obtained in the culture filtrate, indicating that the immediate fusion between the VSI signal peptide and the mTNF protein also resulted in efficient secretion of mTNF. These values are about 10 to 100 times higher than when using the α-amylase regulatory sequences and signal peptide (Table 5) and equalize the highest values obtained for eukaryotic proteins secreted by S.lividans. Example 5: Secretion of hfl. 0 inS. lividans mediated by the regulatoiy sequences and signal sequence of vsi. S. lividans TK24 [pCBS2hIL10] transfonnants were cultured in NNI medium for 30 and 40h. Culture filtrates were checked for hIL 10 production by SDS-PAGE. immunoblot analysis and bioassay. After SDS-PAGE of proteins of the culture filtrate, the concentration of hlLIO obtained was below the detection limit of Coomassie staining. On the otlier hand the immunoblot with anti-ILlO antibodies revealed 2 immunoreactive bands of about 18 and 24 kDa (see Fig. 10). Biological hlLIO activities measured in the culture filtrates were up to 1600 U/l after 30 h of growth. Example 6: Secretion of HIT 2 by S.lividans mediated by the regulatory' sequences and signal sequence of vsi. S.lividans TK24 [pCBS2hIL2] and [pCBShlL2] transfonnants were grown in NM- medium during 28h to 48h on a rotary shaker (280rpm. 27°C). Proteins piesent in the culture filtrates were separated by SDS-PAGE { 12.5%) and subsequently, blotted on a nitrocellulose membrane (BioRad). Immunoblot analysis ofthe blotted proteins revealed the presence of two immunoreactive bands. The proteins of about 15kDa con esponded to the processed hlL2 (with or without 2 additional amino acids of mature vsi). the protein with the size of 18kDa was likely to be unprocessed vsi-hIL2 fusion preprotein. By comparison of the intensity of bands on immunoblots containing a known amount of mature recombinant hIL2 and the immunoreactive proteins, the amount of hIL2 secreted by S.lividans TK24 was estimated to be 20 to 40μg hIL2/l culture. Example 7: Secretion of hGM-CSF bv SAividans mediated by the regulatory sequences and signal sequence of vsi. Transformants of S.lividans TK24 containing pCBS2hGM-CSF were grown in NM- medium for 24h to 48h on a shaker (280 rpm. 27°C) Proteins present in the filtrates of these cultures were analysed by SDS-PAGE and subsequent immunoblot assay . Binding of anti- hGM-CSF antibodies occurred on several proteins with a molecular weight varying fiom 14 to 18 kDa The protein with die lowest M, corresponded to mature hGM-CSF, the origin of the larger immunoreactive proteins has still to be investigated but were supposed to be aggregates of hGM-CSF. By comparison on unmunoblot with a known amount of recombinant hGM-CSF standard the amount of hGM-CSF protein secreted by S.lividans TK24 was estimated to be up to 125μg hGM-CSF/1 culture. Example 8: Secretion of BLA by SAividans mediated by the regulatoiy sequences and signal sequence of vsi. After transformation of S.lividans TK24 protocols with pCBS2sBLA and pCBSsBLA, respective transformants were grown in phage medium (Anne el ai, 1984) or in 1/2 YPD (0.5% bacto yeast extract, 1% bacto peptone and 1% glucose) on a shaker (280 rpm. 27°C). At different time intervals, samples of culture filfrates were taken and analysed for the presence of BLA by immuno radiometric assays. BLA levels of 2 to 2.5mg/l culture could be obtained. Example 9: Influence of positive chaiges in the signal peptide on protein secretion To investigate the importance ofthe three positive charges in the N-tenninal region of the signal peptide for effecting secretion and to analyse whether the level of secreted mTNF could still be improved several mutants of pCBS2mTNF were made by in vitro mutagenesis as indicated in Figure 1 1. As arginine residues aie more commonly used than lysines in streptomycete signal peptides, extra positive charges were added by introducing CGT or CGC. The three positive charges of the WT signal peptide were gradually replaced by glutamine residues. In this way, we obtained signal peptide mutants with a positiv e charge ranging from 0 to 5. After transformation of S. lividans with the different constructs. mTNF was measured both by a biological activity test and by Western blotting. For each ofthe mutants 8 different tiansformants were sampled for the presence of intracellular and extracellular mTNF after 30 and 48 hours of growth. The average results after 48 hours of growth aie shown in Figure 12A. It is clear that charge variation does not inhibit export of mature biologically active mTNF. Nevertheless, the differences in amounts of secreted mTNF are significant. Taking the amount of mTNF secreted under the influence of the wild type signal peptide as a reference (100 %). we found a change in the amounts of secreted mTNF to values of 16 % for zero charge, 80 % for +1, 320 % for +2. 80 % for -t and 50 % for -5. This variation was also visualised on SDS-PAGE followed by Coomassie staining (Figure 12B). We can conclude from this that the presence of positive charges in the N-region is not absolutely required to obtain export. Immunoblot analysis ofthe recombinant mTNF produced by Λ lividans fransfomiants each carrying a different mutant VSI signal peptide showed the mature fonn and a few degradation products in the culture fluid. Cell lysates contained both the processed and unprocessed form of mTNF together with a protein of intermediate size which could be a degradation product ofthe precursor protein (Figure 1 ). Whether the mature mTNF present in the cell extracts is originating from the cytoplasm or is attached to the cell wall or cell membrane was not further investigated. Calculating the real secretion efficiencies (Figure 12C) reveals that, although the wild type is most efficient in directing export of mTNF, the +2 mutant is able to secrete more in absolute terms. Respectively 76 % and 60 % of the total synthesized mTNF is secreted by the wild type and the +2 mutant. Example 10: Positive secretion effect coirclates with increased mRNA level To analyse a possible effect of the introduced mutations at the level of tianscription, total RNA was prepared from S. lividans cultures harbouring the different mutant constructs including the wild type. Results obtained after slot-blotting and hybridisation with a mTNF cDNA specific probe are shown in Figure 14 together with an internal control to ensure that the cultures were approximately at the same position on the growth curve. Therefore a chromosomal stil probe was used As can be concluded from this figure in combination with Figure 3 A. for each mutant, the level of mTNF mRNA is different and seems to be tightly conelated with the total amount (intracellular and extracellular) of synthesized mTNF. REFERENCES Anne, J., W. Wohlleben. H.J. Burkardt, R Springer and A. Piihler ( 1984). 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Claims: CLAIMS 1. A polynucleic acid in substantially isolated fonn comprising a sequence of at least 10 contiguous nucleotides selected from: (a) the polynucleic acid sequence as shown in Fig. 1 (SEQ ID NO 1 ). or (b) d e polynucleic acid sequences which are degenerate as a result of the genetic code to die polynucleic acid sequence as defined in (a) and which code for a polypeptide showing a subtilisin inhibitory activity, or (c) die polynucleic acid sequences which hybridize to the polynucleic acid sequences as defined in (a) or (b). 2. A recombinant DNA molecule comprising a polynucleic acid according to claim 1. 3. A recombinant DNA molecule according to claim 2. comprising the gene expression unit of any of die polynucleic acid sequences as defined in claim I. or part of it. said gene expression unit comprising the promoter sequence, the ribosome binding site, the sequence coding for the secretion signal, die sequence coding for the mature protein, and the termination sequence. 4. A recombinant DNA molecule according to any of claims 2 to 3. comprising the regulatory sequence of any of the polynucleic acid sequences as defined in claim 1. or part of it, operably linked to a heterologous coding sequence, said regulatoiy sequence comprising the promoter sequence, the ribosome binding site and the nucleotide sequence encoding die secretion signal. 5. A recombinant DNA molecule according to any of claims 2 to 4. comprising the regulatory sequence of tfie polynucleic acid sequence represented by SEQ ID NO 1. or part of it, operably linked to a heterologous coding sequence, said regulatoiy sequence comprising - the promoter sequence located in the region extending from pos (-686) to pos (-38) in Fig.1 (SEQ ID NO 1), more specifically, located in the region extending from nucleotide pos (-81 ) to pos (-38) in Fig. 1, and/or, - the ribosome binding site located in tlie region extending from pos (-37) to pos (- 1 ) in Fig. 1 (SEQ ID NO 1), more specifically, located in the region extending from pos (-15) to (-1) in Fig. 1, and/or , - the nucleotide sequence encoding d e secretion signal extending from position (+1 ) to pos (+84) in Fig. 1 (SEQ ID NO 1) . 6. A recombinant DNA molecule according to any of claims 2 to 5. comprising the regulatory sequence of the polynucleic acid sequence represented by SEQ ID NO 1. or part of it, operably linked to the coding sequence: of SEQ ID NO 1. or part of it. wherein said coding sequence is fused in frame with a second heterologous codbig sequence, said coding sequence of SEQ ID NO 1 extending fiom pos (+85) to pos (+439) in Fig. I . 7. A recombinant DNA molecule according to any of claims 2 to 6. wherein the sequence encoding the signal peptide has been specifically mutated resulting in one at least of the following amino acid substitutions in the signal peptide: R substituted by Q at amino acid position 2 of Fig 1. andor R substituted by Q at amino acid position 3 of Fig. I. andor T substituted by R at amino acid position 4 of Fig. I . andor L substituted by R at amino acid positton 5 of Fig. 1. andor K substitued by Q at amino acid position 6 of Fig. 1. 8. A recombinant vector capable of transforming a host cell, and capable of replication andor directing expression in said host cell, wherein said recombinant v ector comprises a recombinant DNA molecule according to any one of claims 2 to 7 9. A host cell transformed by a recombinant vector according to claim 8. said host cell being chosen from among eucaryotic organisms, such as plant cells, animal cells, or yeast cells, or chosen from among procaryotic organisms, such as bacteria. 10. A host cell according to claim 9, said host cell belonging to the genus Streptomyces. 1 1. Process for producing a heterologous protein fiom a host cell, comprising the steps of: - growing a culture of a host cell according to any of claims 9 to 10. under conditions enabling expression and possibly secretion of the heterologous protein . - recovering said heterologous protein from said culture. 12. Polypeptide in substantially pure fonn comprising a sequence of at least 9 contiguous amino acids selected from (a) me polypeptide sequence encoded by any of the polynucleic acid sequences as defined in claim 1, or (b) die polypeptide sequence of 146 amino acid residues as represented in Fig. 1 (SEQ ID NO 2), or (c) polypeptide sequences resulting from the modification of one or several amino acids in the polypeptide sequence represented by SEQ ID NO 2. said modification comprising substitution and/or deletion and/or addition of one or seveial amino acids, pro ided that said modification does not abolish the subtilisin inhibitoiΛ' activity ofthe polypeptide encoded by SEQ ID NO 2. 13. A fusion protein comprising a polypeptide according to claim 12 fused to a heterologous protein. 14. A polypeptide according to claim 12 for use as a medicament. 15. Use of a polypeptide according to claim 12 for treating medical distresses. 16. Use of a polypeptide according to claim 12 in the non-medical field more specifically in food and feed processing. 17. A probe hybridizing specifically witii any of the polynucleic acid sequences as defined in claim 1. 18. A primer amplifying specifically me polynucleic acid sequences as defined in claύn 1

Inventors: ANNE JOZEF, LAMMERTYN ELKE, SCARCEZ THIERRY, VAN BROEKHOVEN ANNIE, VAN MELLAERT LIEVE, ANNE, JOZEF, LAMMERTYN, ELKE, SCARCEZ, THIERRY, VAN BROEKHOVEN, ANNIE, VAN MELLAERT, LIEVE
Applicants: ANNE JOZEF, BROEKHOVEN ANNIE VAN, INNOGENETICS NV, LAMMERTYN ELKE, MELLAERT LIEVE VAN, SCARCEZ THIERRY, ANNE, JOZEF, INNOGENETICS N.V., LAMMERTYN, ELKE, SCARCEZ, THIERRY, VAN BROEKHOVEN, ANNIE, VAN MELLAERT, LIEVE
Application year: 1996
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N1, C12N15