Fusion polypeptides for target peptide production

Technical field

The present invention relates to fusion polypeptides, nucleic acid molecules encoding such fusion polypeptides and genetically modified cells comprising such nucleic acid molecules. Additionally, the present invention relates to a method for preparing a target peptide and target peptide mixtures. Further aspects of the present invention become apparent when studying the attached patent claims and the specification, including examples.

Background of the invention

The need for peptides and proteins with a high purity for various industrial and research applications rose continuously over the past years. It is therefore highly desired to produce peptides with a predetermined sequence in an economically efficient way. To date, the industrial peptide production relies on two options: the production via chemical synthesis and the production via biotechnological methods. The chemical synthesis has the drawback that it is a costly and time-consuming process and not all desired peptides can be produced at an industrial scale. For example, peptides with a high content of hydrophobic amino acids are not producible by chemical synthesis and it is especially challenging to produce peptides with an N-terminal proline. Furthermore, the chemical synthesis requires the use of harsh reagents and costly purification steps. On the other hand, these drawbacks can be overcome by biotechnological synthesis. Biotechnological systems such as genetically modified E. coli are suitable for producing desired target peptides or proteins at a cost- efficient and highly scalable basis. Nevertheless, purification processes are highly inefficient and costly as cells such as E. coli produce a variety of different metabolites, which are also proteins and peptides, and need to be separated specifically from the target peptides or proteins.

WO 2006/113957 discloses a method for recombinant preparation of a heterologous polypeptide comprising the expression of a fusion polypeptide, the fusion polypeptide comprising a mutant of the autoprotease N ^pro of a pestivirus and a second C-terminally connected polypeptide, wherein the second polypeptide may be cleaved autoproteolytically. Moreover, further fusion domains may be present at the N-terminus required for binding to an affinity chromatography system, e g. poly(amino acids) such as polylysin or epitope tags, i.e. short peptide sequences for which a specific antibody is available. Nevertheless, the method discloses complex purification steps such as affinity chromatography and HPLC. As toxic and costly reagents are used for the affinity chromatography, the disclosed process is not easily scalable and cost-efficient. The resulting peptides also need to be further purified to exclude the toxic compounds from the affinity chromatography.

WO 2008/052387 discloses starch-binding domains and recombinant polypeptides including the same, wherein the starch-binding domains are arranged in N-terminal and/or C-terminal direction of the target polypeptide. The fusion polypeptides may be purified by chromatography on a starch carrier. The disclosed method only offers the use of well- known starch binding sites, whereas the binding domain used for purification cannot be separated easily.

More specific, EP 2746390 and AU 2011253661 disclose fusion polypeptides to be used in an affinity chromatography system with an autoprotease N ^pro from Pestivirus. Both documents do not disclose methods to overcome the drawback of using an N ^pro in terms of controlling the autoprotease activity in a very specific pH range and a high dependency on the reaction conditions such as the settled temperature. In addition, minor changes in the reaction environment would lead to the activation or deactivation of the autoproteolytic domain.

WO 2019/138125 discloses also fusion polypeptides with an autoprotease domain from N ^pro . Furthermore, fusion polypeptides with a CBM affinity domain are disclosed. The international application does not disclose specific architectural concepts of designing the CBM or autoproteolytic domain to overcome the drawbacks of the instability and high dependency of the fusion polypeptides to work on fixed reaction conditions. The high dependency to work on fixed reaction conditions can lead to product loss or impurities in the product. Purification processes involving the use of N ^pro as an autoprotease are limited in their performance. Whenever the autoprotease N ^pro as such or as part of a fusion polypeptide is expressed in a standard E. coli expression system, it is deposited in inclusion bodies. As soon as the autoprotease N ^pro is recovered from inclusion bodies and is refolded into its native conformation, autoproteolysis will proceed as the conditions needed for inclusion body refolding and the conditions for autoproteolysis of N ^pro match. Accordingly, the limiting factor of the purification process are the binding kinetics and affinity of the CBM affinity domain for the purification and the yield at the same time. A trade-off decision has to be made between high yield and low purity (N ^pro is not fully activated, but the polypeptide can be regained from the inclusion bodies) or high purity and low yield of the product (N ^pro is fully activated, but not all polypeptide can be regained from the inclusion bodies). The primary object of the present invention was therefore to provide improved fusion polypeptides, which can be used in a method to produce target peptides, which can be or can only be inefficiently produced with commonly available chemical or biotechnological methods. Preferably, such polypeptides or methods allow to avoid one or more, preferably, all of the above mentioned drawbacks of previous methods known from the prior art. Further objects of or underlying the present invention can be derived from the specification, including examples, and the advantages mentioned herein.

Summary of the invention

This primary object is solved by providing fusion polypeptides comprising or consisting of in direction from the N-terminus to the C-terminus a purification domain, an autoprotease domain, a target peptide domain, optionally a signal sequence, and optionally a linker sequence, wherein the purification domain (i) binds to a carbohydrate matrix and comprises or consists of at least one of the amino acid consensus motive sequence according to SEQ ID No: 1 , SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4, SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.: 9.

Furthermore, nucleic acids encoding such fusion polypeptides and genetically modified cells comprising such fusion polypeptides are provided.

In another aspect of the present invention, a method for producing a target peptide are provided. This method comprises the steps of providing a genetically modified cell according to the present invention, culturing the cell under conditions suitable for expression of a fusion polypeptide according to the invention, obtaining the fusion polypeptide and optionally, unfolding of the obtained fusion polypeptide and directed refolding of said fusion polypeptide, contacting the obtained fusion polypeptide with a carbohydrate matrix, cleaving the fusion polypeptide by activating the autoprotease domain of the fusion polypeptide, thereby obtaining a target peptide and collecting a mixture comprising the target peptide.

In yet another aspect of the present invention, a mixture comprising a target peptide is provided, producible with a method according to the invention.

Short description of the sequences

SEQ ID Nos.: 1 to 9 are artificial amino acid sequences encoding consensus motifs of the purification domain.

SEQ ID Nos.: 10 to 13 are artificial amino acid sequences encoding the purification domain.

SEQ ID Nos.: 14 to 116 are amino acid sequences encoding carbohydrate-binding modules of different microorganisms.

SEQ ID No.: 117 is an artificial amino acid sequence encoding the consensus domain of the autoprotease domain.

SEQ ID Nos.: 118 to 120 are artificial amino acid sequences encoding the autoprotease domain. SEQ ID Nos.: 121 and 122 are artificial amino acid sequences encoding preferred linker sequences.

SEQ ID Nos.: 123 to 130 are artificial amino acid sequences encoding signal sequences for intracellulartargeting of the fusion polypeptide and for recovery of the fusion polypeptide in a preferred environment according to the invention.

SEQ ID Nos.: 131 , 133, 135, 137, 139, 141 and 143 are artificial amino acid sequences encoding fusion polypeptides according to the invention. SEQ ID Nos.: 132, 134, 136, 138, 140, 142 and 144 are artificial nucleic acid sequences encoding fusion polypeptides according to the invention. Description of the figures

Figure 1 illustrates the results of a GFP measurement in the inclusion body fraction.

Figure 2 shows the binding of the fusion polypeptide according to the invention to different starch samples. Figure 3 shows an SDS-PAGE with the expression of fusion polypeptides with SEQ ID No.: 131 and GFP as target polypeptide in comparison to a fusion polypeptide constructed according to WO’125 with GFP as target polypeptide.

Gel a) shows uninduced (lane 1) and induced (lane 2) BL21 cells carrying a pET vector with a fusion polypeptide according to SEQ ID No.: 131 with GFP as product. In gel a) one asterisk denominates the fusion polypeptide including the product GFP at 70 kDa. Two asterisks show the fusion polypeptide without the product at 43 kDa. Three asterisks show the product GFP at 27 kDa. A small amount of polypeptide has already been activated and autoproteolysed.

Gel b) shows uninduced (lane 2) and induced (lane 3) BL21 cells carrying a pET vector with a fusion polypeptide from WO’125 carrying GFP. One asterisk denominates the fusion polypeptide including the product GFP at 75 kDa. Two asterisks show the fusion polypeptide without the product at 48 kDa. Three asterisks show the product GFP at 27 kDa. In the case of this fusion polypeptide, no intact fusion polypeptide was produced in inclusion bodies under the same conditions that were used for fusion polypeptide according to SEQ ID No.: 131.

The band at 24 kDa is the enzyme that is needed for chloramphenicol resistance.

Figure 4 shows an SDS-PAGE with different fractions from the purification process for obtaining GFP as target polypeptide. The fusion polypeptide with SEQ ID No.:131 and GFP as target polypeptide is compared to fusion polypeptide constructs according to WO’125. These gels show that it is difficult to keep the fusion polypeptides of WO’125 inactive during downstream processing when compared to fusion polypeptide with SEQ ID No.: 131. The fusion polypeptide needs to be inactive during lysis as any activity of the autoprotease before the planned activation step will result in loss in product yield. Gel a) shows samples of the purification process using the fusion polypeptide according to SEQ ID No.: 131. Gel b) shows a fusion polypeptide construct from WO’125 and gel c) shows another fusion polypeptide construct from WO’125. Lane 1 of each gel shows the first supernatant after cell lysis, lane 2 shows the second supernatant after the second washing step in wash buffer. Lanes 3 and 4 show the wash steps in bidestilled water and lane 5 show the inclusion body pellet fraction. One asterisk denominates the fusion polypeptide including the product GFP at 70 kDa in a), 75 kDa in b) and 85 kDa in c). Two asterisks show the fusion polypeptide without the product at 43 kDa in a) 48 kDa in b) und 58 kDa in c). Three asterisks show the product GFP at 27 kDa. A small amount of polypeptide has already been activated and autoproteolysed in all species from a) to c). However, only the fusion polypeptide according to SEQ ID No.: 131 allows for desirable processing of the inclusion bodies as opposed to the fusion polypeptides of WO’125.

Figure 5 shows an SDS-PAGE depicting the stability of the fusion polypeptide according to SEQ ID No.: 131 with GFP as target polypeptide in denaturation and activation buffer at different pH values. Lane 1 shows the lysate pellet before washing. Lane 2 shows the washed inclusion bodies that were dissolved in denaturation buffer (III) including 2 % sodium dodecyl sulphate (SDS). The sample in lane 2 was taken two weeks after the inclusion bodies were dissolved in denaturation buffer (III) and stored at room temperature. The sample in lane 3 was taken from the same fusion polypeptide sample two weeks after the SDS had been removed and the sample had been stored at 4°C. In lane 2 and 3, the pH value was well above 9.0. Lane 4 and 5 show samples that were derived from the same sample as the one in lane 3. SDS free denaturation buffer was diluted with activation buffer (II) with pH 7.2 with a low concentration of arginine and sucrose in the activation buffer (lane 4) and high concentration of arginine and sucrose (lane 5) with a pH of 7.2. The samples were immersed in the buffer for 60 minutes at room temperature. Lane 6 shows the same sample as lane 3. It was used as a reference sample in the gel at hand. In lane 7, the same sample as in lane 6 was diluted with activating buffer (I) with pH 10.

Figure 6 shows the purification using the fusion polypeptide according to SEQ ID No.: 131 and GFP as target peptide with an elugram detecting the amount of polypeptide and the corresponding SDS-PAGE. The sample is loaded in the buffer at pH 9.4 and GFP is released from the fusion polypeptide and the column with activating buffer at pH 7.2

Detailed description A first aspect of the present invention relates to specific fusion polypeptide comprising or consisting of in direction from the N-terminus to the C-terminus

(i) a purification domain, (ii) an autoprotease domain,

(iii) a target peptide domain,

(iv) optionally: a signal sequence, and

(v) optionally: a linker sequence, wherein the purification domain (i) binds to a carbohydrate matrix and comprises or consists of at least one, i.e. one, two, three, four, five, six, seven, eight or nine, amino acid consensus motive (i.e. a motive common to all purification domains used in connection with the present invention) sequence according to SEQ ID No.: 1 , SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4, SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.: 9, as described herein, in particular as described in the claims.

Thus, the present invention primarily relates to a fusion polypeptide comprising or consisting of in direction from the N-terminus to the C-terminus

(i) a purification domain,

(ii) an autoprotease domain,

(iii) a target peptide domain,

(iv) optionally: a signal sequence, and

(v) optionally: a linker sequence, wherein the autoprotease domain (ii) comprises or consists of an amino acid sequence according to SEQ ID No.: 117 or an amino acid sequence having a sequence identity of 90

%, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more to SEQ ID No.: 117, and wherein the purification domain (i) binds to a carbohydrate matrix and comprises or consists of at least one amino acid consensus motive sequence according to SEQ ID No.: 1 , SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4, SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.: 9.

One preferred embodiment of the fusion polypeptide according to the invention relates to a fusion polypeptide comprising or consisting of in direction from the N-terminus to the C- terminus (i) a purification domain,

(ii) an autoprotease domain,

(iii) a target peptide domain,

(iv) optionally: a signal sequence, and

(v) optionally: a linker sequence, wherein the purification domain (i) is active in the absence of or at guanidiniumhydrochloride concentrations of up to 2 M or urea concentrations of up to 4 M and at a pH of above 7.9 and binds to a carbohydrate matrix (e.g. corn starch, potato starch and/or wheat starch) and/or comprises or consists of at least one, i.e. one, two, three, four, five, six, seven, eight or nine, amino acid consensus motive (i.e. a motive common to all purification domains used in connection with the present invention) sequences according to SEQ ID No.: 1 , SEQ ID No.: 2, SEQ ID No.: 3, SEQ ID No.: 4, SEQ ID No.: 5, SEQ ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8 or SEQ ID No.: 9. Preferably, placing a signal sequence according to SEQ ID No.: 126, SEQ ID No.: 127, SEQ ID No.: 128 and SEQ ID No.: 129 in an N-terminal position to the sequences as described above will enhance the fusion polypeptide ability to be deposited in inclusion bodies and be refolded under basic conditions. The purification domain confers the binding of the fusion polypeptide to a carbohydrate matrix. It was surprisingly found, that the carbohydrate binding modules (CBM) of naturally occurring amylases can be used as the basis for constructing a set of building blocks having a consensus motive sequence, namely the sequences SEQ ID No.:1 to SEQ ID No.:9, which can be combined with each other to adapt the purification domain to the desired reaction conditions. By combining the single building blocks with each other, the binding strength of the purification domain to the carbohydrate matrix can be enhanced, the binding can be stabilized under specific reaction conditions (e.g. a high ionic strength) and the size of the purification domain can be varied to fit the desired target peptide domain.

Different CBMs fulfil different functions as they bind different polysaccharide bonds or motifs within polysaccharides. The functional purpose of CBMs is the binding of the fusion polypeptide to polysaccharides, in which the monomers are connected via glycosidic bonds between D- or L-glucose or other carbohydrate monomers. Preferably, the purification domain comprises or consist of at least one consensus sequence selected from the group of the CBM classes 26, 53, 41 , 35, 48 or 58.

The ability of the purification domain to be deposited in inclusion bodies and to be active in basic environments can be influenced by the choice of the signal sequence and the choice of the domains of the carbohydrate binding moiety. The signal sequences according to SEQ ID No.: 126 to 129 influence the solubility of the N-Terminus during expression as well as during refolding.

The autoprotease domain exhibits the function of an autoproteolytic cleavage site, which separates the target peptide from the purification domain and the autoprotease domain. This domain is activated under certain reaction conditions. The autoprotease domain according to the invention has the advantage that it can be constructed based on different naturally occurring autoproteases, but with a limited window of activation, which can be precisely controlled. Therefore, the autoprotease domain according to the invention has a very low activity outside its reaction conditions for cleaving of the target peptide from the purification domain, and by using such an autoprotease, losses based on an autoproteolytic side-activity can be significantly reduced.

Preferably (and advantageously, in particular in connection with preferred embodiments as described herein), the autoprotease domain is activated at a pH value of 6.8 or above (i.e. is not activated below), more preferably at a pH value of from 6.8 to 7.2. Preferably, when assessing whether the autoprotease domain is activated or not at a specific pH, the skilled person may initiate the binding to starch first. The supernatant of the binding sample that has a pH above 7.2 is removed or eluted and replaced with an equal volume of activating buffer at pH 6,8 to 7.2, preferably at pH 7.2. The change in pH will in turn start the autoproteolysis. This is observable by protein analysis of the eluent or supernatant fraction by analytics well known by the skilled person. Temperature does not play a role in activation of the autoprotease.

The autoprotease N ^pro can be modified such that the pH of its environment is the activating trigger rather than the chaotrope concentration. Consequently, no trade-off between purity and product yield has to be taken into account. The target peptide domain comprises or consists of an amino acid sequence of a target peptide or polypeptide to be produced. The domain can consist of any amino acid sequence having between 2 and more than 1000 amino acids. Preferably, the target peptide consists of an amino acid sequence of 2 to 1000 amino acids, preferably 2 to 500 amino acids, more preferably of 2 to 100 amino acids, especially preferably of 2 to 50 amino acids. In one embodiment of the present invention, the target peptide may have an amount of hydrophobic amino acids of >10 %, based on the total number of amino acids, more preferably of >20 %, especially preferably of >30 % and even more preferably of >40 %. In another embodiment, the target peptide may have an amount of hydrophilic amino acids of >10 %, preferably of >20 %, especially preferably of >30 % and even more preferably of

>40 %, again based on the total number of amino acids. In yet another embodiment, the target peptide may have an amount of hydrophobic and hydrophilic amino acids of >10 %, more preferably of >20 %, especially preferably of >30 % and even more preferably of >40 %, based on the total number of amino acids. One embodiment of the present invention relates to a fusion polypeptide according to the invention, wherein the purification domain (i) comprises or consists of an amino acid sequence selected from the group consisting of sequences according to SEQ ID No.: 10 to SEQ ID No.:116 and sequences having a sequence identity of 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more to any one of said sequences. Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each other these values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program or the EMBOSS Water Pairwise Sequence Alignments (protein) program for amino acid sequences. Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1 ): 195-197). When conducting an alignment, the default parameters defined by the EMBL-EBI are used. Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap open penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5. The skilled person is well aware of the fact that, for example, a sequence encoding a polypeptide can be "codon-optimized" if the respective sequence is to be used in another organism in comparison to the original organism a molecule originates from.

The purification domain comprises or consists of a combination of at least one functional sequence of CBMs as stated above. This design of the purification domain was surprisingly found to offer several advantages, which are not exhibited in the naturally occurring form of carbohydrate binding enzymes. It was shown that a purification domain with the claimed sequences shows a higher binding activity over a broad temperature range, whereas naturally occurring carbohydrate-binding polypeptides, such as the human amylase, are only active in a tight temperature range. In combination to this, when operating bioprocesses, another important factor is the pH-value. It was also shown that the purification domain according to the invention offers a high binding activity also in a broad pH range and, even more surprisingly, in a combination of harsh temperatures of 0 to 80 °C and harsh pH values of down to pH 3.5 in the acidic regime and up to pH 12.0 in the basis regime.

In general, enzymes or especially the active sites of enzymes, such as the CBM of the human amylase are highly sensitive to chaotrope or detergent concentrations. It was shown in connection with the present invention, that the purification domain according to the invention is stable over a broad chaotrope and detergent concentration range.

As described above, the autoprotease domain (ii) of a fusion polypeptide according to the invention comprises or consists of an amino acid sequence according to SEQ ID No.: 117 or an amino acid sequence having a sequence identity of 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more to SEQ ID No.: 117.

The autoproteolytic activity of the autoproteolytic domain is based on the catalytic diade of histidine and cysteine in the active site of auto protease enzymes. These enzymes are the basis for an autoproteolytic domain according to the invention. The basis for such an autoproteolytic domain can be the autoprotease N ^pro from the pestivirus or an autoprotease from a potyvirus, picornavirus or any other viral autoprotease. Through targeted recombination or re-design of these sequences, autoprotease domain building blocks can be designed, which exhibit alone or in combination several advantages over their natural counterpart. On the one hand, the pH sensitivity of the autoprotease can be adjusted precisely. This exhibits the advantage, that the activity of the autoprotease can be controlled to fit the desired reaction conditions. Either with a very tight pH value range to precisely activate the autoprotease at the desired pH and avoid the early release of the target peptide or also at harsh pH values, where naturally occurring autoproteases are not stable anymore.

In a further embodiment, the present invention relates to a fusion polypeptide according to the invention, wherein the autoprotease domain (ii) comprises or consists of an amino acid sequence selected from the group consisting of sequences according to SEQ ID No.: 118, SEQ ID No.: 119 or SEQ ID No.: 120 and sequences having a sequence identity of 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more to any one of said sequences. Through combination of the autoprotease building blocks, several preferred autoprotease domain sequences are obtained, which exhibit a high pH and temperature stability and the ability to be precisely activated over the adjusted pH-value. In a preferred embodiment, the autoprotease may be active at a pH value between 7.5 and 6 and/or in a temperature range between 4°C and 40°C.

Another embodiment of the present invention relates to a fusion polypeptide according to the invention, comprising a signal sequence (iv), wherein the signal sequence (iv) is an inclusion body promoting sequence or a secretion sequence, preferably of secretion type IV ortype II of gram-negative bacteria. Preferably, signal sequences with recurring arginine motifs in the N-terminal signal peptide that were flanked by threonine, lysine and leucine are used in the context of the present invention.

A signal sequence in context of the present invention describes a functional sequence, which guides the fusion polypeptides to specific cell compartments. Several signal sequences are known in the art. Preferably, a signal sequence selected from the group consisting of SEQ ID No.: 123, SEQ ID No.: 124, SEQ ID No.: 125, SEQ ID No.: 126, SEQ ID No.: 127, SEQ ID No.: 128, SEQ ID No.: 129 or SEQ ID No.: 130 is used. Surprisingly it was found, that these sequences do not only control inclusion body promotion but also direct the refolding process in strongly basic environments.

Signal sequences are always selected based on their influence on the reprocessing of the target peptide. In one embodiment, an inclusion body signal sequence is used, guiding the target polypeptide to inclusion bodies. It is well known in the art that polypeptides that are produced in inclusion bodies need to be refolded before further downstream processing. For example, the signal sequence Cry4AaCter (SEQ ID No.: 126) may be used, which enhances the alkaline processing of inclusion bodies. In one embodiment of the present invention, an N-terminal Tat signal may be used, which is a secretion signal of the bacterial secretion system. Using this signal sequence, the target polypeptide is secreted.

Yet another embodiment of the present invention relates to a fusion polypeptide according to the invention, comprising a linker sequence (v), wherein the linker sequence comprises an N-terminal alpha helix and/or a C-terminal sequence of a random coil structure.

A linker sequence in the context of the present invention means a sequence between the functional domains and also between the autoprotease and the target peptide. The length of the linker is preferably 1 to 50 or more than 50 amino acids. In another embodiment, the purification domain and the autoprotease domain are directly fused, i.e. without a linker. In one embodiment of the present invention, a linker sequence selected from SEQ ID No.: 121 or SEQ ID No.: 122 may be used. One aspect of the present invention relates to a recombinant nucleic acid molecule encoding a fusion polypeptide according to the invention.

One embodiment of the present invention relates to a recombinant nucleic acid molecule selected from the group consisting of SEQ ID No.: 132, SEQ ID No.: 134, SEQ ID No.: 136, SEQ ID No.: 138, SEQ ID No.: 140, SEQ ID No.: 142 and SEQ ID No.: 144.

Yet another embodiment of the present invention relates to a recombinant amino acid molecule selected from the group consisting of SEQ ID No.: 131 , SEQ ID No.: 133, SEQ ID No.: 135, SEQ ID No.: 137, SEQ ID No.: 139, SEQ ID No.: 141 and SEQ ID No.: 143.

Another aspect relates to a genetically modified cell, including a recombinant nucleic acid molecule according to the invention, wherein the cell is capable of expressing a fusion polypeptide according to the invention.

One embodiment of the invention relates to a genetically modified cell according to the invention, wherein the cell is selected from the group consisting of Escherichia coli, Vibrio natrigens, Saccheromyces cerevisiae, Aspergillus niger, green algae, microalgae, HEK T293 and Chinese hamster ovary cells (CHO).

Another aspect of the present invention relates to a method of preparing a target peptide comprising the steps of:

(a) providing a genetically modified cell according to the invention,

(b) culturing the cell under conditions suitable for expression of a fusion polypeptide according to the invention,

(c) obtaining the fusion polypeptide and optionally, unfolding of the obtained fusion polypeptide and directed refolding of said fusion polypeptide,

(d) contacting the fusion polypeptide obtained in step (c) with a carbohydrate matrix,

(e) cleaving the fusion polypeptide by activating the autoprotease domain of the fusion polypeptide, thereby obtaining a target peptide,

(f) collecting a mixture comprising the target peptide.

Step (a) comprises providing a genetically modified cell expressing a fusion polypeptide. Such cell is obtainable by introducing a nucleic acid molecule including a sequence encoding a fusion polypeptide, preferably in the form of a vector, into the cell by known methods such as for example by transfection or transformation. In step (b), the cell is cultured under conditions suitable for expressing a fusion polypeptide according to the invention, preferably in a high-density culture. Culture conditions and especially conditions to achieve a high-density culture and corresponding media are well known to the person skilled in the art. In one embodiment of the present invention, the expression of the fusion polypeptide is achieved with a subsequent transport to inclusion bodies using a suitable signal sequence. Step (c) comprises obtaining the fusion polypeptide from the culture broth and optionally unfolding of the obtained fusion polypeptide and directed refolding of said fusion polypeptide, if the fusion polypeptide is present in inclusion bodies. Solubilization conditions for the processing of inclusion bodies and conditions for the directed refolding are well known in the art. Preferably, inclusion bodies are solubilized by using 6 M guanidinium chloride, 8 M urea or 2 % sodium dodecyl sulfate and are refolded under neutral or mildly basic conditions.

In step (d), the solubilized fusion polypeptide is contacted with a carbohydrate-based matrix such that the fusion polypeptide binds to the matrix by its purification domain. This step is performed under conditions, wherein the autoprotease domain (ii) is inactive, preferably by controlling the pH value rather than the chaotrope or denaturant concentration in order to avoid premature cleavage of the target peptide domain (iii) on the one hand and induce activity of the purification domain (i) on the other hand. Under these conditions, the amount of cleaved fusion polypeptide is preferably <10 %, more preferably <5 %, especially preferably <3 % or even more preferably <1 %, based on the total amount of fusion polypeptide. The underlying mechanism of the inactivity of the autoproteolytic domain can be described with two cases:

(1) conditions, wherein the autoprotease domain is constitutionally inactive and is only activated by a change of the environmental conditions, such as by an adaption of the temperature, the pH and/or the ionic strength, preferably by adapting the pH; or

(2) conditions wherein the autoprotease domain is constitutionally active, however, having insufficient activity to achieve a premature cleavage of the target peptide domain during the period of time necessary for performing the method step (d), i.e. is kinetically inactive, preferably for up to 10 min, more preferably for up to 20 min and especially preferably up to 30 min.

In one embodiment, step (d) is performed under native conditions, i.e. under conditions wherein the autoprotease is constitutionally active. Surprisingly, it was found that even if the fusion polypeptide is present in its native state, the autoprotease domain remains sufficiently inactive during step (d). This effect was especially present when using the hybrid N ^pro autoprotease according to SEQ ID NO.: 120. Preferably, an insoluble carbohydrate matrix is used in step (d), which facilitates the separation of impurities.

In step (e), the fusion polypeptide is cleaved by the autoprotease domain and the target peptide (iii) is released. Cleavage of the fusion polypeptide may result from addition of an autoproteolysis buffer, i.e. a buffer providing conditions under which the autoprotease is active, e.g. acidic or alkaline conditions.

Step (f) results in obtaining a mixture by eluting the cleaved target peptide from the column. Preferably, the elution is done by using a buffer selected from the group consisting of HEPES, PBS, and TrisHCI at concentrations between 1 and 100 mM and 30 mM KCI, at a pH of 6.5 to 7.5. Furthermore, the preferred buffer may be supplemented by arginine at a concentration of 10 to 100 mM or by sucrose at a concentration of 2 to 20 mM.

One embodiment of the method according to the invention relates to a method, wherein the carbohydrate matrix in step (d) consists of or comprises a substance selected from the group consisting of starch, lignin carbohydrate polymers, copolymers with alpha-1 ,4- and alpha-1 ,6 glycosidic bonds of glucose or other sugars and mixtures thereof and is preferably present in a packed column, as a packed substrate or as starch grains consisting of amylose and amylopectin.

Starch is a complex mixture of carbohydrates from different sugar polymers. Plant cells collect the sugars they produce in a storage organelle called a vacuole. When the cells and organelles are mechanically destroyed, the starch granules are released. Depending on the plant species, there are differences in the raw starch. Starches can have different grain sizes ranging from less than 25 pm to more than 100 pm in diameter. The higher the proportion with diameters of over 75 pm, the higher the probability of non-specific adsorption and thus the retention of impurities in the products after starch purification. In addition, there are starch granules, such as wheat, which are porous and can absorb amylases in internal channels. Starch consists of the components amylose and amylopectin. In contrast to amylopectin, amylose is water-soluble. The swelling behavior of the respective starch in water also depends on the proportions of the two species. Thus, unpurified cornstarch in water acquires a cement-like consistency, whereas table potato starch remains water-permeable. All carbohydrate-binding enzymes have a high affinity to their substrate, which is also present under harsh conditions. Preferably, the starch grains are insoluble in water. It is furthermore preferred, if the soluble amylose parts and polypeptides have been removed from the starch.

Another embodiment of the method according to the invention relates to a method, wherein the activation of the autoprotease domain in step (e) is performed at pH 6 to pH 8, preferably at pH 6.5 to 7.5, especially preferably at pH 6.8 to pH 7.2 and even more preferably at pH 7 to pH 7.4.

One aspect of the present invention relates to a recombinant nucleic acid molecule, encoding a fusion polypeptide according to the invention and a cloning site for incorporation of a recombinant nucleic acid molecule according to the invention, optionally operatively linked to an expression control sequence. Preferably, an expression control site selected from the group consisting of IPTG controlled promotors, preferably T5 orT7, and rhamnose controlled promotors as well as an ensemble of extra tRNAs is used.

Yet another aspect of the present invention relates to a mixture comprising or consisting of a target peptide, preferably of a synthetic target peptide and a total amount of 0,001 to 1 wt.-% sodium and/or potassium, based on the total weight of the sum of sodium (if present), potassium (if present) and target peptide, wherein the mixture is obtained or obtainable by a method according to the present invention. The mixture obtained in step (f) of the method according to the invention, comprises besides the produced target peptide also specific amounts of sodium and/or potassium.

In another aspect, the present invention relates to a synthetic target peptide, wherein the peptide comprises an N-terminal proline, obtained or obtainable by a method according to the invention or to a mixture according to the invention, wherein the target peptide is a peptide comprising an N-terminal proline. It was surprisingly found, that it was able to produce synthetic target peptides with an N-terminal proline with the method according to the invention. In general, it is an exceptional challenge to produce synthetic peptides with an N-terminal proline as the proline sterically hinders the production processes of peptides of methods known in the art.

In the following, the invention is further characterized by non-limiting examples. Examples

Example 1 : Production of the target peptide mellitin

A. Cloning of a plasmid comprising the target polypeptide nucleic acid sequence

The fusion polypeptide according to any one of the sequences SEQ ID No.: 132, SEQ ID No.: 138, SEQ ID No.: 140 or SEQ ID No.: 142 is cloned into the expression vector pET28a together with a sequence for the target peptide mellitin and an inclusion body promoting sequence. The genetic information of the fusion polypeptide is constructed in such a way, that every building block (e.g. the purification domain or the autoprotease domain) can be interchanged easily. The restriction sites and corresponding enzymes are listed in Table 1. A standard cloning protocol using the restriction sites Ncol or Ndel and EcoRI of the pET28a vectorto insert the fusion polypeptide gene is used. The obtained plasmid solution is stored for further processing.

Table 1: Restriction sites for the building blocks of the fusion polypeptide B. Peptide production of mellitin

Step 1) 5 pi of plasmid solution obtained in Example 1 comprising a DNA concentration of 100 ng/mI are transferred into a 2 ml reaction vessel containing 50mI of pre thawed E. coli BL21 pLys. The mixture of plasmid and cells is incubated on ice for 30 minutes. After the incubation, the mixture of plasmid and cells is exposed to a one minute heat shock at 42°C in a water bath. After the heat shock, the cells are relaxed on ice for 2 minutes and then treated with 950 mI of warm SOC medium. The cells including SOC medium are then incubated with continuous mild agitation at 37°C for one hour. The cells are then spun down at 5000 rpm for two minutes at room temperature and 950 mI of the supernatant are removed. The cells are resuspended in the remaining liquid and transferred to a solid LB-agar plate on a petri dish with 25 ml of LB agar and 34 pg/ml Chloramphenicol and 38 pg/ml Kanamycin. The agar plate is then incubated over night at 37°C.

Step 2) After 24 hours, a colony is picked and transferred to 10 ml of LB medium with 34 pg/ml Chloramphenicol and 38 pg/ml Kanamycin and incubated for eight hours at 37°C and mild agitation. 8 ml of the culture are transferred to 500 ml of LB medium with 34 pg/ml Chloramphenicol and 38 pg/ml Kanamycin in a shaking bottle and incubated with mild agitation overnight. 2 ml of the culture are held back for plasmid preparation and verification of the plasmid and insert. Step 3) The overnight incubated culture is suspended 1 :5 (v/v) in a suitable minimal medium in a 2 I shaking bottle. The culture is grown to an Oϋboo of 5 and then induced with IPTG of a final concentration of 1 mM. The expression culture is run for six hours and discretely supplemented with glucose or glycerol (both 20 g/l), thiamine, citric acid and a suitable trace element solution. The solutions are added at a rate of 0.1 ml/min for 3.5 hour and an additional hour at 0.2 ml/min. The culture is harvested by centrifugation at 4°C and 3000 rpm for 20 minutes and the cells are either promptly used for further downstream processing or shock frozen in dried ice and then stored at -80°C.

Step 4) The cells are resuspended in a lysis buffer. This buffer may contain between 0 mM and 75 mM sodium acetate, 0 mM to 20 mM HEPES, 2 mM Magnesium chloride and 1 % Triton X-100. The cells are weighed in a tared reaction vessel after harvesting them by centrifugation. Ultrasonic lysis requires a fourfold excess of lysis buffer to cell mass. The lysis is carried out with sonication and a 12” cup horn tip. The protocol is performed on ice at 80 W with 15 seconds of pulsing and 20 seconds pause for 8 minutes. The soluble parts of the lysate mixture are separated from the inclusion body carrying solid phase by centrifugation at 4°C and 5000 rpm for 25 minutes. The supernatant and the pellet are checked for their inclusion body containment via polyacrylamide gels. When the majority of the fusion polypeptide as identified by the polyacrylamide gel is found in the pellet, the gel is also checked for contaminations of the pellet. If these contaminations are significant, the pellet needs to be washed. It is resuspended in the same lysis buffer again and kept on ice for 10 minutes. The suspension is homogenized by vortexing every two minutes. After the incubation, the procedure is repeated once more. After the third centrifugation step, the pellet is resuspended in water and incubated for ten minutes on ice including regular vortexing as described above. The suspension is centrifuged for 25 minutes at 4°C and 5000 rpm. The pellet is now weighed a second time and prepared for the next steps.

Step 5) The washed inclusion bodies of step 4 are unfolded. The ratio of the inclusion bodies to the used buffer (w/v) is not lower than 1 :10. The chaotropic buffers contain at least either 6 M guanidinium chloride or 8 M urea. The detergent based buffer contains 5 % (w/v) Natriumlaurylsulfat (SDS) and optionally up to 20 mM

HEPES. The pH-value is controlled with sodium acetate, sodium hydroxide and/or potassium hydroxide. When resuspended, the mixture is incubated at room temperature for 40 to 60 minutes while being vortexed for 30 seconds every 10 minutes. Step 6) A column is packed with starch as column material (which is the substrate of fusion polypeptide binding). The starch may be unaltered or sieve filtered such that the starch grains have a grain size of between 25 and 50 pm. The starch is washed several times with water and/or buffer for protein stabilization in order to remove protein and soluble amylose from the starch. The starch is preferably wheat or potato starch. The latter can be used as a column material easily. The washed and sieved starch grains are packed in a column in the case of wheat and potato starch together with liquid. The fusion polypeptide still immersed in the unfolding buffer is now diluted with an activating buffer. The dilution is performed such that only the binding moiety will refold and allow for binding to the column material. For guanidinium chloride, the final concentration of the chaotrope should be between

4.5 M and 5 M. For urea, the dilution concentration should be between 5 M and 6 M for any of the fusion polypeptides. SDS is not necessarily to be removed from the mixture of unfolded fusion polypeptide and denaturation buffer. SDS is removed from the solution and precipitated by either titrating 30 mM potassium chloride solution to the mixture, cooling the mixture to 0 to 4°C or taking both SDS removal measures at the same time. One can decrease the SDS concentration to 0.5 % SDS in the mixture without activating the autoprotease. Then, the mixture is brought into contact with the column material and excess SDS can be removed using potassium chloride or cooling or using both methods without clogging the column material. Step 7) The diluted buffer is released and eluted. The wash elution is discarded. The column is washed with one column volume of water. The fusion polypeptide is bound to the column material and can be left on the column while the autoproteolytic domain becomes activated. As soon as the residues of the denaturing buffer are removed, the bound fusion polypeptides are active. The column material is immersed in a little more than one volume of activating buffer

(Table 2), which amounts to 5 ml of buffer to 500 mg of column material. The column is then incubated at temperatures of between 2°C and 37°C. The autoprotease is now active in the starch matrix of both methods. The incubation period can last from 80 minutes at between 2°C to 8°C to four hours from 9°C to 22°C and 12 hours from 23°C to 37°C. During this period, the target peptide is released from the fusion polypeptide that is bound to the starch material.

Step 8) The target peptide, which has been released in the previous step, is collected. 50 % column volume of the activating buffer or water are added to wash the total amount of target peptide from the column. The eluate or supernatant that contains the target peptide is precipitated in 1 :3 (v/v) ethanol. The mixture of product and ethanol is centrifuged at 5000 rpm and 4°C for ten minutes. The supernatant is discarded and the precipitate is freeze-dried overnight. The freeze-dried sample is then dissolved in a suitable solvent (e.g. 60 % to 80 % methanol, DMF, deionized water) and sonicated at 40 W in an ultrasonic bath for one hour at room temperature. The sample is then centrifuged for 10 minutes at 5000 rpm and 25°C.

The supernatant contains the pure target peptide. Table 3 shows different target yields of mellitin used together with different fusion polypeptides in comparison with the achieved cell masses.

Table 2: Composition of the activating / autoproteolytic buffer

Table 3: Yields of fusion polypeptides

Example 2: Production of the target peptide GFP A. Cloning of a plasmid comprising the target polypeptide nucleic acid sequence

The fusion polypeptide according to any one of the sequences SEQ ID No.: 132, SEQ ID No.: 138, SEQ ID No.: 140 or SEQ ID No.: 142 is cloned into the expression vector pET28a together with a sequence for the target peptide green fluorescent polypeptide (GFP) and an inclusion body promoting sequence. The fusion polypeptide gene consists of three major and two optional building blocks that are organized in a certain order from N-terminus to C-terminus. All building blocks are separated by restriction sites on a genetic level. The restriction sites and corresponding enzymes are listed in Table 4. A standard cloning protocol using the restriction sites Ncol or Ndel and EcoRI of any vector of the pET family to insert the fusion polypeptide gene is used. The obtained plasmid solution is stored for further processing.

Table 4: Restriction sites for the building blocks of the fusion polypeptide

B. Polypeptide production of GFP

The following production protocol was executed for fusion polypeptides with SEQ ID Nos.: 131 and 137 having an amino acid sequence for GFP as target polypeptide. In comparison to the fusion polypeptides according to the invention, fusion polypeptides according to WO2019138125 (WO’125) were constructed. These fusion polypeptides have the following domain architecture:

• ssTorrA (inclusion body promoting sequence, SEQ ID No.: 2 of WO’125) -3x-CBM Aspergillus niger (Binding domain, SEQ ID No.:10 of WO’125) - N ^pro (autoproteolytic domain, SEQ ID No.: 12 of WO’125) · ssTorrA (inclusion body promoting sequence, SEQ ID No.: 2 ofWO’125)-Amylase- homo-sapiens (Binding domain, SEQ ID No.: 5 of WO’125) - N ^pro (autoproteolytic domain, SEQ ID No. 12 of WO’125) Step 1) Transformation:

5 pi of plasmid solution obtained in Example 2 A comprising a DNA concentration of 100 ng/mI are transferred into a 2ml reaction vessel containing 50mI of pre thawed E. coli BL21 pLys or any other BL21 derivative. The mixture of plasmid and cells is incubated on ice for 30 minutes. After the incubation, the mixture of plasmid and cells is exposed to a one minute heat shock at 42°C in a water bath. After the heat shock, the cells are relaxed on ice for 2 minutes and then treated with 950 mI of warm SOC medium. The cells including SOC medium are then incubated with continuous mild agitation at 37°C for one hour. The cells are then spun down at 5000 rpm for two minutes at room temperature and 950 mI of the supernatant are removed. The cells are resuspended in the remaining liquid and transferred to a solid LB-agar plate on a petri dish with 25 ml of LB agar and 30 pg/ml Chloramphenicol and 30 pg/ml Kanamycin. The agar plate is then incubated over night at 37°C.

Step 2) Colony picking and inoculation culture preparation: After 24 hours, a colony is picked and transferred to 10 ml of LB medium with 30 pg/ml Chloramphenicol and 30 pg/ml Kanamycin and incubated for eight hours at 37°C and mild agitation. 8 ml of the culture are transferred to 500 ml of LB medium with 30 pg/ml Chloramphenicol and 30 pg/ml Kanamycin in a shaking bottle or used as an inoculation culture immediately and incubated with mild agitation over a suitable amount of time, ranging from three hours of expression to overnight expression. 2 ml of the culture are held back for plasmid preparation and verification of the plasmid and insert.

Step 3) Fusion polypeptide expression:

The expression culture is grown in a 2 I shaking bottle and induced with a suitable amount of a chemical inducer, including lactose, rhamnose and Isopropyl-p-D- thiogalactopyranosid (IPTG). The culture is grown at a temperature of 37°C, agitated at shaking between 150 and 250 rpm to the exponential growth phase, and induced. The expression culture is run for six hours and discretely supplemented with glucose or glycerol (both 20 g/l), thiamine, citric acid and a suitable trace element solution. The solutions are added at a rate of 0.1 ml/min for 3.5 hour and an additional hour at 0.2 ml/min. The culture is harvested by centrifugation at 4°C and 3000 rpm for 20 minutes and the cells are either promptly used for further downstream processing or shock frozen in dried ice and then stored at - 80°C. Step 4) Cell lysis and inclusion body preparation:

The cells are resuspended in a lysis buffer. This buffer may contain between 0 mM and 75 mM sodium acetate, 0 mM to 20 mM 2-[4-(2-hydroxyethyl)piperazin-1- yljethane sulfonic acid (HEPES), 2 mM Magnesium chloride and 1 % Triton X-100. The pH of the lysis and washing buffer is setto a pH above 9.0. The cells are weighed in a fared reaction vessel after harvesting them by centrifugation. Lysis can be performed using sonication, dispersion or French press. The soluble parts of the lysate mixture are separated from the inclusion body carrying solid phase by centrifugation at 4°C and 8000 rpm for 10 minutes. The last washing step is performed in water to eliminate Triton X-100 traces and to remove DNA and cytoplasmic polypeptide residues. The supernatant and the pellet are checked for inclusion bodies and active fusion polypeptide via polyacrylamide gels to test for contaminations. If these contaminations are significant, the pellet needs to be washed. The pellet is resuspended in the same lysis buffer again and kept at 4°C to 0°C for 10 minutes. The suspension is homogenized by vortexing (e.g. for 30 seconds every 5 minutes). The incubation and centrifugation of the pellet is repeated once. Finally, the pellet is resuspended in water and incubated for ten minutes on ice including vortexing (e.g. as described above). The suspension is centrifuged for 25 minutes at 4°C and between 6000 and 7000 rpm. The contaminations including DNA and undesired polypeptide are less dense than the inclusion body fraction and can be discarded as part of the supernatant. The pellet is now weighed a second time and prepared for the following step.

Step 5) Inclusion Body Denaturation:

The washed inclusion bodies of step 4 are denatured by unfolding them. The ratio of the inclusion bodies to the denaturation buffer (w/v) is not lower than 1 :10 if urea

(II) and guanidinium chloride (III) containing chaotropic buffers are used. The chaotropic buffers contain at least either 6 M guanidinium chloride or 8 M urea. The detergent based buffer contains up to 2 % (w/v) sodium dodecyl sulphate (SDS) and optionally up to 20 mM HEPES. The pH-value is controlled with sodium acetate, acetate, sodium hydroxide and/or potassium hydroxide. The denaturation buffer (III) is set to pH 9.0 or higher. The ratio of inclusion bodies to SDS buffer is 1 :8 (g/ml). When resuspended, the mixture is incubated at room temperature for 40 to 60 minutes while being vortexed for 30 seconds every 10 minutes. The fusion polypeptide is denatured by SDS that is present in the denaturation buffer (III). The fusion polypeptide can be stored at room temperature dissolved in the denaturation buffer (III) for at least two weeks (Figure 5). The SDS in the buffer is removed from the buffer that contains the fusion polypeptide by cooling the solution down to 4°C for at least one hour. The SDS is precipitated removing a small percentage of the polypeptide that is bound to SDS. SDS crystals are removed by centrifugation at 4°C, 2000rpm to 5000rpm and 4 minutes to 8 minutes. The supernatant is collected and stored at 4°C. If further SDS precipitates, the suspension is centrifuged once more as described above. The pH value of the SDS free buffer is checked. The observed pH range was between 9.0 and 11 .0 forthe SDS deprived buffer containing fusion polypeptide. According to figure 5, the fusion polypeptide did not decay when stored in the SDS free buffer at 4°C for at least two weeks. Denaturation buffer (I) and (II) can also be used. However, the denaturation buffer

(III) exhibits several advantages, i.e. easy removal of the detergent SDS, low costs of SDS (SDS can be recycled after precipitation) and immediate, detergent-free use or storage. In opposite to the denaturation buffer (I) and (II), no loss in product is observed. Table 5: Composition of denaturating buffers

Step 6) Preparation and Packing of Binding Material:

Potato starch and corn starch, both comprising a grain diameter smaller than 32pm are used to pack a column. The starches are washed with a polypeptide collection buffer that removes soluble starch associated polypeptides. The starch to buffer ratio is 1 :2 w/v. The buffer consists of 50 mM 2-Amino-2-(hydroxymethyl)propane-1 ,3-diol (Tris) and 50 mM sodium chloride (NaCI), pH 7.2. The starches are constantly agitated at 37°C for one hour to prevent sedimentation. The incubation is followed by centrifugation at 20°C and 6000 rpm for 12 minutes. The supernatants are removed and the starches are dried at 45°C for eight hours. The dried prepared starches are stored at room temperature. Columns are packed using a mixture of the prepared corn starch and potato starch. Suitable mixtures of corn starch to potato starch include 1 :1 , 2:3 and 1 :4. The dried starches are mixed in the desired ratio by vortexing. A starch slurry for column packing is prepared by suspending the starch mixture in an activation buffer. The activation buffer that is used for column packing is also used for loading the fusion polypeptide sample. The activation buffer that is used to prepare the starch slurry is called activation buffer (I) (Table 7). Activation buffer (I) allows for the binding domain of the fusion polypeptide to bind the starch material without the autoprotease of the fusion polypeptide being activated.

The starch slurry that is described above can be brought into contact with the fusion polypeptide in different ways. A gravity flow column may be packed, an FPLC column may be packed or the contact between the fusion polypeptide and the starch slurry is made in a centrifugation beaker and activation buffer (I) is removed via centrifugation.

Table 6: Composition of the starch washing buffer (pH 7.2) Table 7: Composition of the activation buffer (I) (pH 9.4)

Table 8: Composition of the activation buffer (II) (pH 7.2) Step 7) Purification:

The fusion polypeptide is dissolved in detergent free denaturating buffer (III) (Table 5). The column material that is used in one of the ways described in step 6 is equilibrated. Fusion polypeptide bearing detergent free solution is mixed with activating buffer (I) at a ratio of 1 ml fusion polypeptide solution to between 4 and

6 ml of activating buffer (I). This mixture is loaded on the starch material.

The bound fusion polypeptide will be activated in the presence of activating buffer (II) (Table 8) that is used for elution. This is not possible when using the method and fusion polypeptide described in WO2019138125. When working with the approach of WO2019138125, the chaotropic substances have to be removed completely, to activate the carbohydrate-binding moiety. In the meantime, the autoprotease is already active, which accounts for a significant loss of product. In the current invention, a way has been found, to get an active purification domain (i) allowing for easy removal of any contaminant before the autoprotease is activated. A further observation refers to the temperature dependence of native N ^pro and the EDDI mutant. Both have been found to be most active between 2°C and 8°C. This is not the case for the new purely pH-sensitive autoprotease of SEQ ID No.: 131. The pH range of the autoprotease of the fusion polypeptide according to the SEQ ID No.: 131 is very narrow (pH 6.8 to 7.2). In a purification performed at 23°C, the product GFP is released 15 minutes after the first contact with pH 7.2. The complete GFP that has been loaded on the column as part of the fusion polypeptide is released within thirty minutes. For the previously known N ^pro the incubation period can last from 80 minutes at between 2°C to 8°C to four hours from 9°C to 22°C and 12 hours from 23°C to 37°C. Step 8) GFP collection

The target peptide, which has been released in the previous step, is collected. 50 % column volume of the activating buffer (II) or water are added to wash the total amount of target peptide from the column. The eluate or supernatant that contains the target peptide is precipitated in 1 :1 (v/v) ethanol. The mixture of product and ethanol is centrifuged at 5000 rpm and 4°C for ten minutes. The supernatant is discarded and the precipitate is freeze-dried overnight. The freeze-dried sample is then dissolved in a suitable solvent and sonicated at 40 W in an ultrasonic bath for one hour at room temperature. The sample is then centrifuged for 10 minutes at 5000 rpm and 25°C. The supernatant contains the pure target peptide. For GFP, 500 mg per liter culture were a typical yield. Table 9 shows the yields of fusion polypeptide according to the invention (SEQ ID Nos.: 131 and 137) in comparison to fusion polypeptide constructs according to WO’125. An SDS gel depicting the results from the downstream processing of a fusion polypeptide according to SEQ ID No.: 131 with GFP as target protein in comparison to the fusion polypeptide construct ssTorrA-3x-CBM Aspergillus niger-N ^pro from WO’125 is depicted in Figure 4.

Table 9: Yields of different fractions in the purification process of different target polypeptides

Example 3: Binding kinetics of fusion polypeptides according to the invention

The following Table 10 shows a comparison of five different fusion polypeptides in terms of their purification using different binding materials. The inclusion bodies are denatured with 2 % SDS or secreted fusion polypeptide was used. One volume of 30 mM KCI is used for elution. The same fusion polypeptide is investigated under the same conditions and brought in contact with either maize starch, wheat starch and centrifugation, wheat starch in a column and potato starch in a column. The table shows the target peptide yield (mellitin peptide) in mg/I culture for each construct. Table 10: Comparison of five fusion polypeptides

Example 4: Assessing different production process steps using GFP

The efficiency of inclusion body production is assessed using GFP. As the absorption and fluorescence characteristics of GFP is known under various conditions, GFP absorption and fluorescence are used to investigate production steps. Super folder GFP is used as a target polypeptide. A pre culture of bacteria carrying the expression plasmid with the GFP carrying fusion polypeptide as an insert is grown overnight. This pre-culture is used to inoculate the expression culture. Prior to induction the culture is split and only one of the cultures is induced with IPTG. The non-induced culture is used as a blank for the induction culture. 1 ml of both cultures is collected to measure the Oϋboo at each time point. For both cultures, a dilution series is created in TNG buffer, which consists of 100 mM Tris HCI, 50 mM sodium chloride and 10 % glycerol (w/v). The non-induced cells of the dilution series are used as a blank for the induced cells of the same dilution. The same samples are used for measurements of the absorption at 600nm and the fluorescence in the range of GFP. Each sample is treated with the same amount of rhodamine as an internal standard. The cell aliquots, where the fluorescence has been measured, are centrifuged in fared vessels and the supernatant is removed. The weight of the cell mass is used to relate the cell mass to absorption and fluorescence. The remaining aliquots of the 1ml samples are diluted in the same way as the samples for optical measurements. This new dilution series is subjected to cell lysis. The supernatant and pellet are separated by centrifugation. The supernatants are treated with rhodamine as before. After the measurement, the samples are precipitated in ethanol and the mass is determined. In this way, the GFP in the pellet and the supernatant can be compared to each other. Super folder GFP can be also determined in inclusion bodies. The fluorescence measurement is performed at 491 nm as an excitation wavelength and 512nm as emission wavelength.

In figure 1 GFP fluorescence of two different samples is shown. To the left hand side, the fluorescence in the lysate sample is shown, where no or little fluorescence is expected. In the figure to the right hand side, a clear fluorescence is accounted for GFP production is shown. This figure shows that GFP has been produced.

Example 5: Testing of fusion polypeptide binding

Testing of binding of the fusion polypeptide to the starch matrix is tested. Therefore, an aliquot of the column or centrifuge beaker pellet material (100 mg) is transferred to a 2 ml reaction vessel and treated with 500 pi of a solution of 1 % SDS and 10 mM mercaptoethanol in water. The mixture is agitated at 800 rpm and 37°C for ten minutes. The sample is then centrifuged for five minutes at 10000 rpm and room temperature. 15 pi of the supernatant are used for SDS polyacrylamide gel sample preparation and successive polyacrylamide gel analysis. As the mass of the fusion polypeptide is known, it is possible to identify the right mass in the gel.

Figure 2 shows a 12 % polyacrylamide gel depicting the kinetical observation of the binding of fusion polypeptide to a wheat column. Nine wheat starch samples are loaded with activated fusion polypeptide and the supernatant is eluted. The sample starches are extracted after a certain time point and the resulting samples are loaded on a 12 % polyacrylamide gel. Figure 2 therefore shows that the fusion polypeptide binds to the starch.