Chimeric proteins and expression systems

Abstract

A novel chimeric protein that reduces oxidative stress in a host cell is described, comprising a Bol3 polypeptide operably linked to a Lip5 polypeptide. The chimeric protein may comprise a linker between the Bol3 polypeptide and the Lip5 polypeptide. The linker may be a polyhistidine linker. Also described are a polynucleotide encoding the chimeric protein, a vector incorporating the polynucleotide, and a host cell transformed with the vector. A method of expressing a target polypeptide having at least one disulfide bond (e.g., at least three disulfide bonds), such as a target polypeptide having ICK, in a eukaryotic host cell, comprising transforming the host cell with a polynucleotide encoding the chimeric protein and culturing the host cell under conditions in which the chimeric protein and the polypeptide are both expressed.

Description

[Technical field]

[0001] Technical Field The present invention relates to a chimeric protein and a eukaryotic expression system using the chimeric protein for the production of complex disulfide-linked polypeptides. The expression system is particularly useful for the heterologous expression of "complex" post-translationally modified protein products, i.e., disulfide-rich proteins. Co-expression of the chimeric protein with the complex protein of interest enhances cellular fitness and significantly mitigates ("rescues") the deleterious effects associated with their expression. The chimeric proteins are particularly useful when the host cell also expresses a target polypeptide having at least one disulfide bond. Co-expression of the novel fusion proteins has been shown to increase yeast replication (i.e., growth rate) and / or yield of the target polypeptide having at least one disulfide bond. [Background technology]

[0002] 2. Background of the Invention Polypeptides containing disulfide-bonded secondary structures have generally been demonstrated to have significantly increased chemical, thermal and enzymatic (e.g., resistance to proteolytic digestion) stability, which aids in the biological activity (i.e., longer half-life and target affinity) of the molecule (Hayward et al., 2017, Journal of Biological Chemistry, 292(38), 15670-15680; Sermadiras et al., 2013, PLoS ONE, 8(12), 1-11). Particular examples include toxin-derived peptides that typically contain a complex disulfide-rich (3+ bond) structure collectively referred to as the inhibitor cystine knot ("ICK") motif. The beneficial stability and bioactivity traits observed with ICK motifs have led to numerous attempts to recombinantly express polypeptides bearing ICK motifs and utilize them as novel therapeutic agents (Cao et al., 2003, Peptides, 24(2), 187-192; Schmoldt et al., 2005, Protein Expression and Purification, 39(1), 82-89; Sermadiras et al., 2013, supra; Zhong et al., 2014, PLoS ONE, 9(10), 2-7). It should be noted that ICK peptides are often present in only trace amounts (e.g., in venom secretions), making their study and industrial scale-up extremely difficult, costly and unpredictable (Sermadiras et al., 2013, supra).

[0003] Although many studies have demonstrated that polypeptides containing ICK motifs can be produced successfully in both bacterial and eukaryotic systems (Sermadiras et al., 2013, supra), their expression in eukaryotic host cells has been less successful. The budding yeast Saccharomyces cerevisiae is a well-studied and genetically tractable eukaryotic microorganism with a long and successful track record in the biotechnology industry. As in other eukaryotes, disulfide bond formation in yeast is carried out within the endoplasmic reticulum (ER) by the concerted action of the 58 kDa protein disulfide isomerase (PDI) and its cognate partner, the thiol oxidase Ero1 (65 kDa). To catalyze bond formation, PDI first removes an electron from a cysteine ​​thiol on the target protein, which is then shuttled via Ero1 to the final acceptor, usually oxygen (Frand & Kaiser, 1998; Tyo et al., 2012). This shuttle also generates a stoichiometric amount of the oxidant hydrogen peroxide (H2O2) for each disulfide bond generated (Tyo et al., 2012, supra). In addition, the unfolded protein response (UPR), a yeast proteostasis mechanism that maintains and ensures the "proper" folding of proteins, may be activated under such high folding demands, resulting in additional metabolic costs and impacts on host fitness (Karagoz, et al., 2019 Cold Spring Harbor perspectives in biology vol. 11,9).

[0004] Consequently, while disulfide-bonded proteins are attractive biologically active targets for the biotechnology industry, commercial production of recombinant disulfide-bonded proteins requires new strategies to alleviate the metabolic burden (e.g., oxidative stress) imposed on host cells. As a direct consequence of the metabolic stress caused by the generation of disulfide bonds, expression of such polypeptides poses problems that are particularly exacerbated when the polypeptides contain multiple disulfide bonds. In the case of heterologous expression of "complex" disulfide-containing peptides, such as those containing the ICK motif, these stresses (e.g., oxidative stress) can have many deleterious consequences, ranging from reduced host growth indices (e.g., growth rate, doubling time, etc.), to exponential increases in process time (and expenditure), and reduced end product quality, bulk biomass (wet cell mass, g / L), and yield. Regarding product quality, if the generation of oxidants is not inhibited, it increases the possibility of protein oxidation, especially the formation of adducts via carbonylation, which may adversely affect the quality of the final product (Yang, et al., 2014 Analytical Chemistry, 86(10), 4799-4806). Protein adducts are covalent modifications resulting from the reaction of nucleophilic and electrophilic sites in proteins, such as the N-terminus or amino acid side chains containing sulfhydryl or amine functional groups. The addition of a carbonyl group to a protein is one example of an adduct. Summary of the Invention

[0005] The present invention addresses such problems. In particular, the present invention provides chimeric proteins (or "chimeras") that significantly alleviate the poor growth (growth rate, number of generations per hour) of transgenic host cells expressing a target polypeptide having at least one disulfide bond, e.g., a target polypeptide that includes an ICK motif. The present invention also provides methods for expressing a target polypeptide having at least one disulfide bond, e.g., a target polypeptide comprising an ICK motif, that result in improved host cell fitness and / or improved target polypeptide yield. Expression systems for producing a target polypeptide having at least one disulfide bond, e.g., a target polypeptide comprising an ICK motif, are also described.

[0006] Summary of the Invention The present invention provides novel chimeric proteins comprising a Bol3 polypeptide operably linked to a lipoyl synthase ("Lip5") polypeptide. The chimeric proteins may include a linker between the Bol3 and Lip5 polypeptides. The linker advantageously ensures flexibility and facilitates separation of the Bol3 domain from the Lip5 domain in the chimeric protein. The present invention further provides a polynucleotide encoding this chimeric protein, a vector incorporating this polynucleotide, and a host cell transformed with this vector.

[0007] In a further aspect, the invention provides a method for expressing a target polypeptide having at least one disulfide bond (e.g., a target polypeptide having at least three disulfide bonds, e.g., a target polypeptide having at least three disulfide bonds in the form of an ICK motif) in a eukaryotic host cell, the method comprising transforming the host cell with a polynucleotide encoding a chimeric protein and culturing the host cell under conditions in which the chimeric protein and the target polypeptide are expressed. It is further noted that the expression of the chimeric protein itself is well tolerated by the host, as its expression alone does not adversely affect cell growth rate. The present invention further provides an expression system for the expression of a target protein of interest, which comprises an expression vector comprising a chimeric protein according to the invention and a cloning site for the insertion of a polynucleotide encoding the target polypeptide of interest. Generally, the target polypeptide has at least one disulfide bond (e.g., the target polypeptide has at least three disulfide bonds, e.g., the target polypeptide has at least three disulfide bonds in the form of an ICK motif). [Brief description of the drawings]

[0008] [Figure 1] A: TAE gel of amplification; and B: Schematic of OE-PCR. [Diagram 2]Plasmid map representation showing multiple cloning sites (MCS) 1 and 2. MCS-1 contains a chimeric open reading frame. [Diagram 3] Box plot of growth rate of chimeric proteins according to the invention (chimera) and control strain (control). The data show that there is no significant difference (i.e., no reduction in fitness) when yeast express the chimera. N=12, one-way ANOVA used for significance, no significance indicated by "NS". [Figure 4] Purification of chimeric proteins according to the invention. A single band at the approximate molecular weight of the chimeric protein was isolated in fractions 3 and 4. 12% SDS-PAGE gel, 20 μL loading volume with 5 μL of PageRuler Prestained Protein Ladder. [Diagram 5] Box plot of the growth rate of the chimeric protein according to the invention (chimera-1) and the control strain (control) under oxidative stress. The data show that expression of the chimera promotes resistance to hydrogen peroxide up to 5 mM. N=6 per condition. One-way ANOVA was used for significance. **=p<0.01, NS.=not significant. [Figure 6] Box plot of the growth rate of the chimeric protein according to the invention (chimera) and the control strain (control) under reducing stress. N=6 per condition, one-way ANOVA used for significance. ***=p<0.001, ns=not significant. [Figure 7] A: Gel image of the evasin gene (SEQ ID NO: 19) (EVA); and B: Gel image of the polypeptide product (SEQ ID NO: 18 (EVA) purified by NiNTA affinity chromatography. [Figure 8] A: Multiple sequence comparison and structure of C8 evasin. Eight evasin variants show the (8) conserved cysteine ​​residues. B: Structure of the C8 evasin family shows the cystine knot (ICK motif). [Figure 9] Box plot showing maximum growth rate of evasin-expressing yeast ("evasin-2") and its rescue by co-expression of chimera (chimera; + evasin-2). Significance was determined using one-way ANOVA. ***=p<0.001, NS.=not significant. [Figure 10] Schematic diagram of peptides showing the location of the cystine knots present in each peptide. Cystine is indicated by a "C" followed by its location in the primary sequence. [Figure 11] Competitive lateral flow assay of polyhistidine tagged polypeptides, Protoxin-1, Psarmotoxin-1 and Evasin-2. The banding patterns indicate successful expression of the desired products. [Figure 12] Box plots showing the effect of other ICK polypeptides (protoxin-1, psalmotoxin-1) on the growth rate of yeast (S. cerevisiae). One-way ANOVA was used for significance. ***=p<0.001, *=p<0.05, NS.=not significant. [Figure 13] Fermentation of Chimera;+EVA. Batch mode, results for each batch (g wet cell mass / L, final OD600 and time to dissolved oxygen setpoint). [Figure 14] Fermentation of EVA (Evasin-2). Batch mode, results for each batch (wet cell mass g / L, final OD600 and time to dissolved oxygen setpoint). [Figure 15] Fermentation of Protoxin-1 co-expressing chimeras. Batch mode, results for each batch (g wet cell mass / L, final OD600 and time to dissolved oxygen setpoint). [Figure 16] Fermentation of Protoxin-1. Batch mode, results for each batch (wet cell mass g / L, final OD600 and time to dissolved oxygen setpoint). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description of the Invention The chimeric proteins, polynucleotides and vectors encoding the chimeric proteins, expression systems and methods of the present invention are described in further detail below. As used herein, the term "and / or" is to be understood as specifically disclosing each of the two specified features or components without regard to the presence or absence of the other. As used herein, the term "comprising" should be interpreted as encompassing both "including" and "consisting of," both meanings being specifically intended and therefore separately disclosed embodiments in accordance with the present invention.

[0010] As used herein, the term "polypeptide" refers to a polymer composed of amino acids linked by peptide bonds, and does not refer to a specific length of the polymer. A "peptide bond" is a covalent bond between one amino acid in which the α-amino group of one amino acid is attached to the α-carboxyl group of the other amino acid. Polypeptides may be modified, for example, glycosylated, amidated, carboxylated, phosphorylated, etc. Modifications may be in vitro or in vivo. Although amino acid chains less than about 100 amino acids in length are generally considered "peptides" in the art, "peptides" and "proteins" are included within the definition of "polypeptide" as used herein. The terms "amino acid sequence" and "polypeptide sequence" are used interchangeably. All amino acid or polypeptide sequences are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) unless otherwise noted. For convenience of nomenclature, this application refers to a "chimeric protein" (or "chimera") and a "target polypeptide having at least one disulfide bond." However, the designation "protein" in the term "chimeric protein" and "polypeptide" in the term "target polypeptide having at least one disulfide bond" is not intended to imply any information regarding the size or relative sizes of the two polymers.

[0011] The present invention particularly relates to the expression of a target polypeptide having at least one disulfide bond. The disulfide bond is formed by the covalent bonding of the thiol groups of two cysteine ​​residues in the polypeptide. Two cysteine ​​residues are required for each disulfide bond. As explained above, the formation of disulfide bonds leads to oxidative stress in the host cell. Optionally, the target polypeptide has two or more disulfide bonds. Optionally, the target protein has three or more disulfide bonds. Optionally, the target protein having at least one disulfide bond has an ICK as defined in more detail below. Optionally, the target polypeptide may contain another cystine motif, such as a cyclic cystine knot or a growth factor cystine knot. "Inhibitor cystine knot" or "ICK" refers to a motif in a polypeptide that contains at least three pairs of cysteine ​​residues that form three separate disulfide bonds. Two of the disulfide bonds form a loop through which a third disulfide bond (connecting the third and sixth cysteines in the sequence) passes to form a knot.

[0012] As used herein, "conservative substitution" when applied to amino acid sequences refers to the replacement of one amino acid residue with another amino acid residue having a side chain with similar physical and chemical properties. For example, conservative substitutions can be made in amino acid residues with hydrophobic side chains (e.g., Met, Ala, VaL, Leu, and Ile), neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), acidic side chains (e.g., Asp and Glu), basic side chains (e.g., His, Lys, and Arg), or aromatic side chains (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions do not cause significant changes in the conformational structure of the protein, and thus may retain the biological activity of the protein. The term "polynucleotide" refers to a nucleic acid, e.g., a polymer of DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric polynucleotide molecule comprising one of these polynucleotides, alone or in combination. The term "nucleic acid" is used interchangeably with the term "polynucleotide."

[0013] The term "vector" as used herein refers to a genetic construct for facilitating the manipulation of a target polynucleotide. A vector may further comprise a gene, such as a marker gene, that allows the selection of the vector in a suitable host cell and under suitable conditions. Expression of the polynucleotide or vector comprises transcription of the polynucleotide into a translatable mRNA. Usually, a vector comprises a regulatory sequence that ensures the initiation of transcription. Other elements responsible for the initiation of transcription, such as regulatory elements, may also be present. A vector also comprises a transcription termination signal downstream of the target polynucleotide.

[0014] When applied to an amino acid sequence (or nucleic acid sequence), "percent sequence identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those of a reference sequence, compared to the amino acid (or nucleic acid) residues in the candidate sequence, after sequence alignment and, if necessary, introducing gaps to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered as identical residues. The percent sequence identity of an amino acid (or nucleic acid) sequence can be determined by aligning the sequences with tools disclosed in the art. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the alignment, for example, by selecting an appropriate algorithm. The percentage identity between two polypeptide sequences can be easily determined by programs such as BLASTp, which is published at http: / / blast.ncbi.nlm.nih.gov.

[0015] An "isolated" material is altered by human beings from its natural state. When an "isolated" substance or component is present in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal is not isolated, but can be considered "isolated" if the polynucleotide or polypeptide is sufficiently separated from coexisting substances in its natural state and exists in a sufficiently pure state. In some embodiments, the polynucleotide or polypeptide is at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (e.g., ion exchange chromatography or reverse phase HPLC). The terms "variant", "homolog" or "derivative", in reference to a nucleotide sequence, include any substitution, variation, modification, substitution, deletion or addition of one (or more) nucleic acid(s) from or to that sequence.

[0016] In a first aspect, the present invention provides a novel chimeric protein comprising a Bol3 polypeptide operably linked to a Lip5 polypeptide. The chimeric protein may comprise a linker between the Bol3 polypeptide and the Lip5 polypeptide. Expression of the chimeric protein reduces oxidative stress in a host cell, and is particularly useful when the host also expresses a target polypeptide having at least one disulfide bond. The term "Bol3" is used to refer to the Bol3 protein of yeast (e.g., S. cerevisiae), but is also used herein to refer to homologs of this protein in other species, in particular eukaryotic (such as the Bol3A homologs of mouse, bovine and human cells) and to homologs of the Bol3 protein of Escherichia coli (E. coli).

[0017] In one embodiment, the Bol3 polypeptide comprises at least 50% sequence identity to SEQ ID NO:1. Optionally, the Bol3 polypeptide has more than 50% sequence identity to SEQ ID NO: 1, e.g., at least 55%, 60%, 65% 70%, 75% 80%, 85% or 90% sequence identity to SEQ ID NO: 1. Optionally, the Bol3 polypeptide has more than 90% sequence identity to SEQ ID NO: 1, e.g., 95% or more, e.g., 98% or more sequence identity to SEQ ID NO: 1. SEQ ID NO: 1 is the sequence of the Bol3 protein of S. cerevisiae. Optionally, the Bol3 polypeptide has more than 50% sequence identity to the protein expressed from SEQ ID NO: 4, e.g., at least 55%, 60%, 65% 70%, 75% 80%, 85% or 90% sequence identity to the protein expressed from SEQ ID NO: 4. Optionally, the Bol3 polypeptide has more than 90% sequence identity to the protein expressed from SEQ ID NO: 4, e.g., 95% or more, e.g., 98% or more sequence identity to the protein expressed from SEQ ID NO: 4. SEQ ID NO: 4 is a polynucleotide sequence encoding the S. cerevisiae Bol3 protein without the native stop codon used in the chimeric proteins described in the Examples.

[0018] The term "Lip5" is used to refer to the Lip5 protein of yeast (e.g., S. cerevisiae), but is also used herein to refer to homologues of this protein in other species, in particular homologues of the Lip5 protein in eukaryotes, plants and E. coli. Optionally, the Lip5 polypeptide comprises at least 50% sequence identity to SEQ ID NO: 2. Optionally, the Lip5 polypeptide has more than 55% sequence identity to SEQ ID NO: 2, such as 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity to SEQ ID NO: 2. Optionally, the Lip5 polypeptide has more than 90% sequence identity to SEQ ID NO: 2, such as 95% or more, such as 98% or more, sequence identity to SEQ ID NO: 2. Optionally, the Lip5 polypeptide comprises at least 50% sequence identity to the protein expressed from SEQ ID NO: 5. Optionally, the Lip5 polypeptide has more than 55% sequence identity to the protein expressed from SEQ ID NO: 5, for example, 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity to the protein expressed from SEQ ID NO: 5. Optionally, the Lip5 polypeptide has more than 90% sequence identity to the protein expressed from SEQ ID NO: 5, for example, 95% or more, for example, 98% or more sequence identity to the protein expressed from SEQ ID NO: 5. SEQ ID NO: 5 is a polynucleotide sequence encoding the Lip5 protein of S. cerevisiae without the native start codon, used in the chimeric proteins described in the Examples.

[0019] In one embodiment, the linker sequence is located between the Bol3 and Lip5 polypeptides. The term "linker" as used herein refers to a group or sequence that allows for linking two parts of a chimeric protein. For example, a linker allows for linking a Bol3 polypeptide with a Lip5 polypeptide. The linker serves to link the two components. The linker according to the present invention may be flexible or rigid, but more preferably allows for some flexibility between the Bol3 and Lip5 parts of the chimeric protein. Suitable linkers are known to those skilled in the art. More specifically, the term "linker" refers to a peptide chain consisting of 1 to 50 amino acids that form a peptide bond, or a derivative thereof, the N-terminus and C-terminus of which form a covalent bond with either the Bol3 domain or the Lip5 domain, respectively, thereby linking the Bol3 domain to the Lip5 domain. Optionally, the linker sequence is a polyhistidine linker. For example, the linker sequence can include 6-20 (e.g., 8-16, e.g., 8-12) histidine residues of a polyhistidine linker, i.e., the linker includes 6-20 consecutive histidine residues to form a polyhistidine linker. Other suitable linkers are known in the art, including FLAG tags, Cys tags, GST tags, etc. Another suitable linker is the N-terminal part of the Cia2 protein (see SEQ ID NO: 28), optionally with additional linking amino acids. SEQ ID NO: 28 shows the N-terminal part of the Cia2 protein, and optionally the linker can include further amino acids, for example the sequence of SEQ ID NO: 29 can be used as a linker. Optionally, a polynucleotide sequence encoding this N-terminal part of the Cia2 protein can be used with additional nucleotides to ensure in-frame cloning. For example, SEQ ID NO: 30 shows a polynucleotide encoding a suitable linker sequence, the Cia2 sequence being encoded by nucleotides 19 to 52 (inclusive). SEQ ID NO: 29 shows the amino acid sequence encoded by SEQ ID NO: 30. Thus, the linker can be a sequence comprising the sequence of SEQ ID NO: 29.

[0020] One embodiment of the present invention is a chimeric protein comprising a first amino acid sequence of Bol3 having at least 50% sequence identity to SEQ ID NO:1 or at least 50% sequence identity to a polypeptide encoded by SEQ ID NO:4, a linker peptide, and a second amino acid sequence of Lip5 having at least 50% sequence identity to SEQ ID NO:2 or at least 50% sequence identity to a polypeptide encoded by SEQ ID NO:5. Optionally, the linker sequence is a polyhistidine linker. For example, the linker sequence may comprise 6 to 20 (e.g. 8 to 16, e.g. 8 to 12) histidine residues in the polyhistidine linker. For example, the linker sequence may be the N-terminal portion of a Cia2 protein. Optionally, the linker may comprise or consist of the sequence of SEQ ID NO:28 or SEQ ID NO:29. Optionally, the sequence identity of the Bol3 polypeptide in the chimeric protein to the polypeptide encoded by SEQ ID NO:1 or SEQ ID NO:4 in the chimeric protein is greater than 50%, for example, at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. Optionally, the Bol3 polypeptide in the chimeric protein has greater than 90% sequence identity to the polypeptide encoded by SEQ ID NO:1 or SEQ ID NO:4. Optionally, the sequence identity of the Lip5 polypeptide in the chimeric protein to the polypeptide encoded by SEQ ID NO:2 or SEQ ID NO:5 in the chimeric protein is greater than 50%, for example, at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%. Optionally, the Lip5 polypeptide in the chimeric protein has greater than 90% sequence identity to the polypeptide encoded by SEQ ID NO:2 or SEQ ID NO:5.

[0021] One embodiment of the present invention is a chimeric protein comprising a first amino acid sequence of Bol3 having at least 95% sequence identity to a polypeptide encoded by SEQ ID NO:1 or SEQ ID NO:4, a linker peptide and a second amino acid sequence of Lip5 having at least 95% sequence identity to a polypeptide encoded by SEQ ID NO:2 or SEQ ID NO:5. Optionally, the linker sequence is a polyhistidine linker. For example, the linker sequence may comprise 6 to 20 (e.g. 8 to 16, e.g. 8 to 12) histidine residues in the polyhistidine linker. Optionally, the linker sequence is the N-terminal portion of the Cia2 protein (see SEQ ID NO:28). Optionally, the linker may comprise or consist of the sequence of SEQ ID NO:28 or SEQ ID NO:29. In some embodiments, the first amino acid sequence (Bol3) has at least 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of a polypeptide encoded by SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, the second amino acid sequence (Lip5) has at least 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of the polypeptide encoded by SEQ ID NO:2 or SEQ ID NO:5. Optionally, the chimeric protein comprises at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3, e.g., at least 75%, 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 3. Optionally, the chimeric protein has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:3.

[0022] In one embodiment of the invention, the chimeric protein comprises the amino acid sequence shown in SEQ ID NO:3. In one embodiment of the invention, the chimeric protein has at least 50% sequence identity to the protein expressed from SEQ ID NO: 31. Optionally, the chimeric protein has more than 55% sequence identity to the protein expressed from SEQ ID NO: 31, such as 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity to the protein expressed from SEQ ID NO: 31. Optionally, the chimeric protein has more than 90% sequence identity to the protein expressed from SEQ ID NO: 31, such as 95% or more, such as 98% or more, sequence identity to the protein expressed from SEQ ID NO: 31. Optionally, the chimeric protein is encoded by SEQ ID NO: 31.

[0023] In a second aspect of the present invention, a polynucleotide encoding the chimeric protein described above is provided. Furthermore, the present invention also encompasses a polynucleotide that specifically hybridizes to the polynucleotide encoding the chimeric protein under stringent conditions. For the purposes of this specification, hybridization under stringent hybridization conditions means maintaining hybridization after washing with 0.1×SSC, 0.5% SDS at a temperature of at least 68° C. as described by Sambrook et al. (Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press). Optionally, the present invention provides an isolated polynucleotide. According to an embodiment of the present invention, the isolated polynucleotide encodes a chimeric protein as described above. Thus, the isolated polynucleotide according to the present invention can be used to encode a chimeric protein that reduces oxidative stress in a host cell.

[0024] It will be understood by those of skill in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it will be understood that those of skill in the art can, using routine techniques, make nucleotide substitutions that do not alter the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed. The polynucleotides of the invention may be composed of DNA or RNA. The polynucleotides may be single-stranded or double-stranded. The polynucleotides may contain synthetic or modified nucleotides. Several different types of modifications to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. For the purposes of the invention described herein, it should be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or life span of the polynucleotide of interest.

[0025] Optionally, a polynucleotide of the invention comprises a sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 4. Optionally, a polynucleotide of the invention comprises a sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity to the nucleotide sequence of SEQ ID NO: 4. Optionally, a polynucleotide of the invention comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4. Optionally, a polynucleotide of the invention comprises a sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 5. Optionally, a polynucleotide of the invention comprises a sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity to the nucleotide sequence of SEQ ID NO: 5. Optionally, a polynucleotide of the invention comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to the nucleotide sequence of SEQ ID NO: 5. Optionally, a polynucleotide of the invention has a nucleotide sequence that expresses a chimeric protein having at least 70% sequence identity to SEQ ID NO: 3. Optionally, a polynucleotide of the invention encodes a polypeptide having more than 70%, such as 75%, 80%, 85%, 90% or even more sequence identity to SEQ ID NO: 3. Optionally, a polynucleotide of the invention encodes a polypeptide having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 3.

[0026] Optionally, a polynucleotide of the invention has at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 32. Optionally, a polynucleotide of the invention comprises a sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity to the nucleotide sequence of SEQ ID NO: 32. Optionally, a polynucleotide of the invention comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to the nucleotide sequence of SEQ ID NO: 32. Optionally, a polynucleotide of the invention encodes a polypeptide comprising the nucleotide sequence of SEQ ID NO:3. Optionally, a polynucleotide of the invention encodes a polypeptide comprising the nucleotide sequence of SEQ ID NO:31.

[0027] In a third aspect, the present invention provides vectors comprising such polynucleotides, in particular expression vectors which express or overexpress said polynucleotides. In the present invention, a "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted to allow expression of the protein. A vector can be used to transform, transduce, or transfect (these terms are used interchangeably herein) a host cell so that the genetic elements carried by the vector are expressed in the host cell. A variety of vectors are available. A vector can include various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, signal sequences, one or more marker genes, selection elements, reporter genes, and transcription termination sequences. In addition, a vector can also include an origin of replication. A vector can also include components that aid in the entry of the vector into a cell, including, but not limited to, a viral particle, a liposome, or a protein shell. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., bacteriophage M12 or M13), animal viruses, and the like. In some embodiments, vector systems include mammalian, bacterial, and yeast systems, including, but not limited to, plasmids such as "pENDO-2" and other vectors available from laboratories or commercially available vectors. Suitable eukaryotic vectors include vectors with 2 micron or centromeric origins of replication. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).

[0028] Thus, the present invention provides an expression vector comprising the above polynucleotide. The expression vector of this embodiment can be prepared by subcloning such a polynucleotide into an expression vector by any conventionally known genetic engineering method. The type of expression vector that can be used in this embodiment is not particularly limited, and examples include any expression vector that is suitable for heterologous gene expression in eukaryotes and can drive the expression of a target polypeptide. For example, a eukaryotic vector having a 2 micron or centromeric origin of replication together with a constitutive promoter / terminator cassette, such as the Tef1 promoter, can be advantageously used. The vector containing the polynucleotide encoding the chimeric protein can be introduced into a host cell for cloning (amplification of the DNA) or gene expression using recombinant techniques well known in the art. In another embodiment, the chimeric protein can be made by homologous recombination methods well known in the art.

[0029] Thus, in a fourth aspect, the present invention provides a host cell comprising a vector or polynucleotide as above. In the present invention, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector is introduced. The amino acid sequence of the fusion protein of the present application can be converted into the corresponding DNA coding sequence using genetic engineering techniques well known in the art. Due to the degeneracy of the genetic code, the transformed DNA sequence may not be completely identical, while the encoded protein sequence remains unchanged. Suitable host cells for cloning or expressing the vectors of the invention are the prokaryotic cells, yeast or higher eukaryotic cells mentioned above. Suitable prokaryotic cells for use in the invention include E. coli (e.g., E. coli DH5α and BL21de3).

[0030] In one embodiment, eukaryotic host cells are used for cloning or expression of vectors encoding chimeric proteins. Saccharomyces cerevisiae (S288C) or baker's yeast is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are common and suitable for use in the present invention, such as other members of the Saccharomyces clade (including S. pastorianus, S. eubayanus and S. paradoxus), Komagataella (including K. pastoris), Kluyveromyces (including K. lactis) and Yarrowia (including Y. lipolytica). In another aspect of the invention, the invention provides a recombinant cell or recombinant microorganism comprising a polynucleotide or vector as described above. Thus, a recombinant cell or recombinant microorganism according to the invention may express the chimeric protein of the invention. The invention further relates to a recombinant host cell comprising a polynucleotide, or vector as described above. The polynucleotide or vector of the invention present in the host cell may be integrated into the genome of the host cell or may be maintained extrachromosomally. Once the polynucleotide or vector has been integrated into a suitable "host cell", the host cell is maintained under conditions suitable for high level expression of the polynucleotide or vector.

[0031] Transformed host cells can be propagated according to methodologies known in the art to achieve cell growth. Optionally, once expressed, the chimeric protein can be purified according to standard procedures in the art, including affinity columns, column chromatography such as size exclusion chromatography (SEC), gel electrophoresis, ammonium sulfate precipitation, and the like. The chimeric protein of the invention can then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of the chimeric protein can be by any conventional means, including, for example, preparative chromatographic separation.

[0032] Host cells are transformed with the above-described expression or cloning vectors capable of producing the chimeric protein and then cultured in conventional nutrient media appropriate for inducing the promoter, selecting transformed cells, or amplifying the gene encoding the modified target sequence. The host cells used to produce the chimeric protein in the present invention can be cultured in a variety of media known in the art. The media may also contain other necessary additives known in the art at appropriate concentrations. The conditions of the media, such as temperature, pH, etc., are previously selected for the expression of the host cells and are well known to those skilled in the art.

[0033] The present invention further provides a method for producing a chimeric protein as described above, which method comprises the steps of: suitably culturing a recombinant host cell and expressing a polynucleotide encoding the chimeric protein or a vector encoding the chimeric protein. In a fifth aspect, the present invention provides a method for producing a target polypeptide having at least one disulfide bond in a host cell, said method comprising culturing the host cell under conditions suitable for expression of the target polypeptide and concomitantly expression of a chimeric protein.

[0034] The target polypeptide can be a natural or synthetic polypeptide. Optionally, the target polypeptide has two or more disulfide bonds to create its desired 3D structure, for example, 2, 3, 4 or 5 disulfide bonds. Optionally, these disulfide bonds are formed between non-adjacent cysteine ​​residues, i.e., the disulfide bonds form a complex 3D configuration of a "knot" in the target polypeptide. Optionally, the target polypeptide includes an "inhibitor cystine knot" or "ICK". Optionally, the target polypeptide is a toxin polypeptide, e.g., from the "evasin" family of salivary peptides. Optionally, the target polypeptide has at least 90% sequence identity to any one of SEQ ID NOs:6-13. Optionally, the target polypeptide has at least 90% sequence identity to SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

[0035] Optionally, the target polypeptide is a toxin polypeptide, for example from the "protoxin" of the wolf spider (Alopecosa marikovskyi) (encoded by SEQ ID NO:20, or SEQ ID NO:21) and / or the "psalmotoxin-1" of the Trinidad chevron tarantula (Psalmopoeus cambridgei) (UniProt ID: TXP1_PSACA) (encoded by SEQ ID NO:22, or SEQ ID NO:23). Alternatively, the target polypeptide can be, for example, Limulus polyphemus factors "C" and "B" and / or Tachypleus tridentatus "coagulogen-1" (UniProt ID: COAG_TACTR), or Megathura crenulata "hemocyanin-1" (UniProt ID: HCY1_MEGCR) and Megathura crenulata "hemocyanin-2" (UniProt ID: HCY1_MEGCR). ID:HCY2_MEGCR), as well as other polypeptides of interest having disulfide bonds, such as bovine serum albumin, BSA, UniProtKB-P02769 (ALBU_BOVIN), human serum albumin, HSA, UniProt-B-P02768 (ALBU_HUMAN), human insulin (including human insulin analogs and human insulin mimetic peptides) UniProtKB-P01308 (INS_HUMAN), human erythropoietin UniProtKB-P01588 (EPO_HUMAN) or human granulocyte-macrophage colony-stimulating factor UniProtKB-P04141 (CSF2_HUMAN). Optionally, the target polypeptide may be an antibody, such as a monoclonal antibody, a humanized antibody or an antibody fragment. Optionally, the target polypeptide can be a glycoprotein, such as a glycoprotein having a secretory sequence (ie, a glycoprotein that is secreted from a host cell).

[0036] A host cell can be genetically engineered to express said target polypeptide. For example, a host cell can be transformed with an expression vector comprising a polynucleotide sequence encoding the target polypeptide. The expression vector can be integrated into the genome of the host cell or maintained extrachromosomally. Once the vector is introduced into a suitable "host cell", the host cell is maintained under conditions suitable for high level expression of the target polypeptide. The host cell can be genetically engineered to express the target polypeptide and then further genetically engineered to express the chimeric protein, or vice versa. Optionally, the host cell naturally expresses the target polypeptide and is only transformed to express the chimeric protein as described above. Optionally, a single vector can be formed that contains polynucleotides encoding both the chimeric protein and the target polypeptide, and then a host cell is simply transformed with this vector that is capable of expressing both the target polypeptide and the chimeric protein, optionally under the control of the same promoter / inducer / enhancer.

[0037] In a further aspect, the present invention provides an expression vector comprising a cloning site for the insertion of a polynucleotide encoding a chimeric protein as described above and a polynucleotide encoding a target polypeptide. The cloning site may be defined by suitable restriction sites that allow easy insertion of the polynucleotide encoding the target polypeptide. For example, the expression vector may comprise a multiple cloning site with up to 20 different restriction sites to facilitate easy insertion of different constructs of the target polypeptide.

[0038] In yet a further aspect, the present invention provides an expression system for expressing a target polypeptide of interest in a host cell, said system comprising an expression vector comprising a polynucleotide encoding a chimeric protein as described above and a vector comprising a cloning site for the insertion of a polynucleotide encoding a target polypeptide. Optionally, the cloning site can be provided on the vector encoding the chimeric protein of the present invention. Alternatively, a cloning site for the expression of the target polypeptide can be provided on another vector. The cloning site can be defined by suitable restriction sites that allow easy insertion of the polynucleotide encoding the target polypeptide. For example, the expression vector can comprise a multiple cloning site with up to 20 different restriction sites to facilitate easy insertion of different constructs of the target polypeptide.

[0039] The target polypeptide can be a natural or synthetic polypeptide. Optionally, the target polypeptide has at least one disulfide bond, and may contain more than one disulfide bond to create its desired 3D structure, for example, 2, 3, 4 or 5 disulfide bonds. Optionally, the disulfide bonds are formed between non-adjacent cysteine ​​residues, i.e., the disulfide bonds form a complex 3D configuration of a "knot" in the target polypeptide. Optionally, the target polypeptide includes an "inhibitor cystine knot" or "ICK". Optionally, the target polypeptide is a toxin polypeptide, e.g., from the "evasin" family of salivary peptides. Optionally, the target polypeptide has at least 90% sequence identity to any one of SEQ ID NOs:6-13. Optionally, the target polypeptide has at least 90% sequence identity to SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

[0040] Optionally, the target polypeptide is a toxin polypeptide, such as from the "protoxin" of the wolf spider (Alopecosa marikovskyi) (see SEQ ID NOs: 20 and 21) and / or the "psalmotoxin-1" of the Trinidad chevron tarantula (Psalmopoeus cambridgei) (UniProt ID: TXP1_PSACA) (see SEQ ID NOs: 22 and 23). Optionally, the target polypeptide is from factor "C" or "B" of the American horseshoe crab (Limulus polyphemus), "coagulogen-1" of the horseshoe crab (Tachypleus tridentatus) (UniProt ID: COAG_TACTR), or "hemocyanin-1" of the giant keyhole limpet (Megathura crenulata) (UniProt ID: HCY1_MEGCR) and "hemocyanin-2" of the giant keyhole limpet (Megathura crenulata) (UniProt ID: HCY1_MEGCR). ID:HCY2_MEGCR), bovine serum albumin (BSA, UniProtKB-P02769 (ALBU_BOVIN), human serum albumin (has) UniProt-B-P02768 (ALBU_HUMAN), human insulin UniProtKB-P01308 (INS_HUMAN), human erythropoietin UniProtKB-P01588 (EPO_HUMAN), or human granulocyte-macrophage colony-stimulating factor UniProtKB-P04141 (CSF2_HUMAN).

[0041] Optionally, the target polypeptide may be an antibody, such as a monoclonal antibody, a humanized antibody or an antibody fragment. Optionally, the target polypeptide can be a glycoprotein, such as a glycoprotein having a secretory sequence (ie, a glycoprotein that is secreted from a host cell). Optionally, once expressed, the target protein can be purified according to standard procedures in the art, including affinity columns, column chromatography such as size exclusion chromatography (SEC), gel electrophoresis, ammonium sulfate precipitation, and the like. The target polypeptide can then be isolated from the growth medium, cell lysate, or cell membrane fraction. Isolation and purification of the target polypeptide can be by any conventional means, including, for example, preparative chromatographic separation.

[0042] All documents mentioned herein are incorporated by reference. Any modifications and / or variations to the described embodiments that would be apparent to one of ordinary skill in the art are also encompassed herein. Although the invention has been described herein with reference to certain specific embodiments and examples, it should be understood that the invention is not intended to be unduly limited to these specific embodiments or examples. Preferred or alternative features of each aspect or embodiment of the invention apply mutatis mutandis to each other aspect or embodiment of the invention (unless the context requires otherwise). EXAMPLES

[0043] method Strains, culture conditions and materials Oligonucleotides and sequences for this study were first designed using in silico cloning software with reference to the Saccharomyces genome database and then purchased from ThermoFisher custom oligo order service. Templates for PCR were purified from fresh overnight cultures of Saccharomyces cerevisiae (BY4741, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) by digestion with 20mg / mL Lyticase (Sigma Aldrich, UK) in digital dry block (ThermoFisher, UK) at +37°C followed by a New England Biolabs Total Genomic Spin Prep Kit (Monarch, New England Biolabs, UK). For subcloning, 10μL of electrocompetent Escherichia coli (DH5α) cells (New England Biolabs, UK) were routinely used and plasmid selection was performed under positive antibiotic selection in Luria Bertani (LB) medium supplemented with 100μg / mL ampicillin. Transformation of E. coli was performed according to the manufacturer's instructions and transformants were incubated statically at +37°C for at least 16 h. All aliquots and buffers were briefly centrifuged at maximum speed (15,500 rcf) for at least 60 s before use. Creation of fusion ORF Two methodologies were used to generate the fusion ORFs; the first methodology used was adapted from Hilgarth & Lanigan, (2020), MethodsX, 7 (October 2019), 100759. The latter construct used conventional restriction enzyme cloning.

[0044] Stage 1: High Fidelity DNA polymerase for PCR was used as a master mix (Hot Start Q5 High Fidelity, NEB UK) containing 4 mM MgCl2 and 2 mM dNTP mix (2x). PCRs were typically performed in 50 μL in clean thin-walled 0.2 mL tubes (ThermoFisher, UK), prepared on ice and mixed thoroughly by pulse vortexing at maximum speed (Stuart, UK). Thermocycling was performed using a 24-well Prime 3 The reaction was run for 2 hours and 30 minutes in a thermocycler (Techne, UK) with a preheated lid (+105°C). Typical thermocycling conditions for stage 1 and stage 3 of OE-PCR were an initial denaturation at +98°C for 10 seconds, 35 cycles of +98°C for 10 seconds, +60°C for 60 seconds, followed by an extension step of +72°C for 2 minutes. A total of 35 cycles were performed. Finally, a final extension step of +72°C for 10 minutes was performed. The reaction was then held at +20°C. After thermocycling, the amplicons were briefly reconstituted by pulse centrifugation at 15,000 rcf for approximately 10 seconds. After this, the reaction was checked by TAE gel electrophoresis using 0.7% w / v agarose (FisherSci, UK) and 0.05% v / v EtBr (Sigma-Aldrich, UK) as DNA intercalators. The gel was run at 150V, 400mA for 30 minutes using a small gel tank (Alpha labs, UK) and an electrophoresis power pack (FisherSci, UK). After completion, the gel was carefully visualized under blue light (proBLUEView, Alpha Labs, UK). To determine the approximate molecular weight of the amplicons, 10 μL of GeneRuler 1kb (ThermoFisher, UK) was used as a standard. As a result of PCR, two single bands of approximately 350 bps and 1200 bps were obtained, which were consistent with the reference molecular weights of Bol3 and Lip5, respectively (Saccharomyces genome database, yeastgenome.org). The gel fragments were then excised with a clean scalpel and purified by a commercially available spin column protocol (GeneJet Gel Extraction Kit, ThermoFisher, UK) according to the manufacturer's instructions.

[0045] Stage 2: The second stage fused both ORFs using the built-in complementarity between each amplicon. This was achieved by adding a 30-mer polyhistidine (10x) sequence with a melting temperature (+68°C) slightly higher than that of the annealing sequence. This region formed a "linker" between each ORF. The histidine codons were staggered to avoid tRNA depletion. Unlike stage 1, a touchdown PCR protocol was used in this stage to obtain higher sensitivity to the polyhistidine linker region. The templates for the touchdown PCR consisted of equimolar (1:1) concentrations (ng / μL) of the amplicons generated in stage 1. Calculations were performed using a ligation calculator, and the shorter sequence was considered as the "insert". The PCR was performed again using a pre-heated lid to reduce evaporation. Thermocycling consisted of 9 cycles of denaturation at +95°C for 30 s, followed by annealing at +72°C for 15 s for 3 min, decreasing the annealing temperature by 0.5°C after each cycle. This was followed by 5 cycles of denaturation at +95°C for 30 s, followed by annealing at +67.5°C for 30 s, followed by an extension step at +68°C for 3 min for 30 s. Finally, an extension step at +68°C for 10 min was performed.

[0046] Stage 3: In the final stage of OE-PCR, the unpurified PCR product from the second stage was used as a template. Thermocycling was performed with the same program as in stage 1, except that different oligonucleotides were used. Here, oligonucleotides against the 5' (forward) and 3' (reverse) of the first and second ORFs were used to amplify only the fusion sequence generated in stage 2. The oligonucleotides used are shown in Table 1 below. As described above, TAE gel electrophoresis was performed to confirm the success of the fusion reaction. Here, a single band representing the combined molecular weight of both ORFs was detected. The gel slice was then excised and the DNA was purified using a commercial gel extraction kit as described above. [Table 1]

[0047] Restriction enzyme digestion and subsequent cloning Restriction enzymes (Xba I, Not I and Sac II) were obtained from New England Biolabs CutSmart range (New England Biolabs, UK) and digestions (50 μL) were performed in 1× CutSmart buffer for 2 h at +37°C in a digital dry batch according to the manufacturer's instructions. After digestion, any condensation was removed by pulse centrifugation at maximum speed (15,500 rcf) in a benchtop centrifuge (SciQuip, UK). DNA ligations (20 μL) were similarly performed using the Quick Ligation Kit (New England Biolabs, UK) according to the manufacturer's instructions at a 5:1 insert to vector molar ratio (standardized at 27 fmol for vector ratio 1). DNA (ng / μL) was routinely quantified using a Spectro (260–700 nm) UV / Vis spectrophotometer (SpectroStar Nano, BMG Labtech, UK). After incubation, 2 μL of the ligation reaction was transformed into electrocompetent DH5α E. coli cells on ice. After at least 16 hours of incubation at +37°C (to allow colony growth), individual colonies were analyzed for successful ligation by diagnostic digestion and colony PCR. Approximately 20 colonies were screened per ligation in a final reaction volume of 20 μL. The protocol was repeated as above for stage 3, except that one colony (marked) was used per reaction. Successful amplifications were reverted to individual colonies and plasmids were purified with a commercial MiniPrep kit (GeneJet MiniPrep Kit, ThermoFisher). After purification, the plasmid eluate was labeled and stored at -20°C.

[0048] Rapid Yeast Transformation Introduction of the newly constructed plasmids into the S. cerevisiae host was performed using an overnight culture of BY4741 and pre-dried uracil drop-out plates (Kaiser minimal drop-out media, Formedium, UK). Prior to the reaction, 1 mL of 1 mg / mL single-stranded DNA (Ultrapure Salmon sperm, Sigma Aldrich, UK) was boiled at +95°C for 10 min and immediately placed on ice. Transformation was performed using 240 μL of 50% w / v polyethylene glycol (PEG 2000) and 100 μL of PEG 2000. 4000 The reaction mixture consisted of 36 μL of 1 M lithium acetate (Sigma Aldrich, UK), 10 μL of freshly boiled single-stranded carrier DNA, 7.2 μL of 5 M DTT (Melford, UK), 2 μL of plasmid and finally 69.5 μL of sterile Milli-Q water. All solutions and buffers were sterilized by autoclaving before use. After assembling the reaction mixtures, they were thoroughly vortexed at maximum speed for at least 1 min per transformation, incubated at room temperature for 20 min, and then heat shocked at +42°C for an additional 20 min. After this, the reaction mixtures were pelleted by gentle centrifugation at 2000 r.cf for 2 min, gently resuspended in 200 μL of sterile deionized water, and plated onto pre-dried dropout plates. Plates were sealed and incubated at +30°C for 4 days after which colonies appeared.

[0049] High-resolution growth rate analysis Yeast strains were incubated overnight (at least 16 hours) in 10 mL of defined synthetic Kaiser dropout medium (uracil dropout, Formedium) at +30°C and 175 rpm. After incubation, the density of each culture was measured by spectroscopy (SpectroStar Nano, BMG Labtech, UK) at 600 nm (OD ) in a 1 mL cuvette (BMG Labtech, UK). 600nmCultures were then loaded onto an OT-2 liquid handling robot (Opentrons, USA) and plated in sterile flat-bottom 96-well plates (360 μL well volume, Greiner CELLSTAR® 96-well plates, Sigma Aldrich UK) at optical density (OD 600nm ) was diluted back to 0.1. Growth of each strain was then continuously monitored until it reached saturation, at which point the experiment was terminated and data collected.

[0050] Batch fermentation Fermentations were performed using cultures of recombinant yeast grown overnight in defined synthetic uracil dropout medium (Kaiser, Formedium, UK). Typically, a working volume of 100 mL (total volume 250 mL) was used, consisting of 0.67 g of Yeast Nitrogen Base without amino acids (Formedium, UK), 0.19 g of relevant amino acid supplement, and 2 g of anhydrous D-glucose (Melford, UK). The reactor (MiniBio total volume 250 mL, Applikon Biotechnology, NL) was assembled and its contents sterilized in a Prestige Medical Classic autoclave (+121 °C, 104 kPa, 30 min). After this, the reactor was connected to a MiniBio fermentation control system (Applikon Biotechnology, NL), tubing (alkali, air) was connected, and probes (pH and dissolved oxygen) were left to polarize overnight at room temperature. This step was also used as a sterility control. Overnight cultures (5 mL) of yeast were prepared in the appropriate dropout medium and incubated for 16 h at +30°C, 175 rpm as described above. Meanwhile, the probes were calibrated as follows: Dissolved oxygen (DO2) was calibrated to read 100% DO2 in uninoculated medium (approximately 70 nA at +30°C). pH was calibrated with pH 4.0 and pH 7.0 standards (20 mL) (Sigma, UK). The following morning, strains were assayed for OD 600nmThe culture was subcultured (5 mL) until the OD reached 0.2 and then incubated again at 175 rpm and +30°C for 4 hours. After this time, the bioreactor was inoculated with the previously calculated inoculum volume (mL) until the OD reached 0.1 (OD 600nm ) and fermentations were monitored using Lucullus Process Information Management Software (SecureCell, CH). Setpoints were 35% DO2 ± 5%, pH 5.0 ± 0.5 and 1 vvm sparging with compressed air (Bambi PT5 UK). Total fermentation time was typically 20 h.

[0051] Downstream Processing After fermentation, all samples (approximately 120 mL, OD 600nm Approximately 35–55% of the culture medium was extracted and decanted into two 50 mL Falcon tubes (ThermoFisher, UK). 15 mL of the culture was then pelleted by centrifugation at 15,500 rcf for 10 min, weighed (g wet cell mass / L), and dissolved in Yeast Protein Extraction Reagent (YPER, Pierce, UK) according to the manufacturer's instructions. Chemical cell lysis (protein extraction) Lysis of samples was performed as described above under "Downstream processing". Cell pellets were placed in an appropriate volume of YPER (following manufacturer's instructions) and agitated at 1800 rpm (Stuart Vortex, UK) for 20 min at room temperature with Pierce Protease Inhibitor Tablets (Thermo Scientific, UK). Samples were then clarified by centrifugation to remove insoluble debris and the supernatant was aspirated into a clean 1.5 mL Eppendorf tube (Eppendorf, UK) for further analysis.

[0052] Affinity purification Immobilized metal affinity chromatography (IMAC) was performed to confirm the expression of the 60 kDa fusion protein. The individual molecular weights of the Bol3 and Lip5 polypeptides are 13 and 46 kDa, respectively (Source: Saccharomyces Genome database, yeastgenome.org). For purification, 1 mL of HisPur Nickel chromatography resin (Sigma, UK) was dispensed into an empty PD-10 (10 mL, Sigma-Aldrich, UK) column and equilibrated with one column volume of denaturing binding buffer (8 M urea, 10 mM imidazole, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 20% v / v glycerol pH 7.4). To this, the sample prepared in "Chemical cell lysis (protein extraction)" was carefully added by pipette and allowed to flow out by gravity. The flow-through was collected and labeled "RT" for further analysis. Washing was performed with (8 M urea, 50 mM imidazole, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 20% v / v glycerol, pH 7.4). Protein was eluted with elution buffer B (8 M urea, 50 mM imidazole, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 20% v / v glycerol, pH 7.4) and collected in 15 × 1 mL fractions (1.5 mL Eppendorf tubes). All samples were kept on ice during purification. Protein concentration (mg / mL) was quantified using UV / Vis spectroscopy at 280 nm (SPECTROstar Nano, UK) with LVis plates (BMG, UK).

[0053] Detection of recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Fractions from the affinity purification steps described above were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for 10 min and 180 V, 200 mA for 50 min. Polyacrylamide gels were prepared according to the Laemmli stacking method using a 10% v / v resolving gel (Surecast, Resolving Buffer pH 8.8, Thermo Fisher, UK) and a 4% v / v stacking gel (Surecast Stacking Buffer, pH 6.8, Thermo Fisher, UK), and polymerization was induced with 50 μL of 10% w / v ammonium persulfate (Sigma, UK) and 5 μL of 100% v / v tetramethylethylenediamine (TEMED, Melford, UK). Samples were denatured in 4x Laemmli reducing buffer, diluted to working concentration (1x) in a final volume of 20 μL, and heated to +100°C for 5 min in a thermocycler (Prime3, Techne, UK). 20 μL of diluted (1:20) sample per sample was added to the wells along with 5 μL of PageRuler prestained molecular weight marker (ThermoFisher, UK). After electrophoresis, gels were removed and stained with SimplyBlue protein stain (Invitrogen, UK) according to the manufacturer's instructions. After staining, gels were documented using white light transillumination (proBLUE View, Cleaver Scientific).

[0054] Competitive lateral flow assay for polyhistidine-tagged polypeptides Expression of recombinant polyhistidine-tagged polypeptides was confirmed using the Pro-Detect™ Rapid His competitive assay kit (Thermo Scientific, UK) according to the manufacturer's instructions. 4. Data Analysis Data analysis Analysis and graphing were usually performed in RStudio (RStudio, V4.0.01) and then visualized in Adobe Illustrator CC (2020). Growth rates were calculated using the “GrowthCurver” script available at https: / / cran.rproject.org / web / packages / growthcurver / vignettes / Growthcurver-vignette.html. In general, we used the Graphics Grammar (ggplot2) available at https: / / ggplot2.tidyverse.org / for graphic design. Fermentation data were recorded and visualized using Lucullus Process Information Management software (Applikon, Getinge, NL and SecureCell, CH).

[0055] result Example 1: Episomal expression of recombinant Bol3-Lip5 chimeras Using overlap extension PCR, the two open reading frames of bol3 and lip5 were fused into a single fusion open reading frame that lacked the natural stop and start codons of bol3 and lip5, respectively (Figure 1B), resulting in a single fusion transcript. Upon amplification, the final chimeric amplicon migrated with an approximate molecular mass of 1632 bps, which was consistent with the expected mass of both bol3 and lip5 together (Figure 1A). The amplicon was then gel excised, digested, ligated to a centromeric yeast expression construct, and transformed into chemically competent DH5α E. coli according to the manufacturer's instructions (New England Biolabs, UK). The resulting construct (Figure 2) was then transformed into Saccharomyces cerevisiae (BY4741) using 100 mM DTT and a modified LiAC / PEG method (Gietz, et al., 2002 Methods in Enzymology, Volume 350, pp. 87-96). Due to the presence of a polyhistidine motif that serves to link both ORFs, it was possible to purify the fusion polypeptide from the yeast lysate by nickel column chromatography. This yielded a single polypeptide that separated at an approximate molecular weight of about 60 kDa, which corresponds to the combined molecular weight of both Bol3 (13 kDa) and Lip5 (46 kDa) polypeptides (Figure 4).

[0056] Expression of the chimeric protein does not adversely affect the yeast's growth rate, helping to alleviate pressure from environmental stimuli. High-resolution growth rate analysis showed that the cellular fitness of the newly generated chimera-expressing yeast strains was statistically indistinguishable (CI = 95%) from the control (Figure 3). These data suggest that the chimera is well tolerated in the yeast system. Further experiments under sublethal to lethal oxidizing conditions (hydrogen peroxide) showed that chimeric expression conferred a significant degree of protection up to peroxide concentrations of 10 mM (Figure 5). Importantly, this finding was specific to the chimeric-expressing yeast. In contrast, experiments under reducing conditions demonstrated that expression of the chimera was more sensitive to the disulfide bond disrupting agent dithiothreitol (DTT) than the control (FIG. 6).

[0057] Example 2: Expression of recombinant toxin-derived peptide EVA The synthetic polypeptide "EVA" (SEQ ID NO: 18) is derived from a secreted protein of the tick "evasin" family of bioactive salivary peptides (Figure 8) (Hayward et al., 2017, supra). In the host organism (including Amblyomma cajennense, "Cajun tick"), evasins are secreted from the salivary glands and promote parasite survival by targeting and sequestering CXXC and CXC chemokines and cytokines released by the host to eliminate the parasite (Denisov et al., 2019, Journal of Biological Chemistry, 294(33), 12370-12379). By sequestering these molecules, the tick effectively weakens the host's defenses (chemotaxis of immune cells) and thus prolongs the feeding and parasitic life cycle (Denisov et al., 2019, supra). Recently, this mechanism has attracted attention as a potential therapeutic agent for treating (calming) otherwise deadly “cytokine storms” associated with both viruses and other pathologies (Darlot et al., 2020, The Journal of Biological Chemistry, 295(32), 10926-10939). After determining that the yeast cultures contained the recombinant EVA gene (SEQ ID NO: 19), as shown in Figure 7A, protein expression was confirmed using nickel chromatography and SDS-PAGE. The results are shown in Figure 7B, which shows that a strong band was isolated that corresponds approximately to the predicted molecular weight of EVA (approximately 30 kDa). An antibody-based assay was used to confirm the presence of a polyhistidine-tagged polypeptide within the fractions (Figure 11).

[0058] Example 3: EVA-bearing yeast exhibits a significant temperature-dependent decrease in growth rate compared to controls High-resolution growth rate analysis at both +30 and +32 degrees Celsius showed that expression of EVA resulted in a significant decrease (p<0.001) in growth rate compared to controls (wild type and empty vector) (Figure 9). These data (summarized in Table 2) showed that expression of EVA resulted in approximately a 60 and 40% decrease in relative growth rate compared to wild type and empty vector controls, respectively. This was exacerbated by increasing the incubation temperature to +32°C, demonstrating linearity. [Table 2]

[0059] Example 4: Rescue of reduced EVA proliferation rate by enhanced mitochondrial antioxidant system Co-expression of the chimera with EVA significantly rescues the EVA-dependent decrease in proliferation rate Being a potent antioxidant and a key regulator of metabolism via lipoylation of both PDH and aKDH enzymes, we hypothesized that overproduction of lipoic acid might increase resistance to oxidative stress in cells. Cell proliferation rate was used as a measure of cellular fitness under various oxidative conditions (Figure 5). As shown above, expression of the mite polypeptide analog EVA resulted in a significant (approximately 60% of wild type and 40% of control) decrease in cellular fitness (growth rate). EVA and the chimeric protein were co-expressed with the hypothesis that an enhanced antioxidant system might serve to rescue the observed phenotype.

[0060] Table 3 details the experimental design of the growth rate rescue experiment. A total of four independent yeast strains were compared for growth rate. A "control" strain carrying an "empty" plasmid was used to examine the background metabolic effects of maintaining a low-copy (centromeric) plasmid in which no recombinant protein was expressed. All yeasts had the same genetic background. [Table 3]

[0061] The data from these experiments are summarized in FIG. [Table 4]

[0062] The data in Figure 9 and Table 4 also show that when rescued, proliferation of chimera;+EVA cultures was not statistically significant compared to control (+empty vector, or non-EVA expressing) cultures, suggesting that expression of the chimera was sufficient by itself to preserve cell fitness. These experiments also showed that expression of the chimera reversed the temperature-dependent decrease in growth rate in EVA-expressing yeast.

[0063] Co-expression of the chimeric fusion with two other ICK-peptides, Protoxin-1 and Psarmotoxin-1, elicits a response similar to EVA As the data showed that the chimeric strains could significantly rescue the growth of one ICK polypeptide, we further investigated whether they could rescue two ICK expressing strains. Expression constructs for the venom peptides "protoxin" (UniProt ID: TXPR1_ALOMR) (see SEQ ID NOs: 20 and 21) from the wolf spider Alopecosa marikovskyi and "psalmotoxin-1" (UniProt ID: TXP1_PSACA) (see SEQ ID NOs: 22 and 23) from the Trinidad chevron tarantula Psalmopoeus cambridgei were transformed into yeast cultures and their growth rates were monitored as described above. Both peptides have well-documented therapeutic potential as analgesics or antimalarials and, together with other ICK peptides, contain four and three disulfide bonds, respectively. A schematic representation of each peptide is shown in Figure 10. Expression of both polypeptides was confirmed by antibody-based assays (Figure 11).

[0064] A total of four independent yeast strains were compared for growth rate (see Table 5). A "control" strain carrying an "empty" plasmid was used to examine the background metabolic effects of maintaining a low-copy (centromeric) plasmid in which no recombinant protein was expressed. All yeasts had a BY4741 background. [Table 5] [Table 6]

[0065] The data presented in Table 6 above (and shown graphically in FIG. 12) again show that co-expression of the chimeric fusions was sufficient to restore the growth rate of cells expressing either of the ICK peptides. Compared to the expression of evasin (EVA), there was a greater variation within these data sets. This was especially true for the growth of psalmotoxin-1 expressing yeast (psalmotoxin-1), possibly reflecting the tolerance to the relatively uncomplex (in terms of disulfides) polypeptide. Taken in combination (Figs. 10 and 12), the number of disulfides (SS) present in each toxin peptide (Table 6) appears to predict how well yeast cultures will (or will not) respond (growth rate) to their expression. This effect appears to be independent of the molecular weight of each peptide (Table 5). Higher numbers of disulfides (evasin and protoxin, four) caused a greater reduction in growth rate than lower numbers (psalmotoxin, three). Similarly, the chimera-dependent rescue of growth rate mirrored this finding, with a stronger rescue seen with higher molecular weight (26 kDa) and higher number of disulfides (four) (Fig. 10 and Table 6).

[0066] Pilot-scale 100 mL fermentations were then performed to determine whether the growth findings described above (i.e., protoxins and evasins) could be transferred to a commercially suitable batch fermentation system. These data are shown in Figures 13-16. In four independent batch fermentations, expression of the chimeras increased significantly with increasing culture (optical) density (OD 600 ) and wet cell mass, as well as final yield. Of particular note is that these batch fermentations are consistent with the growth measurements obtained in Figures 9 and 12, i.e., chimeric expression favors higher molecular weight evasins over protoxin-1 in the yeast system. We speculate that recombinant expression of disulfide-rich ICK increases the flux of oxidative protein folding pathways, resulting in increased production of radical species. Since each disulfide bond forms a stoichiometric amount of radical oxygen species through Ero1-dependent oxidation of cysteine ​​thiols (Tyo et al., 2012, BMC Biology, 10.), we hypothesized that heterologous expression of ICK peptides might also result in "increased" production of oxidants, placing a greater burden on the protein folding machinery (including the UPR), with disastrous consequences for the rate of collection and final product yield. This is evidenced by the difference in growth rates of evasin, protoxin-1 and psalmotoxin, where the peptide with the lowest number of disulfides (psalmotoxin, disulfide n=3) appears to be better tolerated (has less impact on growth rate) than evasin or protoxin-1 (four disulfides each).

[0067] We were able to demonstrate that by co-expressing modified versions of key antioxidant pathways via chimeras, ICK-expressing yeast no longer exhibited slower growth rates and their fitness appeared to be restored. We suggest that this was due to an indirect antioxidant "buffering" effect of the expression of the chimeric proteins. This would have the effect of preventing radicals from damaging key biomolecules (nucleic acids, lipids, proteins, etc.) and compromising cellular fitness, allowing the yeast host to tolerate the folding "cost" of recombinant ICK.

Claims

1. A chimeric protein comprising a Bol3 polypeptide operably linked to a Lip5 polypeptide, wherein the Bol3 polypeptide has at least 50% sequence identity to the amino acid sequence of SEQ ID NO: 1, and the Lip5 polypeptide has at least 50% sequence identity to the amino acid sequence of SEQ ID NO:

2.

2. The chimeric protein of claim 1, wherein the chimeric protein comprises a linker sequence between the Bol3 polypeptide and the Lip5 polypeptide, and the linker sequence may comprise a polyhistidine peptide, and the linker sequence may comprise 6 to 20 consecutive histidine residues in the polyhistidine linker.

3. The chimeric protein of claim 1, wherein the protein has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

3.

4. The chimeric protein of claim 2, wherein the linker sequence comprises a Cia2 sequence, and the linker sequence may comprise the amino acid sequence of SEQ ID NO: 28, or the linker sequence may comprise the amino acid sequence of SEQ ID NO:

29.

5. The chimeric protein of claim 4, wherein the chimeric protein has at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

31.

6. The chimeric protein of claim 1 expressed from the polynucleotide of SEQ ID NO:

32.

7. 2. A polynucleotide encoding the chimeric protein of claim 1, wherein the polynucleotide may comprise a sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 4 and / or a sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO:

5.

8. 8. The polynucleotide of claim 7, which encodes a polypeptide having a sequence that has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3, or has at least 70% sequence identity to SEQ ID NO:

32.

9. A vector comprising the polynucleotide of claim 7.

10. A host cell transformed with the polynucleotide of claim 7.

11. A host cell transformed with the vector described in claim 9.

12. 10. An expression system for expressing a target polypeptide in a host cell, comprising the expression vector of claim 9, wherein the vector also comprises a cloning site for insertion of a polynucleotide encoding the target polypeptide, wherein the target polypeptide may have at least one disulfide bond, wherein the target polypeptide may comprise two to five disulfide bonds, and wherein the disulfide bonds of the target polypeptide may form an ICK.

13. 10. An expression system for expressing a target polypeptide in a host cell, comprising the expression vector of claim 9 and an expression vector encoding the target polypeptide, wherein the target polypeptide may have at least one disulfide bond, or may contain two to five disulfide bonds, and the disulfide bonds of the target polypeptide may form an ICK.

14. A method for producing a target polypeptide having at least one disulfide bond in a host cell, comprising culturing the host cell described in claim 10 under conditions suitable for expression of the target polypeptide and the chimeric protein.

15. 15. The method of claim 14, wherein the target polypeptide has at least one disulfide bond, the target polypeptide may contain two to five disulfide bonds, the disulfide bonds of the target polypeptide may form an ICK, and the target peptide may be a toxin peptide.

16. 16. The method of claim 15, wherein the host cell is genetically engineered to express the target polypeptide.

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