Protection of SpyTag-containing periplasmic fusion proteins from degradation by the proteases Tsp and OmpT

Abstract

The present invention provides a periplasmic fusion protein, a nucleic acid construct encoding the periplasmic fusion protein, a vector comprising the nucleic acid construct, and a method for producing the periplasmic fusion protein, wherein the periplasmic fusion protein comprises a binding motif connected to the C-terminus of a first protein or embedded in the amino acid sequence of the first protein. A protease-deficient host cell for producing the periplasmic fusion protein is also provided.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 819,758, filed on March 18, 2019, which is incorporated herein by reference in its entirety. Background Art

[0002] There are several technologies that allow polypeptides to be covalently coupled or linked at specific predetermined sites. One example is the SpyTag / SpyCatcher (Reddington et al., 2015) system, in which the concept of spontaneous isopeptide bond formation in natural proteins has been used to covalently link one polypeptide to another. A domain of the Streptococcus pyogenes protein FbaB that contains such an isopeptide bond is divided into two parts. One part is SpyTag (SEQ ID NO: 1), which is a 13 amino acid peptide that contains a portion of the autocatalytic center (e.g., an aspartic acid residue). The other part is SpyCatcher, which is a 116 amino acid protein domain that contains another portion of the center (e.g., lysine) and a nearby catalytic glutamic acid or aspartic acid residue. Mixing the two polypeptides restores the autocatalytic center and leads to the formation of an isopeptide bond, thereby covalently linking SpyTag to SpyCatcher (Zakeri et al., 2012). Further engineering resulted in a shortened version of SpyCatcher, which has only 84 amino acids, and optimized versions SpyTag002 (SEQ ID NO: 2) and SpyCatcher002, which have accelerated reactions (Li et al., 2014 and Keeble et al., 2017; the entire texts of which are hereby incorporated by reference). Further engineering resulted in another optimized version, SpyTag003 (SEQ ID NO: 36) and SpyCatcher003, whose reactions are close to the diffusion limit (Keeble et al., 2019), which are now incorporated by reference in their entirety.

[0003] The two polypeptides to be linked to each other are usually produced as fusion proteins, where each polypeptide has a portion of the autocatalytic center from FbaB, so that when the polypeptides are mixed, an isopeptide bond is formed. For example, a first polypeptide connected to SpyTag and a second polypeptide connected to SpyCatcher are produced separately as fusion proteins, and when the first and second polypeptides are mixed together, an isopeptide bond is formed between SpyTag and SpyCatcher. Such fusion proteins can be produced in bacteria. Many fusion proteins can be produced in the bacterial cytoplasm, but due to reducing conditions, disulfide bridged proteins do not form disulfide bonds and therefore do not fold correctly when expressed in the reducing environment of the cytoplasm. However, many important protein classes do contain disulfide bonds. An example is an antibody fragment. Functional expression of antibody Fv and Fab fragments is achieved by transferring the expressed protein chains to the periplasm of Gram-negative bacteria such as Escherichia coli, which have oxidizing conditions and are able to form disulfide bonds (Plückthun A., 1990, Escherichia coli antibodies, Nature 347, 497–498), paving the way for the field of antibody engineering. Although bacterial recombinant expression of many SpyTag fusion proteins is described in the literature, they are almost exclusively expressed in the bacterial cytoplasm (Keeble et al., 2019). There are only a few examples of periplasmic bacterial expression of SpyTag fusion proteins (Alves et al., 2015; Alam et al., 2017), and the yields are low or unclear. Keeble et al. (2019), Alves et al. (2015) and Alam et al. (2017) are hereby incorporated by reference in their entirety. Summary of the invention

[0004] The present invention provides periplasmic fusion proteins, nucleic acid constructs encoding periplasmic fusion proteins, vectors comprising the nucleic acid constructs, and methods for producing the periplasmic fusion proteins, wherein the periplasmic fusion proteins include a binding motif (e.g., SpyTag, SpyTag002, or SpyTag003) linked to a first protein (e.g., an antigen binding fragment) or embedded in the amino acid sequence of the first protein. Also provided are mutant protease-deficient E. coli cells for producing periplasmic fusion proteins.

[0005] In one embodiment, the periplasmic fusion protein comprises a binding motif attached to a first protein or embedded within an amino acid sequence of the first protein, and the binding motif comprises SEQ ID NO: 1 or a sequence having at least 60% sequence identity to SEQ ID NO: 1. In certain embodiments, the binding motif comprises SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2. In certain embodiments, the binding motif comprises SEQ ID NO: 36 or a sequence having at least 70% sequence identity to SEQ ID NO: 36. In some embodiments, the binding motif is attached to the N-terminus or C-terminus of the first protein directly or through a linker sequence. In some embodiments, the first protein is a protein domain. In certain embodiments, the linker sequence comprises a purification tag. In some embodiments, the binding motif comprises SEQ ID NO: 1 or a sequence having at least 60% sequence identity to SEQ ID NO: 1 and is attached to the C-terminus of the first protein directly or through a linker sequence. In some embodiments, the binding motif is proteolytically sensitive. In some embodiments, the binding motif is proteolytically resistant. In some embodiments, the first protein is an antigen binding fragment. In some embodiments, the first protein is an antigen binding fragment, and the antigen binding fragment is Fab, scFv or scFab. In some embodiments, the antigen binding fragment is Fab. In certain embodiments, the periplasmic fusion protein further comprises a purification tag connected to the N-terminus or C-terminus of the binding motif. In certain embodiments, the binding motif is a linker sequence connecting the C-terminus of the first protein and the N-terminus of the second protein or the N-terminus of the first protein and the C-terminus of the second protein.

[0006] Also provided is a nucleic acid construct comprising a polynucleotide sequence encoding a periplasmic fusion protein. Also provided is a vector comprising the nucleic acid construct.

[0007] The present invention also provides a method for producing a periplasmic fusion protein, the periplasmic fusion protein comprising a binding motif linked to a first protein or embedded within the amino acid sequence of the first protein, the binding motif comprising SEQ ID NO: 1 or a sequence having at least 60% sequence identity to SEQ ID NO: 1, the binding motif comprising SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2, or the binding motif comprising SEQ ID NO: 36 or a sequence having at least 70% sequence identity to SEQ ID NO: 36. In some embodiments, the method comprises culturing an E. coli host cell transformed with a vector containing a nucleic acid encoding the periplasmic fusion protein in a culture medium under conditions effective to express the periplasmic fusion protein, and recovering the periplasmic fusion protein from the E. coli host cell. In some embodiments, the binding motif of the fusion protein expressed in the E. coli host cell is resistant to proteolysis. In certain embodiments, the binding motif of the fusion protein expressed in the E. coli host cell is sensitive to proteolysis. In these embodiments, the E. coli host cell is a mutant cell deficient in one or more periplasmic proteases. In some embodiments, the mutant E. coli cells used in the method are deficient in the functional chromosomal gene tsp encoding the protease Tsp (tail-specific protease). In some embodiments, the mutant E. coli cells used in the method are deficient in the functional chromosomal genes tsp and ompT encoding the protease Tsp and OmpT (outer membrane protein T).

[0008] Also provided are E. coli TG1, TG1F-, XL1 Blue, MC1061, SS320, BL21, JM83, JM109, HB2151, W3110 or Cmax5α strains that are deficient in the functional chromosomal gene tsp encoding the protease Tsp. In some embodiments, such mutant E. coli strains comprise a nucleic acid encoding a periplasmic fusion protein comprising a binding motif, and the binding motif comprises SEQ ID NO: 1 or a sequence having at least 60% sequence identity to SEQ ID NO: 1. Also provided are E. coli TG1, TG1 F-, XL1 Blue, MC1061, SS320, BL21, JM83, JM109, HB2151, W3110 or Cmax5α strains that are deficient in the functional chromosomal genes tsp and ompT encoding the proteases Tsp and ompT. In some embodiments, such mutant E. coli strains comprise a nucleic acid encoding a periplasmic fusion protein comprising a binding motif, and the binding motif comprises SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2. In certain embodiments, such mutant E. coli strains comprise a nucleic acid encoding a periplasmic fusion protein comprising a binding motif, and the binding motif comprises SEQ ID NO: 36 or a sequence having at least 70% sequence identity to SEQ ID NO: 36.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, and 1P show various periplasmic fusion protein constructs of the embodiments.

[0011] Figure 2 Partial nucleotide and amino acid sequences of the Fab-SpyTag construct used for the periplasmic expression studies described in Example 1 are shown. CH1: the last 7 amino acid residues of the CH1 domain of human IgG1. Hinge: the first 4 amino acid residues of the hinge domain of human IgG1. EcoRI linker: 2 amino acids introduced by restriction sites. Linker: a short linker sequence of 1-4 amino acid residues. SpyTag (Spy): sequence AHIVMVDAYKPTK. His-tag: 6 histidine residues. X-linker: (GGGS)2 linker. Flag: sequence DYKDDDDK. Sx2 tag: two Strep-tags (SAWSHPQFEK) connected by a linker.

[0012] Figure 3 Western blot results from periplasmic expression studies as described in Example 1 are shown.

[0013] Figure 4 Partial nucleotide and amino acid sequences of the MBP-SpyTag construct used for periplasmic expression studies described in Example 2 are shown.

[0014] Figure 5 is an image of an SDS-PAGE gel showing the reaction of Fab-SpyTag-His and Fab-FLAG-SpyTag-His fusion proteins with SpyCatcher over time (see Example 5). When SpyTag is coupled to SpyCatcher, the heavy chain (HC) band disappears and a new band corresponding to the SpyTag fusion-SpyCatcher coupling product appears.

[0015] Figure 6 Partial nucleotide and amino acid sequences of the Fab-SpyTag002 construct used for the mutant E. coli TG1 F-strain studies described in Example 8 are shown. CH1: the last 7 amino acid residues of the human IgG1 CH1 domain. Hinge: the first 4 amino acid residues of the human IgG1 hinge domain. EcoRI linker: 2 amino acids introduced by restriction sites. Linker: a short linker sequence of 1-4 amino acid residues. SpyTag002 (Spy002): sequence VPTIVMVDAYKRYK. His-tag: 6 histidine residues. X-linker: (GGGS)2 linker. Flag: sequence DYKDDDDK.

[0016] Figure 7 Shown are SpyTag-containing constructs used to test various strategies for protecting SpyTag during periplasmic expression in non-protease-deficient bacterial strains as described in Example 9.

[0017] Figure 8 Partial nucleotide and amino acid sequences of the MBP-SpyTag constructs used for the periplasmic expression studies described in Examples 2, 6, and 10 are shown.

[0018] Fig. 9 Partial nucleotide and amino acid sequences of the scFv-SpyTag constructs used for the periplasmic expression studies described in Examples 3, 7, and 11 are shown. DETAILED DESCRIPTION

[0019] The present invention provides periplasmic fusion proteins, nucleic acid constructs encoding periplasmic fusion proteins, vectors comprising the nucleic acid constructs, and methods for producing periplasmic fusion proteins, wherein the periplasmic fusion proteins include a binding motif (e.g., SpyTag, SpyTag002, or SpyTag003) that is linked to a first protein (e.g., an antigen binding fragment or a protein domain) or is embedded in the amino acid sequence of the first protein. A protease-deficient host cell for producing periplasmic fusion proteins is also provided.

[0020] It has been found that when expressed in the periplasm of E. coli, the fusion protein comprising SpyTag, SpyTag002 or SpyTag003 binding motif is digested by periplasmic protease. SpyTag is directly connected to the C-terminal or N-terminal (not through the linker sequence) of protein, protein domain or protein domain fragment to produce fusion protein, wherein SpyTag has substantial resistance to periplasmic protease when produced in E. coli. It has also been found that when SpyTag, SpyTag002 or SpyTag003 are connected to the N-terminal or C-terminal of protein or protein domain by the linker sequence, SpyTag, SpyTag002 or SpyTag003 are sensitive to periplasmic protease during expression of fusion protein in bacteria. For this protease-sensitive fusion protein, an E. coli host cell with a defective periplasmic protease for cutting SpyTag, SpyTag002 or SpyTag003 has been established.

[0021] definition

[0022] Unless otherwise indicated, the following terms used in this application (including the specification and claims) have the following definitions. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0023] "Antibody" refers to an immunoglobulin, complex (such as a fusion) or fragment thereof. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG and IgM classes derived from antibody-producing cell lines or from in vitro antibody libraries, including natural forms or genetically modified forms, such as humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted and in vitro generated antibodies. "Antibody" also includes complex forms, including but not limited to fusion proteins with immunoglobulin portions.

[0024] As used herein, the phrase "antigen binding fragment" refers to a protein comprising an antigen binding portion of an antibody (e.g., Fab). Other antigen binding fragments include variable fragments (Fv), disulfide-stabilized Fv fragments (dsFv), single-chain variable fragments (scFv), or single-chain Fab fragments (scFab). Further examples of antigen binding fragments include antigen binding fragments in monovalent form comprising an antigen binding site, which antigen binding site includes a heavy chain antibody variable domain (VHH), a single domain antibody (sdAb), or a shark variable new antigen receptor (VNAR). In addition, non-antibody scaffolds, such as variable lymphocyte receptors (VLRs), affimers, affibodies, DARPins, anticalins, monomers, monobodies, or antigen binding peptides, may also be considered "antigen binding fragments".

[0025] The term "binding motif" refers to a protein sequence that is attached to a polypeptide and can form a covalent bond with another polypeptide. Non-limiting examples of binding motifs include SpyTag, SpyTag002, and SpyTag003 sequences. The SpyTag sequence forms a covalent bond with the SpyCatcher sequence. The binding motif can be fused to the N-terminus, the C-terminus, or can be embedded in the amino acid sequence of the polypeptide. One or more linker sequences (e.g., glycine / serine-rich linkers) or one or more protein tags may be located on both sides of the binding motif to enhance reaction accessibility, enhance the flexibility of the fusion polypeptide, or be used for purification and / or detection of the polypeptide. When the binding motif is connected to two or more proteins, the N-terminus and C-terminus of the binding motif may be flanked by one or more linker sequences to enhance reaction accessibility, enhance the flexibility of the fusion polypeptide, or be used for purification and / or detection of the polypeptide.

[0026] The term "prokaryotic system" refers to prokaryotic cells, such as bacterial cells (e.g., E. coli or Salmonella) or prokaryotic bacteriophages or bacterial spores. The term "eukaryotic system" refers to eukaryotic cells, including cells of animals, plants, fungi and protists, as well as eukaryotic viruses such as retroviruses, adenoviruses, baculoviruses, etc. Prokaryotic and eukaryotic systems are collectively referred to as "expression systems."

[0027] The term "expression cassette" refers to a functional unit constructed in a vector in this article for expressing a recombinant polypeptide in the periplasm. The expression cassette includes one or more promoters, a transcription terminator sequence, one or more ribosome binding sites, and a DNA encoding a fusion protein. Depending on the expression system (e.g., enhancers and polyadenylation signals for eukaryotic expression systems), other genetic components may be added to the expression cassette.

[0028] As used herein, the term "vector" refers to a nucleic acid molecule, preferably self-replicating in a cell, which transfers the inserted nucleic acid molecule into a host cell and / or between host cells. Typically, a vector is a circular DNA comprising a replication origin, a selection marker and / or a viral packaging signal and other regulatory elements. In the description of the present invention, vectors, vector DNA, plasmid DNA, and phage DNA are interchangeable terms. The term includes vectors that primarily function to insert DNA or RNA into a cell, replication vectors that primarily function to replicate DNA or RNA, and expression vectors that function to transcribe and / or translate DNA or RNA. Also included are vectors that provide more than one of the above functions.

[0029] As used herein, the term "expression vector" is a polynucleotide that, when introduced into an appropriate host cell, results in the transcription and translation of one or more polypeptides under appropriate conditions. The term "expression vector" refers to a vector that directs the expression of a polypeptide of interest fused in frame with a binding motif.

[0030] As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after the assembly of the nucleotide polymer.

[0031] As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, D or L optical isomers, amino acid analogs and peptidomimetics.

[0032] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length.

[0033] As used herein, the term "protein domain" or "domain" refers to a semi-autonomous compact folding unit with a single hydrophobic core" (Ezkurdia and Tress, 2011). A domain is a conserved portion of a given protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure that can be stabilized and folded independently. Examples of protein domains include, but are not limited to, the constant domains of human IgG1 and maltose binding protein (MBP) (e.g., CH1, including the first two amino acids of the hinge region). The "C-terminus" of a protein domain refers to the last amino acid that is structured (i.e., visible in a crystal structure) in a crystal structure annotated in the Protein Data Bank (Berman et al., 2000) as having the same or a closely related protein domain (i.e., having at least 70% sequence identity). A protein domain can be shortened by up to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N- or C-terminus without losing the ability to fold, and therefore the ability to maintain the domain structure.

[0034] As used herein, the term "host cell" includes a single cell or cell culture that can be or has been a recipient of a disclosed expression construct. Host cells include progeny of a single host cell. Progeny may not necessarily be identical to the original parent cell due to natural, accidental or intentional mutations.

[0035] When the maximum correspondence is compared as described below, two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues in the two sequences is respectively the same. The term "identical" or "identity" percentage in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides when compared and compared with respect to the maximum correspondence of the comparison window, such as using one of the following sequence comparison algorithms or by manual alignment and visual measurement. When the percentage of sequence identity is used in relation to proteins and peptides, it is believed that different residue positions are usually different due to conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues with similar chemical properties (such as charge or hydrophobicity), and thus do not change the functional properties of the molecule. When the sequences differ due to conservative substitutions, the percentage of sequence identity can be adjusted upward to correct according to the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. It is generally included to score conservative substitutions as part of the sequence identity rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, identical amino acids are scored as 1, non-conservative substitutions are scored as 0, and conservative substitutions are scored as 0-1. Scoring for conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988), for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California, USA).

[0036] Sequences are "substantially identical" to each other if they have a specified percentage of nucleotide or amino acid residues that are identical (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over a specified region or over the entire specified region if a region is not specifically specified) when compared and aligned for maximum correspondence over a comparison window.

[0037] For sequence comparison, generally a sequence is used as a reference sequence compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or other parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0038] As used herein, a "comparison window" includes a reference to a segment of any one of a plurality of consecutive positions selected from 10 to 600, about 10 to about 300, about 10 to about 150, wherein a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. The comparison window can also be the entire length of a reference or test sequence.

[0039] Percent sequence identity and sequence similarity can be determined using the BLAST 2.0 algorithm, which is described in Altschul et al. (J. Mol. Biol. 215: 403-10, 1990). Software for performing BLAST 2.0 analysis is publicly available from the National Center for Biotechnology Information (World Wide Web address: ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighboring word score threshold (Altschul et al., supra). These initial neighboring word hits are used as seeds to initiate searches in order to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be improved. Extension of the word hits in each direction is terminated when: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults: word length (W) 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) 50, expectation (E) 10, M=5, N=-4, and comparison of both strands.

[0040] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if the smallest sum probability when comparing a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0041] Periplasmic fusion protein

[0042] In one embodiment, the periplasmic fusion protein comprises a Figure 1 A-1N) or an amino acid sequence embedded in the first protein (e.g. Figure 1 As used herein, a "periplasmic fusion protein" refers to a protein produced in the periplasm of an appropriate bacterial host cell and comprises two polypeptides, the first being any protein and the second being a SpyTag, SpyTag002 or SpyTag003 binding motif. The binding motif may be directly ( Figure 1 A, 1D, 1G, 1H, 1K, 1N) or through a linker sequence ( Figure 1 In some embodiments, the first protein is a protein domain. In some embodiments, the first protein is an antigen binding fragment (e.g., Fab, scFv, or scFab). As used herein, a "binding motif" refers to a peptide sequence that is connected to a protein expressed in the periplasm and is connected to another binding motif (e.g., SpyCatcher, SpyCatcher002, or SpyCatcher003) through a protein when two binding motifs contact each other, and the other binding motif is connected to another polypeptide. The covalent bond between the two binding motifs is formed spontaneously or with the help of an enzyme. For example, a binding motif can form a covalent bond with another binding motif or a multimeric binding motif on the N-terminus of the Fc fragment. Examples of Fc fragments with another binding motif are described in co-applied U.S. application 62 / 819748 (antigen binding fragments coupled to multiple Fc isotypes and subclasses; filed on March 18, 2019; BRL.123P), and examples of multimeric binding motifs are described in co-applied U.S. application 62 / 819753 (antigen binding proteins, filed on March 18, 2019; BRL.129P), each of which is incorporated herein in its entirety. In some embodiments, the binding motif comprises SEQ ID NO: 1 (i.e., SpyTag) or a sequence having at least 60% sequence identity to SEQ ID NO: 1. In some embodiments, the binding motif comprises SEQ ID NO: 2 (i.e., SpyTag002) or a sequence having at least 70% sequence identity to SEQ ID NO: 2. In certain embodiments, the binding motif comprises SEQ ID NO: 36 (i.e., SpyTag003) or a sequence having at least 78% sequence identity to SEQ ID NO: 36.

[0043] In some examples, the binding motif (e.g., SpyTag) is directly linked to the first protein (i.e., without a linker sequence; Figure 1 A, 1D, 1G, 1H, 1K, 1N) yield a periplasmic fusion protein that is substantially proteolytically insensitive during expression, i.e., resistant to cleavage by periplasmic proteases.

[0044] In some embodiments, the periplasmic fusion protein comprises at least one linker sequence between the first protein and the binding motif. As used herein, a "linker sequence" or "linker" refers to a peptide or polypeptide comprising one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 10 or more amino acid residues) connected by peptide bonds. Such a linker can provide rotational freedom, allowing each component of the fusion protein to interact with its intended target without steric hindrance. These linkers can be a mixture of glycine and serine, for example - (GGGS) n -, wherein n is 1, 2, 3, 4 or 5. Other suitable peptide / polypeptide linker sequences optionally include naturally occurring or non-naturally occurring peptides or polypeptides. Optionally, the peptide or polypeptide linker sequence is a flexible peptide or polypeptide ( Figure 1 B, 1E, 1I, 1L). Exemplary flexible peptides / polypeptides include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser, Ala-Ser, Gly-Gly-Gly-Ser, Gly4-Ser, (Gly4-Ser)2, (Gly4-Ser)3, (Gly4-Ser)4, (Gly4-Ser)2-Gly-Ala-Gly-Ser-Gly4-Ser, Gly-(Gly4-Ser)2, Gly4-Ser-Gly, Gly-Ser-Gly2, and Gly-Ser-Gly2-Ser. Other suitable peptide linker sequences optionally include the TEV linker ENLYFQG, a linear epitope recognized by tobacco mosaic virus protease. Exemplary peptides / polypeptides include, but are not limited to GSENLYFQGSG. Other suitable peptide linker sequences include helical linkers, such as Ala-(Glu-Ala-Ala-Ala-Lys) n -Ala (n=1-5). In some embodiments, the linker sequence is a GAP (Gly Ala Pro) sequence. In some embodiments, the linker sequence comprises a purification tag ( Figure 1 C, 1F, 1J, 1M). Purification tags may include, but are not limited to, polyhistidine or His-tags and - tag (i.e., the amino acid sequence DYKDDDDK, where D is aspartic acid, Y is tyrosine, and K is lysine). In certain embodiments, the linker sequence comprises a binding motif (Figures G and N), and optionally comprises a purification tag and / or a flexible linker sequence attached to one or both of the C-terminus and the N-terminus of the binding motif. In some embodiments, a sequence of 1 to 50 amino acid residues can be used as a linker. In some embodiments, the linker is protease resistant (i.e., periplasmic expression of the polypeptide with the linker occurs in the host cell, and the linker is not cleaved by the protease). If two or more linker sequences are used between the protein and the binding motif, the two or more linker sequences can be the same or different.

[0045] In some embodiments where the periplasmic fusion protein comprises a linker sequence between the first protein and the binding motif, the binding motif (i.e., SpyTag, SpyTag002, and SpyTag003) can be proteolysis sensitive, i.e., during periplasmic expression, the binding motif can be cleaved by one or more E. coli proteases. In certain embodiments, cleavage of the binding motif by the periplasmic protease depends on the linker length (i.e., the number of amino acids), but is independent of the linker amino acid composition.

[0046] In some embodiments, the linker sequence comprises a SpyTag, SpyTag002 or SpyTag003 binding motif. In this embodiment, the binding motif connects the N-terminus of the first protein to the C-terminus of the second protein ( Figure 1 G), or connecting the C-terminus of the first protein to the N-terminus of the second protein ( Figure 1 N). Examples of the second protein include, but are not limited to, antigen binding fragments, fluorescent proteins (e.g., green fluorescent protein), enzymes (e.g., horseradish peroxidase or other peroxidases), alkaline phosphatase, luciferase, split fluorescent protein, and MBP. In certain embodiments, the linker sequence comprising SpyTag, SpyTag002, or SpyTag003 further includes a purification tag or a flexible linker sequence between the binding motif and one or both of the first and second proteins.

[0047] In certain embodiments, the periplasmic fusion protein has a binding motif linked to its N-terminus ( Figure 1 D, 1E), binding motif C-terminal ( Figure 1 K, 1L) or the N-terminal and C-terminal binding motifs ( Figure 1 F and 1M) purification tags.

[0048] Nucleic acid construct

[0049] Also provided are nucleic acid constructs encoding periplasmic fusion proteins with or without a linker between the first protein and the binding motif and / or between the binding motif and the second protein of the periplasmic fusion protein. Such nucleic acids can be present in an expression vector in an appropriate prokaryotic host cell.

[0050] Typically, the polynucleotide sequence encoding a Fab fused to a binding motif at the C-terminus encodes two peptides, i.e., the L chain and the H chain of the Fab. A binding motif such as SpyTag can be fused to the L or H chain directly or through one or more linkers. The Fab expression cassette can comprise a bicistronic vector that produces an mRNA encoding both the L chain and the H chain, at least one of which is fused to the binding motif. In addition, both the H and L chains can have a signal peptide to direct their export to the periplasm.

[0051] Nucleic acid constructs are usually introduced into various vectors. The vectors described herein usually contain transcription or translation control sequences required for expressing the fusion protein. Suitable transcription or translation control sequences include, but are not limited to, origin of replication, promoter, enhancer, repressor binding region, transcription start site, ribosome binding site, translation start site and termination site for transcription and translation.

[0052] The origin of replication (commonly referred to as the ori sequence) allows the vector to replicate in a suitable host cell. The choice of ori depends on the type of host cell and / or gene packaging used. When the host cell is a prokaryotic organism, the expression vector usually contains an ori sequence that directs the autonomous replication of the vector in the prokaryotic cell. Preferred prokaryotic ori can direct the replication of the vector in bacterial cells. Non-limiting examples of such ori include pMB1, pUC and other E. coli origins.

[0053] As used herein, "promoter" is a DNA region that can bind RNA polymerase under specific conditions and initiate transcription of the coding region located downstream (3' direction) of the promoter. It can be constitutive or inducible. Typically, the promoter sequence binds to the transcription start site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. The promoter sequence has a transcription start site and a protein binding domain responsible for RNA polymerase binding.

[0054] The choice of promoter depends largely on the host cell into which the vector is introduced. For prokaryotes, a variety of powerful promoters are known in the art. Preferred promoters are lac promoter, Trc promoter, T7 promoter and pBAD promoter.

[0055] In constructing the vector of the present invention, the terminator sequence combined with the protein coding sequence is also inserted into the 3' end of the sequence to be transcribed, to provide polyadenylation of mRNA and / or transcription termination signal. The terminator sequence preferably comprises one or more transcription termination sequences (e.g., polyadenylation sequence), and can also be extended by comprising additional DNA sequences to further interrupt transcription read-through. The preferred terminator sequence (or termination site) of the present invention has a gene followed by a transcription termination sequence, which is its own termination sequence or a heterologous termination sequence. Examples of such terminator sequences include termination codons coupled to various yeast transcription termination sequences or mammalian polyadenylation sequences, which are known in the art and can be widely available. In the case where the terminator comprises a gene, it may be advantageous to use a gene encoding a detectable or selective marker; thereby providing a means by which the presence and / or absence of a terminator sequence (and the corresponding inactivation and / or activation of the transcription unit) can be detected and / or selected.

[0056] In addition to the above elements, the vector may contain a selective marker (e.g., a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which the selectable gene has been introduced can survive and / or grow under selective conditions. Typical selection genes encode proteins that confer resistance to (a) antibiotics or other toxins such as ampicillin, kanamycin, neomycin, bleomycin, G418, methotrexate, etc.; (b) compensate for auxotrophic deficiencies; or (c) provide key nutrients that cannot be obtained from complex culture media. The choice of a suitable marker gene will depend on the host cell, and suitable genes for different hosts are known in the art.

[0057] In one embodiment, the expression vector is a shuttle vector that can replicate in at least two unrelated host systems. In order to promote this replication, the vector usually contains at least two replication origins, one effective in each host system. Generally, the shuttle vector can replicate in a eukaryotic host system and a prokaryotic host system. This can detect protein expression (expression cell type) in a eukaryotic host and vector amplification (amplification cell type) in a prokaryotic host. Preferably, one replication origin is from SV40 or 2u, and one is from pUC, although any suitable starting point known in the art can be used, as long as it guides the replication of the vector. When the vector is a shuttle vector, the vector preferably includes at least two selection markers, one for expressing cell types and one for amplifying cell types. Any selection marker known in the art or those described herein can be used, as long as it works in the expression system utilized.

[0058] The vector of the present invention can be obtained by using recombinant cloning methods and / or by chemical synthesis. A large amount of recombinant cloning techniques, such as PCR, restriction endonuclease digestion and connection, are well known in the art and do not need to be described in detail at this time. Those skilled in the art can also use the sequence data provided herein or the sequence data in a public or proprietary database to obtain the required vector by any synthetic means available in the art. In addition, suitable sequences can be cut out from various DNA sources using well-known restriction and ligation techniques, and operably integrated with the exogenous sequence to be expressed according to embodiments of the present invention.

[0059] Methods for producing fusion proteins

[0060] Also provided is a method for producing a periplasmic fusion protein comprising a binding motif optionally connected to a first protein or a first and a second protein by one or more linkers. In one embodiment, the method comprises culturing an E. coli host cell transformed with a vector containing a nucleic acid encoding the periplasmic fusion protein in a culture medium under conditions effective to express the periplasmic fusion protein in the host cell. Any suitable E. coli strain can be used to produce the periplasmic fusion protein. One E. coli strain that can be used for expression of a protein (e.g., an antibody, an antibody fragment, or MBP) is the TG1 strain. The TG1 strain is based on E. coli K-12 (genotype glnV44 thi-1Δ(lac-proAB)Δ(mcrB-hsdSM)5(r K –m K -)F'[traD36 proAB+lacIq lacZΔM15]), which is often used to express antibodies and antibody fragments in the periplasm (see Knappik 2009, for example, experiments showing periplasmic expression). In some embodiments, the E. coli cell strain is TG1F-(genotype glnV44 thi-1Δ(lac-proAB)Δ(mcrB-hsdSM)5(r K –m K–)), which is a F pili depleted form. In certain embodiments, the E. coli host cell strain includes, but is not limited to, XL1 Blue, MC1061, SS320, BL21, JM83, JM109, HB2151, W3110, Cmax5α, and any E. coli strain suitable for functional expression of antibody fragments in E. coli. In some embodiments, the E. coli host cell is a mutant cell deficient in one or more periplasmic proteases. In some embodiments, the mutant E. coli cell is deficient in a functional chromosomal gene tsp encoding a protease Tsp (tail-specific protease). In some embodiments, the mutant E. coli cell is deficient in a functional chromosomal gene tsp encoding a protease Tsp and OmpT (outer membrane protein T). The gene for a protease can be "knocked out", for example, by deleting or replacing the gene with an exogenous DNA sequence, such as a gene encoding antibiotic resistance. One method for knocking out a protease gene is described in Datsenko and Wanner (2000), Proc Natl Acad Sci USA, 97(12): 6640–6645. In some embodiments, the gene for the protease is modified to produce a mutant protease without proteolytic activity or with reduced proteolytic activity (Keiler, 1995). In some embodiments, antisense morpholino, antisense peptide nucleic acid or other antisense nucleotide oligomers (Geller, 2005) inhibit the expression of tsp or ompT protease, thereby reducing or eliminating the tsp or ompT protease activity in Escherichia coli, respectively. In some embodiments, both tsp and ompT proteases are inhibited by antisense morpholino, antisense peptide nucleic acid or other antisense nucleotide oligomers, resulting in reduced or eliminated tsp and ompT protease activity. In some embodiments, tsp or ompT protease activity is reduced or eliminated by chemical protease inhibitors (including but not limited to phenylmethanesulfonyl fluoride or p-toluenesulfonyl fluoride (Prouty and Goldberg, 1972)), peptides or small proteins (e.g., aprotinin (Brannon, 2015), or metal cations (Silber, 1991)). In some embodiments, both tsp and ompT protease activities are reduced or eliminated by the above-mentioned chemical inhibitors.

[0061] Mutant E. coli TG1 F-strain SK4 (DSM 33004) and SK13 (DSM 33005) were deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Inhoffenstraβe 7B, 38124 Braunschweig, Germany) on January 8, 2019. Mutant E. coli TG1 F-strain SK4 with DSMZ accession number DSM 33004 is tsp-deficient, and mutant E. coli TG1 F-strain SK13 with DSMZ accession number DSM 33005 is tsp- and ompT-deficient.

[0062] Vectors containing nucleic acids encoding periplasmic fusion proteins can be transformed into cells using standard techniques, for example, by chemical methods (Green R, Rogers EJ. Transformation of chemically competent E. coli, Method Enzymol 2013; 529: 329-36) or by electroporation. In some embodiments where the binding motif is SEQ ID NO: 1 (SpyTag), the periplasmic fusion protein is transformed into a mutant E. coli TG1 F-strain having DSMZ accession number DSM 33004, which is tsp deficient. In some embodiments where the binding motif is SEQ ID NO: 2 (SpyTag002) or SEQ ID NO: 36 (SpyTag003), the periplasmic fusion protein is transformed into a mutant E. coli TG1 F-strain having DSMZ accession number DSM33005, which is tsp and ompT deficient.

[0063] Cells that are able to express one or more markers are able to survive / grow / multiply under certain artificially imposed conditions (e.g., adding toxins or antibiotics to the culture medium) because the polypeptide / gene or polypeptide component of the selection system contained therein confers a characteristic (e.g., antibiotic resistance). Cells that are unable to express one or more markers are unable to survive / grow / multiply under artificially imposed conditions.

[0064] Any suitable selection system can be used in the methods described herein. Typically, the selection system can be based on including one or more genes in the vector that provide resistance to known antibiotics, such as tetracycline, chloramphenicol, kanamycin or ampicillin resistance genes. Cells grown in the presence of the relevant antibiotic can be selected because they express both the gene that confers resistance to the antibiotic and the desired protein.

[0065] In one embodiment, the method further comprises the step of culturing the transformed cells in culture medium to express the periplasmic fusion protein.

[0066] This approach can also use inducible expression systems or constitutive promoters to express periplasmic fusion proteins.

[0067] Any suitable medium may be used to culture the transformed cells. The medium may be adapted to a specific selection system, for example the medium may contain antibiotics to allow only those cells that have been successfully transformed to grow in the medium.

[0068] The expressed fusion protein is then recovered from the periplasm of the host cells by lysing the bacteria, first by whole cell lysis or periplasmic lysis. The method may also include one or more steps of extracting and purifying the periplasmic fusion protein. The periplasmic fusion protein may be isolated from the cell extract by an appropriate purification method, including but not limited to protein A chromatography, protein L chromatography, thiophilic mixed mode resins, nickel nitrilotriacetic acid (Ni-NTA) resins for His tags, of or Resin, -labeling, hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate, ethanol or polyethylene glycol fractionation / precipitation, ion exchange membranes, expanded bed adsorption chromatography, or simulated moving bed chromatography.

[0069] In some embodiments, the method further comprises measuring the expression amount of the periplasmic fusion protein after purification.

[0070] Additional disclosures and requested topics

[0071] Item 1. A periplasmic fusion protein comprising a binding motif linked to a first protein or embedded within the amino acid sequence of the first protein, wherein the binding motif comprises SEQ ID NO:1 or a sequence having at least 60% sequence identity with SEQ ID NO:1.

[0072] Item 2. The periplasmic fusion protein according to Item 1, wherein the binding motif is connected to the N-terminus of the first protein directly or via a linker sequence.

[0073] Item 3. The periplasmic fusion protein according to Item 1, wherein the binding motif is connected to the C-terminus of the first protein directly or via a linker sequence.

[0074] Item 4. The periplasmic fusion protein according to Item-2 or 3, wherein the linker sequence comprises a purification tag.

[0075] Item 5. The periplasmic fusion protein according to Item 3, wherein the binding motif is directly linked to the C-terminus of the protein domain in the first protein, and the binding motif is resistant to proteolysis.

[0076] Item 6. The periplasmic fusion protein according to Item 5, wherein the protein domain is a human scFv single-chain antibody fragment with a C-terminal truncation within the FR4 region.

[0077] Item 7. The periplasmic fusion protein according to Item 3, wherein the binding motif is connected to the C-terminus of the protein domain in the first protein via a linker of 1 or 2 amino acids.

[0078] Item 8. A periplasmic fusion protein according to any one of Item 3, wherein the binding motif is linked to the C-terminus at IMGT position 121 of the human heavy chain CH1 antibody domain via a linker of 2, 3 or 4 amino acids.

[0079] Item 9. A periplasmic fusion protein according to Item 3, wherein the binding motif is linked to the C-terminus at IMGT position 121 of the human constant light chain antibody domain via a linker of 2, 3 or 4 amino acids.

[0080] Item 10. The periplasmic fusion protein according to any one of Items 1-9, further comprising a purification tag connected to the N-terminus or C-terminus of the binding motif.

[0081] Item 11. A periplasmic fusion protein according to any one of Items 1-10, wherein the binding motif connects the C-terminus of the first protein to the N-terminus of the second protein, or connects the N-terminus of the first protein to the C-terminus of the second protein.

[0082] Item 12. A nucleic acid construct comprising a polynucleotide sequence encoding a periplasmic fusion protein as defined in any one of Items 1-11.

[0083] Item 13. A vector comprising the nucleic acid construct described in Item 12.

[0084] Item 14. A method for producing a periplasmic fusion protein, the method comprising:

[0085] An E. coli host cell transformed with a vector containing a nucleic acid encoding a periplasmic fusion protein is cultured in a culture medium under conditions effective to express the periplasmic fusion protein, wherein:

[0086] The periplasmic fusion protein includes a binding motif linked to a first protein or embedded within the amino acid sequence of the first protein;

[0087] The binding motif comprises SEQ ID NO: 1 or a sequence having at least 60% sequence identity to SEQ ID NO: 1; and

[0088] Compared with wild-type cells, the Tsp protein activity of E. coli host cells is reduced or absent due to:

[0089] a) a mutation in a Tsp gene encoding a mutant Tsp protein which reduces or eliminates protease activity; or

[0090] b) a mutation in the Tsp gene or in a Tsp gene regulatory sequence that reduces or eliminates the expression of the Tsp protein; or

[0091] c) one or more deletions in bacterial chromosomal regions that reduce or eliminate Tsp protein activity; or

[0092] d) an inhibitor or inactivator that reduces or eliminates Tsp protease activity or an inhibitor of Tsp protease expression; and

[0093] Recovery of periplasmic fusion proteins from E. coli host cells.

[0094] Item 15. A method for producing a periplasmic fusion protein, the method comprising:

[0095] An E. coli host cell transformed with a vector containing a nucleic acid encoding a periplasmic fusion protein is cultured in a culture medium under conditions effective to express the periplasmic fusion protein, wherein:

[0096] The periplasmic fusion protein includes a binding motif linked to a first protein or embedded within the amino acid sequence of the first protein;

[0097] The binding motif comprises SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 36 or a sequence having at least 78% sequence identity to SEQ ID NO: 36; and

[0098] Compared with wild-type cells, the Tsp and ompT protein activities of E. coli host cells are reduced or absent due to:

[0099] a) a mutation in a Tsp gene encoding a mutant Tsp protein, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protein, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates Tsp protein activity; and

[0100] b) a mutation in an OmpT gene encoding a mutant OmpT protein, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protein, or one or more deletions in a region of the bacterial chromosome that reduces or eliminates OmpT protein activity; and recovering the periplasmic fusion protein from the E. coli host cell.

[0101] Item 16. A method for producing a periplasmic fusion protein, the method comprising:

[0102] An E. coli host cell transformed with a vector containing a nucleic acid encoding a periplasmic fusion protein is cultured in a culture medium under conditions effective to express the periplasmic fusion protein, wherein:

[0103] The periplasmic fusion protein includes a binding motif linked to a first protein or embedded within the amino acid sequence of the first protein;

[0104] The binding motif comprises SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 36 or a sequence having at least 78% sequence identity to SEQ ID NO: 36; and

[0105] Compared with wild-type cells, the Tsp and ompT protease activities of E. coli host cells are reduced or absent due to:

[0106] a) a Tsp protease inhibitor or inactivator or a Tsp expression inhibitor; and

[0107] b) an ompT protease inhibitor or inactivator or an ompT expression inhibitor; and

[0108] Recovery of periplasmic fusion proteins from E. coli host cells.

[0109] Item 17. A periplasmic fusion protein according to any one of Items 14-16, wherein the binding motif is linked to the N-terminus of the first protein directly or via a linker sequence.

[0110] Item 18. A periplasmic fusion protein according to any one of Items 14-16, wherein the binding motif is linked to the C-terminus of the first protein directly or via a linker sequence.

[0111] Item 19. A method according to any one of Items 14-18, wherein the first protein is a protein domain.

[0112] Item 20. A method according to any one of Items 17-19, wherein the linker sequence comprises a purification tag.

[0113] Item 21. A method according to Item 18, wherein the binding motif comprises an amino acid sequence as described in SEQ ID NO: 1 or a sequence having at least 60% sequence identity with SEQ ID NO: 1, and is directly linked to the C-terminus of the first protein.

[0114] Item 22. A method according to any one of Items 14-21, wherein the binding motif is sensitive to proteolysis.

[0115] Item 23. A method according to any one of Items 14-21, wherein the binding motif is resistant to proteolysis.

[0116] Item 24. A method according to any one of Items 14-23, wherein the first protein is an antigen binding fragment, and the antigen binding fragment comprises Fab, scFv or scFab.

[0117] Item 25. The method according to Item 24, wherein the antigen binding fragment is Fab.

[0118] Item 26. A method according to any one of Items 14-25, wherein the periplasmic fusion protein further comprises a purification tag connected to the N-terminus or C-terminus of the binding motif.

[0119] Item 27. A method according to any one of Items 14-25, wherein the binding motif connects the C-terminus of the first protein to the N-terminus of the second protein, or connects the N-terminus of the first protein to the C-terminus of the second protein.

[0120] Item 28. A method according to Item 14, wherein the Escherichia coli host cell is a mutant Escherichia coli TG1 F-strain deposited on January 8, 2019 with DSM accession number 33004.

[0121] Item 29. A method according to Item 15 or 16, wherein the Escherichia coli host cell is a mutant Escherichia coli TG1 strain with DSM accession number 33005 deposited on January 8, 2019.

[0122] Item 30. A strain of Escherichia coli TG1, TG1 F-, XL1 Blue, MC1061, SS320, BL21, JM83, JM109, HB2151, W3110 or Cmax5α, which has reduced or no Tsp protein activity compared to wild-type cells, and the reduced or no Tsp protein activity is caused by a mutation in a Tsp gene encoding a mutant Tsp protein, and the mutation reduces or eliminates protease activity, or is caused by a mutation in the Tsp gene or in a Tsp gene regulatory sequence, and the mutation reduces or eliminates Tsp protein expression, or is caused by the deletion of one or more regions in the bacterial chromosome that reduce or eliminate Tsp protein activity.

[0123] Item 31. An Escherichia coli strain according to Item 30, comprising a nucleic acid encoding a periplasmic fusion protein comprising a binding motif, and the binding motif comprises SEQ ID NO: 1 or a sequence having at least 60% sequence identity with SEQ ID NO: 1.

[0124] Item 32. Escherichia coli TG1, TG1 F-, XL1 Blue, MC1061, SS320, BL21, JM83, JM109, HB2151, W3110 or Cmax5α strains having reduced Tsp protein activity and ompT protein activity or no Tsp protein activity and ompT protein activity compared to wild-type cells, wherein:

[0125] a) a mutation in a Tsp gene encoding a mutant Tsp protein, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protein, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates Tsp protein activity; and

[0126] b) a mutation in an OmpT gene encoding a mutant OmpT protein, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protein, or one or more deletions in a region of the bacterial chromosome that reduces or eliminates OmpT protein activity.

[0127] Item 33. The Escherichia coli strain according to Item 32, comprising:

[0128] A nucleic acid encoding a periplasmic fusion protein comprising a binding motif, and the binding motif comprises SEQ ID NO: 2 or a sequence having at least 70% sequence identity to SEQ ID NO: 2, or

[0129] A nucleic acid encoding a periplasmic fusion protein comprising a binding motif, and the binding motif comprises SEQ ID NO:36 or a sequence having at least 78% sequence identity to SEQ ID NO:36.

[0130] Item 34. An Escherichia coli strain according to any one of Items 30-33, wherein the binding motif is proteolysis-sensitive.

[0131] Item 35. An Escherichia coli strain according to any one of Items 30-33, wherein the binding motif is resistant to proteolysis.

[0132] Item 36. A mutant Escherichia coli strain:

[0133] a) having reduced or no Tsp protein activity compared to wild-type cells due to a mutation in the Tsp gene encoding the mutant Tsp protein, which mutation reduces or eliminates protease activity, or a mutation in the Tsp gene or Tsp gene regulatory sequence, which mutation reduces or eliminates the expression of the Tsp protein, or one or more deletions in the bacterial chromosome that reduce or eliminate the activity of the Tsp protein;

[0134] for expressing a periplasmic fusion protein comprising a binding motif linked to a first protein or embedded within the amino acid sequence of a first protein, wherein the binding motif comprises SEQ ID NO: 1 or a sequence having at least 60% sequence identity to SEQ ID NO: 1; or

[0135] b) having reduced or no Tsp protein activity and ompT protein activity compared to wild-type cells, due to:

[0136] i) a mutation in a Tsp gene encoding a mutant Tsp protein, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protein, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates Tsp protein activity; and

[0137] ii) a mutation in an OmpT gene encoding a mutant OmpT protein, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protein, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates OmpT protein activity;

[0138] For expressing a periplasmic fusion protein, which comprises a binding motif linked to a first protein or embedded within the amino acid sequence of the first protein, wherein the binding motif comprises SEQ ID NO: 2 or SEQ ID NO: 36 or a sequence having at least 70% sequence identity with SEQ ID NO: 2 or a sequence having at least 78% sequence identity with SEQ ID NO: 36, respectively.

[0139] Item 37. A mutant Escherichia coli strain TG1 F-strain, which has DSM accession number 33004 or 33005, both deposited on January 8, 2019.

[0140] Example

[0141] The following examples are provided for illustration only and are not intended to be limiting. It will be readily apparent to those skilled in the art that there are a number of non-critical parameters that can be varied or modified and yield substantially the same or similar results.

[0142] Example 1 - Periplasmic expression of Fab-X-SpyTag fusion protein with X as linker

[0143] The coding gene of the human antibody fragment in the form of Fab with SpyTag at the C-terminus of the truncated heavy chain (i.e., Fab-SpyTag construct) is cloned into an expression vector with a signal sequence on the H and L chains of the Fab, which is transported by bacteria to import the nascent chain into the periplasm. The Fab gene used in this example encodes a non-covalent heterodimer of a light chain (without C-terminal cysteine) with a truncated heavy chain of the first 4 amino acids (at most, but excluding the first hinge cysteine) of the VH domain, the CH1 domain, and the hinge region. This vector was then used to transform Escherichia coli TG1 F- (without F-addition body; Bio-Rad). Fab constructs from five different antibodies were tested. Figure 2 Partial sequences of constructs are shown. Transformants were cultured in 250 ml 2xYT broth containing 0.1% glucose and chloramphenicol. After 1 hour of growth at 37°C, the culture was induced with 0.8 mM IPTG. Expression was allowed to proceed for about 16 hours at 30°C. The culture was centrifuged and the cells were frozen at -80°C. The cells were lysed with BugBuster lysis buffer (Millipore-Sigma). The expression was then purified by affinity chromatography (e.g., Ni-NTA chromatography for fusion proteins with a hexahistidine tag or by HPLC for proteins with a hexahistidine tag). The fusion protein The fusion protein was purified by 4% polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX) and Coomassie gel chromatography using non-reducing conditions and 3xPBS. Staining, the purity of the fusion protein was determined by SDS-PAGE. In addition, all purified Fab fragments were functionally tested by ELISA (2 μg / ml) using Fab antigen (5 μg / ml in PBS, applied to the surface of microtiter plate wells at 4°C overnight). The binding of Fab fragments to their antigens was detected using HRP-conjugated anti-Fab (STAR126P, Bio-Rad) or anti-histidine tag (MCA1396P, Bio-Rad) secondary antibodies and QuantaBlu fluorescent substrate (Thermo-Fisher).

[0144] Initial attempts to purify the Fab-SpyTag fusion protein were unsuccessful. A construct containing the FLAG-SpyTag-His peptide sequence at the C-terminus of the Fab heavy chain (SEQ ID NO: 4) could not be purified, which is consistent with the fact that the SpyTag has a purification tag (His tag or ) were similar to all other constructs (SEQ ID NOs: 3-5 and 8-10) containing the His-SpyTag or His-SpyTag-FLAG peptide sequence at the C-terminus of the Fab heavy chain (SEQ ID NOs: 6 and 7, respectively) could be purified, but those constructs did not react in the subsequent SpyTag-SpyCatcher protein ligation reaction. To test the protein ligation of the SpyTag portion of the fusion protein with SpyCatcher, each fusion protein (final concentration 15 μM) was mixed with SpyCatcher (final concentration 20 μM) in 1xPBS buffer and coupled for 2 hours at room temperature. SpyCatcher was produced by bacterial cytoplasmic expression and purified by Ni-NTA as described by Zakeri et al. (2012). SDS-PAGE was used to test the new band that appeared on the gel, which corresponds to the SpyTag fusion-SpyCatcher coupling product. No new SpyTag fusion protein-SpyCatcher bands of the correct size were observed for any fusion protein, indicating that none of the fusion protein was coupled to SpyCatcher and that the SpyTag portion of the fusion protein was neither fully functional nor complete. Without being bound by theory, applicants hypothesize that SpyTag is susceptible to cleavage by one or more periplasmic proteases.

[0145] To determine the location of cleavage in the fusion protein, the expression products of Fab-FLAG-SpyTag-His (SEQ-ID-NO: 4) and Fab-His-SpyTag-FLAG (SEQ-ID-NO: 7) fusion proteins were analyzed by Western blot analysis (SDS-PAGE with reducing sample buffer (Bio-Rad), AnyKD TGX gel (Bio-Rad), transferred to PVDF membrane (Bio-Rad)). The expression products of fusion proteins were detected by Western blotting using 1% paraformaldehyde (Sigma A8592) or HRP-labeled anti-histidine tag antibody (Bio-Rad MCA1396P). The results of Western blotting are shown in Figure 3 .

[0146] Results: Figure 3 As shown in the Western blot, the expression product of Fab-FLAG-SpyTag-His fusion protein was labeled The expression product of the Fab-His-SpyTag-FLAG fusion protein was recognized by the labeled anti-Histidine tag antibody, but not by the labeled anti-FLAG antibody, indicating that the FLAG-containing portion of the fusion protein was cleaved from the C-terminus of the fusion protein, i.e., cleavage occurred after the FLAG sequence. The expression product of the Fab-His-SpyTag-FLAG fusion protein was recognized by the labeled anti-Histidine tag antibody, but not by the labeled anti-FLAG antibody, indicating that the FLAG-containing portion of the fusion protein was cleaved from the C-terminus of the fusion protein, i.e., cleavage occurred after the His-tag.

[0147] The masses of light and heavy chain peptides of the Fab-His-SpyTag (SEQ ID NO: 6) expression product purified by Ni-NTA were determined by MALDI TOF mass spectrometry (4800 MALDI TOF / TOF analyzer, AB Sciex). The samples were desalted (ZipTip C4, Merck Millipore) and co-crystallized with sinapinic acid. The masses were determined in linear mode between 5000-50000 m / z. One spectrum was loaded with 4000 laser shots. Protein standard I (Bruker) was used for mass calibration. The mass spectrometry results are as follows.

[0148] Light chain:

[0149] Calculated mass (full length): 22691Da

[0150] Measured mass (m / z) 22691Da

[0151] Heavy Chain:

[0152] Calculated mass (full length): 26437Da

[0153] Measured mass (m / z) 25416Da

[0154] Calculated mass (-9aa) 25403Da

[0155] The results of mass spectrometry analysis showed that 9 amino acids were cleaved at the C-terminus of the Fab-His-SpyTag fusion protein. Therefore, under the action of proteases in the periplasm of E. coli, 9 amino acid residues were cleaved from the C-terminus of SpyTag (length 13 amino acids: AHIVMVDAYKPTK; SEQ ID NO: 1) after the valine at amino acid position 4. Without wishing to be bound by theory, the applicant believes that since SpyTag is cleaved in all constructs, the cleavage of SpyTag is independent of the amino acid sequence before and after SpyTag.

[0156] Example 2 - Periplasmic expression of various maltose binding protein-SpyTag fusion proteins

[0157] The gene encoding maltose binding protein (MBP) with a FLAG-SpyTag-His tag (SEQ ID NO: 11) or a His-SpyTag-FLAG tag (SEQ ID NO: 12) at the C-terminus was cloned into an expression vector for periplasmic expression and transformed into Escherichia coli TG1F-. The C-terminus of the MBP used in this example was removed by 4 amino acids. Figure 4 A partial sequence of the construct containing MBP is shown. Expression and purification of the construct were performed as described in Example 1.

[0158] Initial attempts to purify the periplasmic expressed MBP-SpyTag fusion protein were unsuccessful. Similar to the Fab fragment in Example 1, the periplasmic construct containing the FLAG-SpyTag-His peptide sequence at the C-terminus of MBP could not be purified. The construct containing the His-SpyTag-FLAG peptide sequence at the C-terminus of MBP could be purified, but did not react in the subsequent SpyTag-SpyCatcher protein ligation reaction. Western blot analysis of the expression products before purification gave similar results as described in Example 1, i.e., the first tag of each construct could be detected, but the last tag could not be detected.

[0159] MALDI-TOF-mass spectrometry analysis of the MBPΔ4aa-His-SpyTag-FLAG construct (SEQ ID NO: 12) was performed as described in Example 1. The mass spectrometry results are as follows.

[0160] Full-length protein:

[0161] Calculated mass (full length): 44241Da

[0162] Measured mass (m / z): 44273Da (full length)

[0163] Measured mass (m / z): 42032Da (main product)

[0164] Calculated mass (1-382aa) 42011Da

[0165] The mass spectrometry results showed a small amount of full-length protein. However, the main product consisted of amino acids 1-382, indicating that the cleavage occurred after the valine at amino acid position 4 of SpyTag. This is the same position where the Fab fragment in Example 1 was cut. Without wishing to be bound by theory, the applicant believes that the cleavage of SpyTag is independent of the Fab amino acid sequence or structure, because the cleavage also occurs on a structurally completely independent protein (ie, MBP). Mass spectrometry also showed that the ompT signal peptide used for transport to the periplasm was cut, which occurred after the protein was transferred to the periplasm. Since the expression of the full-length SpyTag fusion protein in the cytoplasm is described in Keeble et al. (2019), the applicant assumes that the SpyTag cleavage described in Examples 1 and 2 occurs in the periplasm.

[0166] To test this hypothesis, MBP with FLAG-SpyTag and His tag at the C-terminus was cloned into an expression vector for cytoplasmic expression in the absence of the ompT signal peptide and transformed into E. coli. Expression and purification were performed as described in Example 1. Cytoplasmic expression of the construct resulted in high yields of the full-length product (about 11 mg / L).

[0167] Periplasmic expression of Fab and MBP-SpyTag fusion proteins resulted in truncated proteins with SpyTag cleaved, whereas cytoplasmic expression of MBP with the same amino acid sequence (without the signal peptide) produced a full-length product. Without being bound by theory, applicants hypothesize that cleavage of SpyTag is due to one or more periplasmic proteases.

[0168] Example 3 - Periplasmic expression of scFv-SpyTag fusion protein

[0169] The C-terminus carries FLAG-SpyTag-His (SEQ ID NO: 34; Fig. 9 ) was cloned into an expression vector for periplasmic expression and transformed into Escherichia coli TG1F-. Fig. 9 Partial sequences of scFv constructs are shown. Expression and purification of constructs were performed as described in Example 1.

[0170] Initial attempts to purify the periplasmic expressed scFv-SpyTag fusion protein were unsuccessful. Similar to the Fab fragment in Example 1 and the MBP construct in Example 2, the periplasmic scFv construct containing the FLAG-SpyTag-His peptide sequence at the scFv C-terminus could not be purified via the His tag.

[0171] Example 4 - Periplasmic expression of Fab-X-SpyTag fusion protein with X as linker in various bacterial strains

[0172] The Fab-SpyTag-His (SEQ ID NO: 3) and Fab-FLAG-Spy-His (SEQ ID NO: 4) constructs were transformed into the following E. coli strains for periplasmic expression to determine which periplasmic protease(s) cleave the SpyTag:

[0173] 1.TG1 F-(without F-episome; Bio-Rad)

[0174] 2.Jw0157:degP-(Yale Coli Genetic Repository)

[0175] 3. KS476: degP- (Yale Coli Genetic Repository)

[0176] 4. KS1000: prc-(or tsp-) (New England Biolabs)

[0177] 5.JW3203:degQ-(Yale Coli Genetic Reservoir)

[0178] 6.27C2:degP-,ptr3-,ompT-(ATCC)

[0179] 7.HM130:degP-,ptr-,ompT-,tsp-,eda (University of Texas at Austin)

[0180] Transformants were cultured, expressed and purified as described in Example 1. The concentrations of purified fusion proteins of each E. coli strain were determined, and the expression products of each fusion were analyzed by SDS-PAGE under non-reducing conditions. The expression of full-length Fab (including tags) was shown as heavy and light chains on SDS-PAGE, while SpyTag cleavage resulted in Fab without purification tags, which was not purified. These two types of fusion proteins were expressed as full-length proteins only in KS1000 (tsp-) and HM130 (degP-, ptr-, ompT-, tsp-, eda) strains, indicating that tsp protease is involved in the cleavage of SpyTag.

[0181] Example 5 - Generation of knockout cell lines using TG1 F- strain for Fab-SpyTag constructs

[0182] Since all expression strains except TG1 F- used in Example 4 grew poorly and / or failed to produce high yields of properly folded soluble Fab, a TG1 F-protease knockout strain was constructed to increase the yield of Fab fused to SpyTag.

[0183] As described by Datsenko and Wanner (2000), Proc Natl Acad Sci USA, 97(12):6640–6645, mutant E. coli TG1 F-cell strains were generated in which the tsp gene, the degP gene, or both the tsp and depP genes were knocked out. Briefly, the knockout gene was transformed with a PCR product containing the homologous flanking regions of the FRT site and the antibiotic resistance gene together with a plasmid containing the lambda recombinase. Clones were selected by antibiotic resistance in which the gene was replaced by the PCR product by recombination. Next, these clones were transfected with the Flp recombinase, resulting in excision of the resistance gene.

[0184] Fab-SpyTag-His and Fab-FLAG-Spy-His constructs (i.e., the same constructs as in Example 4) were transformed into strains constructed as above, i.e., TG1 F-Δtsp (SK4, DSMZ accession number DSM33004), TG1 F-ΔdegP, and TG1 F-ΔtspΔdegP double knockout. The expressed fusion proteins were purified and analyzed by SDS-PAGE as described in Example 1.

[0185] Results: Both fusion proteins were expressed as full-length proteins in the TG1 F-Δtsp strain (SK4) and the TG1 F-ΔtspΔdegP double knockout strain, but not in the TG1 F-ΔdegP strain, indicating that degP is not involved in SpyTag cleavage. In addition, the expression of TG1 F-Δtsp and TG1 F-ΔtspΔdegP gave similar yields, leading to the conclusion that only tsp cleaves SpyTag.

[0186] Next, the ability of Fab-SpyTag-His and Fab-FLAG-SpyTag-His constructs to form a covalent bond with SpyCatcher via protein ligation was tested. SpyCatcher was produced by bacterial cytoplasmic expression and purified by Ni-NTA as described by Zakeri et al. (2012). The fusion proteins were expressed and purified using the SK4 strain as described above. Each fusion protein (final concentration 15 μM) was mixed with SpyCatcher (final concentration 20 μM) in 1xPBS buffer and coupled at room temperature for 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and overnight. SDS-PAGE was used to test the new band that appeared on the gel, which corresponded to the SpyTag fusion-SpyCatcher coupling product (see Figure 5). For both fusion proteins, new SpyTag fusion-SpyCatcher bands of the correct size were observed, indicating that both Fab-SpyTag-H and Fab-FLAG-SpyTag-H were coupled to SpyCatcher and that the SpyTag portion of the fusion proteins was intact and fully functional.

[0187] Example 6 - Periplasmic expression of MBP-SpyTag fusion protein in a protease knockout strain

[0188] The C-terminus carries a FLAG-SpyTag-His tag (SEQ ID NO: 11; Figure 4 The gene encoding the maltose binding protein (MBP) of ) was cloned into an expression vector for periplasmic expression and transformed into the E. coli SK4 strain. The most common MBP sequence in fusion proteins used for protein crystallization studies has 4 amino acids truncated at the C-terminus (Waugh, 2016). This sequence was used for these experiments. Expression and purification of the constructs were performed as described in Example 1. A high yield (about 10 mg / L) of the full-length protein was obtained using a tsp protease knockout strain.

[0189] Example 7 - Periplasmic expression of scFv-SpyTag fusion protein in a protease-deficient strain

[0190] The C-terminus carries FLAG-SpyTag-His (SEQ ID NO: 34; Fig. 9 The coding gene of the scFv of ) was cloned into an expression vector for periplasmic expression and transformed into the E. coli TG1F-Δtsp knockout strain. The expression and purification of the construct were performed as described in Example 1. A high yield (about 7 mg / L) of the full-length protein was obtained using the tsp protease knockout strain.

[0191] Example 8 - Generation of knockout cell lines using TG1 F- strain for Fab-SpyTag002 construct

[0192] A human antibody fragment in Fab format with SpyTag002 at the C-terminus of the truncated heavy chain (i.e., Fab-SpyTag002 construct, Figure 6The coding gene of Fab-SpyTag002 was cloned into an expression vector with signal sequences on the H and L chains of Fab, which imports the nascent chains into the periplasm by bacterial transport. This vector was then used to transform E. coli TG1 F- (without the F-episome; Bio-Rad). The construct was expressed and purified as described in Example 1. All attempts to purify the Fab-SpyTag002 fusion protein with a C-terminal His tag (SEQ ID NO: 13 and 14) were unsuccessful. The construct with a His tag between the Fab and the SpyTag (SEQ ID NO: 15) could be purified, but did not carry a functional SpyTag002.

[0193] Fab-SpyTag002-His( Figure 6 The expression of constructs (SEQ ID NO. 13) shown in FIG. 1 (containing Fabs derived from various antibodies) was tested in the following knockout cell lines, where constructs containing SpyTag but not SpyTag002 were successfully expressed:

[0194] 1.KS1000(Δtsp)

[0195] 2.SK4(TG1 F-Δtsp)

[0196] 3.TG1 F-ΔtspΔdegP

[0197] 4. HM130 strain (Δtsp, ΔdegP, ΔompT, Δptr).

[0198] After purification via the His tag, the products were analyzed by SDS-PAGE as described in Example 4. The full-length fusion protein failed in KS1000 (Δtsp), SK4 (TG1 F-Δtsp), and TG1 F-ΔtspΔdegP, but succeeded in the HM130 strain (Δtsp, ΔdegP, ΔompT, Δptr) with acceptable or "high" yields of purified antibody (i.e., about 10 mg / L).

[0199] Next, as described in Example 1, Fab-His-SpyTag002 (SEQ ID NO: 15; Figure 6 ) to determine where the fusion protein was cut. The mass spectrometry results are as follows.

[0200] TG1 F-expression of Fab-His-SpyTag002:

[0201] Light chain:

[0202] Calculated mass (full length): 22691Da

[0203] Measured mass (m / z) 22682Da

[0204] Heavy Chain:

[0205] Calculated mass (full length): 26643Da

[0206] Measured mass (m / z) 25478Da

[0207] Calculated mass (-9aa) 25488Da

[0208] TG1 F-ΔtspΔdegP expression of Fab-His-SpyTag002:

[0209] Light chain:

[0210] Calculated mass (full length): 22691Da

[0211] Measured mass (m / z) 22680Da

[0212] Heavy Chain:

[0213] Calculated mass (full length): 26643Da

[0214] Measured mass (m / z): 26626Da (full length)

[0215] Measured mass (m / z) 26182Da (-3aa)

[0216] Calculated mass (-3aa) 26195Da

[0217] Results: Based on the results of mass spectrometry analysis of non-protease-deficient strains, a 9-amino acid portion was cut from the C-terminus of the fusion protein. This is the same as the observed cleavage site of SpyTag, which is cleaved by the tsp protease. According to the results of mass spectrometry analysis of strains deficient in tsp and degP proteases (and with ompT protease), a 3-amino acid portion was cut from the C-terminus. According to all mass spectrometry analysis results, SpyTag002 (14 amino acids in length: VPTIVMVDAYKRYK; SEQ ID NO: 2) is cleaved by the tsp protease after the valine at amino acid position 5 and by the second protease after the lysine at amino acid position 11. Therefore, two different proteases are involved in the cleavage of SpyTag002, one of which is tsp, and based on the expression results in the four strains, it can be assumed that the second is ompT or ptr.

[0218] The following TG1 F-knockout strains were prepared using the same process as described in Example 4: TG1F-ΔompT strain and TG1F-ΔtspΔompT strain (SK13, DSMZ accession number DSM 33005). The Fab-SpyTag002-His construct ( Figure 6 ; SEQ ID NO: 13) and Fab-FLAG-SpyTag002-His construct ( Figure 6 ; SEQ ID NO:14). Expression in the TG1F-ΔompT strain did not produce significant amounts of full-length protein because the tsp cleavage site of SpyTag was still present in SpyTag002. Expression of the construct in the TG1 F-1ΔtspΔompT strain (SK13) was successful, and a high protein yield (i.e., about 10 mg / L) of full-length protein was obtained after purification. Therefore, both tsp and omp T proteases are involved in cleaving SpyTag002.

[0219] Example 9 - Testing of various strategies to protect SpyTag during periplasmic expression of Fab-SpyTag fusion proteins in non-protease-deficient strains

[0220] Experiments were performed to determine whether SpyTag could be protected from cleavage during expression of SpyTag fusion proteins in a non-protease-deficient E. coli TG1 F- cell line.

[0221] The following strategies were tested to prevent SpyTag cleavage:

[0222] 1.ST2- A linker with two cysteine ​​residues (CXC) was introduced between the tag and SpyTag to form a disulfide bridged loop (Wu et al., 2012). Without being bound by theory, the applicants speculated that this non-linear linker might prevent protease binding: Fab heavy chain-Flag-CXC-SpyTag-His6.

[0223] 2. ST3-Introduction of polyproline linkers in fusion proteins. Polyproline linkers form polyproline helices (Qi et al., 2018). Without being bound by theory, the applicants speculate that the polyproline helices can prevent protease binding. Two fusion proteins with polyproline linkers were prepared:

[0224] a. Strong: PPPPPT

[0225] b. Weak: PLPPPF

[0226] 3. ST4 - Two amino acids protruding from the globular folded domain of the Fab heavy chain (PDB structure accession number 2JB5). These two amino acids were removed and the SpyTag was attached to a truncated Fab heavy chain (ending with a conserved valine (IMGT position number 121) and two hinge amino acids glutamic acid and proline) that does not contain an unfolded linker sequence (see "b" below). Without being bound by theory, the applicants speculated that moving the SpyTag closer to the folded domain could prevent protease binding.

[0227] a.Fab heavy chain: ...VEPKS-COOH

[0228] b.ST4:…VEP-SpyTag-His

[0229] 4. ST5 - The idea is similar to ST2, but includes FLAG and SpyTag in a disulfide bridged loop: Fab heavy chain-C-Flag-Spy-C-His6.

[0230] The above constructs were expressed in non-protease-deficient E. coli TG1 F-cell strain, purified via the His tag, and analyzed by SDS-PAGE as described above. Only ST4 (SEQ ID NO: 16; Figure 7 ) was expressed without SpyTag cleavage. As described in Example 5, the coupling of ST4 to SpyCatcher was tested. SDS-PAGE analysis showed that within about 2 hours, there were no new bands in the SpyTag fusion-SpyCatcher coupling product. Therefore, the fusion protein with two amino acids removed from the Fab heavy chain was expressed without SpyTag cleavage, but was not coupled to SpyCatcher. Without wishing to be bound by theory, the applicant believes that the close proximity of SpyTag to the folded antibody structure prevents protease binding and cleavage of SpyTag, but also spatially hinders the binding of SpyCatcher to SpyTag, which is necessary for the SpyTag-SpyCatcher reaction.

[0231] Further constructs based on the ST4 design were prepared and tested as described previously, such as Figure 7 (SEQ ID NO:17-32) to determine whether SpyTag is cut. As described in Example 5, the SpyTag and SpyCatcher coupling abilities of these constructs were also tested. Table 1 summarizes the full-length expression yields and connection results of all ST4 constructs. "High" means that the fusion protein expression yield is about 5-10 mg / L, "low" means that the expression yield is about 2-4 mg / L, and "very low" means that the expression yield is less than about 2 mg / L.

[0232] Table 1

[0233]

[0234]

[0235] The results in Table 1 show that SpyTag directly connected to the C-terminus of the Fab heavy chain (or ST4+2) has the best overall performance, where the fusion protein shows a high full-length protein yield (ie, about 5-10 mg / L) and is connected to SpyCatcher. Fusion proteins with 1, 2, or 3 amino acid linkers between the C-terminus of the Fab heavy chain and SpyTag (ie, ST4+3 and ST4+4 / 5) are significantly cleaved by proteases, resulting in low or very low yields of full-length proteins. Without being bound by theory, the applicant believes that the longer the linker between the C-terminus of the folded Fab globular domain and the SpyTag, the easier it is for the SpyTag to be cut and connected to the SpyCatcher. The applicant also believes that the fusion protein directly connecting the SpyTag to the C-terminus of the Fab heavy chain is the best compromise between connecting the SpyTag to the folded domain close to the Fab to avoid periplasmic protease cleavage and leaving enough space for the SpyTag to be spatially connected to the SpyCatcher.

[0236] Example 10 - Testing of strategies to protect SpyTag during periplasmic expression of maltose binding protein-SpyTag fusion proteins in non-protease-deficient strains

[0237] Experiments were performed to test the hypothesis that moving SpyTag closer to the folded domain in maltose binding protein (MBP) would protect SpyTag from periplasmic protease digestion. MBP was chosen because it is a different protein than Fab. The MBP sequence most commonly used for crystallization studies is truncated by 4 amino acids at the C-terminus (Waugh, 2016). Since crystallization experiments and structure determination generally benefit from increased protein rigidity (i.e., a folded domain without a flexible linker), and the stability of SpyTag to protease cleavage is expected to benefit from this, the same sequence was used in this example. A SpyTag-His tag (SEQ ID NO:33; Figure 8 The coding gene for MBP of 100 μg / mL was cloned into an expression vector for periplasmic expression and transformed into E. coli TG1 F-strain. Expression and purification of the construct were performed as described in Example 1. High yields (about 7 mg / L) of the full-length protein were obtained in the non-protease-deficient TG1 F-strain. As described in Example 5, the construct was tested for the ability of SpyTag to connect to SpyCatcher and found to be true.

[0238] Directly connecting SpyTag to the folded domain of MBP results in a fusion protein in which SpyTag is protected from periplasmic protease cleavage and SpyTag is still functional, similar to the Fab fusion protein in Example 9, in which SpyTag is directly connected to the heavy chain. In contrast, the MBP-Flag-SpyTag-His and MBP-His-SpyTag-Flag fusion proteins in Example 2 contain tags as linkers with 13 and 12 amino acids, respectively, which makes SpyTag susceptible to protease cleavage. Without wishing to be bound by theory, the applicant believes that these results indicate that the cleavage of SpyTag fused to the C-terminus of Fab without a linker (i.e., the Fab-SpyTag fusion protein tested in Example 9) is independent of the Fab structure, because cleavage also occurs when SpyTag is fused to the C-terminus of MBP via a linker, which is structurally unrelated to Fab.

[0239] Example 11 - Testing of strategies to protect SpyTag during periplasmic expression of scFv-SpyTag fusion proteins in non-protease-deficient strains

[0240] Experiments were performed to test the hypothesis that moving SpyTag closer to the folded domain in the scFv would protect SpyTag from periplasmic protease digestion. The scFv was chosen because it is a different protein and has a different structure than Fab or MBP. The scFv used in this example had 6 amino acids removed from the C-terminus, resulting in the scFv being truncated in the FR4 region of its light chain (encoded by the J gene). The scFv (Δ6aa)-SpyTag-His fusion construct (SEQ ID NO: 35; Fig. 9 ) was cloned into an expression vector for periplasmic expression and transformed into E. coli TG1F-; wherein SpyTag-His was directly fused (i.e., without a linker sequence) to the C-terminus of the C-terminally truncated scFv. Expression and purification of the fusion protein were performed as described in Example 1. A high yield of full-length protein (about 8 mg / L) was produced, indicating that direct attachment of SpyTag to scFv protects SpyTag from periplasmic cleavage.

[0241] Example 12 - Periplasmic expression of fFab-SpyTag003 fusion protein in a protease-deficient strain

[0242] The genes encoding human antibody fragments in Fab format with SpyTag003 at the C-terminus of the truncated heavy chain (i.e., constructs of SEQ ID 3 and 4 with SpyTag002 replaced by SpyTag003 (SEQ ID NO: 36)) were cloned into expression vectors for periplasmic expression as described in Example 1. The plasmids were transformed into E. coli TG1 F- or SK4 strains, and expression and purification of the constructs were performed as described in Example 1. All attempts to purify the Fab-SpyTag003 fusion protein were unsuccessful.

[0243] The plasmid was transformed into the SK13 strain and expressed and purified as described in Example 1. The expression of the construct in the TG1F-1ΔtspΔompT strain (SK13) was successful, and the full-length protein was obtained in good protein yield (i.e., about 6 mg / L) after purification. Therefore, both tsp and omp T proteases are involved in the cleavage of SpyTag003.

[0244] All patents, patent applications, and other published references cited in this specification are hereby incorporated by reference in their entirety.

[0245] SEQ ID NO: 1 (SpyTag)

[0246] AHIVMVDAYK PTK

[0247] SEQ ID NO:2 (SpyTag002)

[0248] VPTIVMVDAY KRYK

[0249] SEQ ID NO: 3 (Fab-Spy-His; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0250] Human CH1-EPKSEFGAHIVMVDAYKPTKGAPHHHHHHH

[0251] SEQ ID NO:4 (Fab-FLAG-Spy-His; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0252] Human CH1-EPKSEFDYKDDDDKGGSAHIVMVDAYKPTKGAPHHHHHH

[0253] SEQ ID NO: 5 (Fab-X-Spy-His; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0254] Human CH1-EPKSEFGGGSGGGSAHIVMVDAYKPTKGAPHHHHHHH

[0255] SEQ ID NO:6 (Fab-His-Spy; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0256] Human CH1-EPKSEFHHHHHHGAPGAHIVMVDAYKPTK

[0257] SEQ ID NO:7 (Fab-His-Spy-FLAG; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0258] Human CH1-EPKSEFHHHHHHGAPGAHIVMVDAYKPTKGGSDYKDDDDK

[0259] SEQ ID NO: 8 (Fab-Spy-Sx2; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0260] people

[0261] CH1-EPKSEFGAHIVMVDAYKPTKGAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0262] SEQ ID NO:9 (Fab-FLAG-Spy-Sx2; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0263] people

[0264] CH1-EPKSEFDYKDDDDKGGSAHIVMVDAYKPTKGAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0265] SEQ ID NO: 10 (Fab-X-Spy-Sx2; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0266] people

[0267] CH1-EPKSEFGGGSGGGSAHIVMVDAYKPTKGAPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0268] SEQ ID NO:11(MBP(Δ4aa)-FLAG-Spy-His)

[0269] MKKTAIAIAVALAGFATVAQAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKA FQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKA GLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTEFDYKDDDDKGGSAHIVMVDAYKPTKGAPHHHHHH

[0270] SEQ ID NO:12(MBP(Δ4aa)-His-Spy-FLAG)

[0271] MKKTAIAIAVALAGFATVAQAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKA FQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKA GLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKD KPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTEFHHHHHHGAPGAHIVMVDAYKPTKGGSDYKDDDDK

[0272] SEQ ID NO: 13 (Fab-Spy2-His; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0273] Human CH1-EPKSEFGVPTIVMVDAYKRYKGAPHHHHHHH

[0274] SEQ ID NO: 14 (Fab-FLAG-Spy2-His; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0275] Human CH1-EPKSEFDYKDDDDKGGSVPTIVMVDAYKRYKGAPHHHHHHH

[0276] SEQ ID NO: 15 (Fab-His-Spy2; partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121)

[0277] Human CH1-EPKSEFHHHHHHGAPGVPTIVMVDAYKRYK

[0278] SEQ ID NO: 16 (Fab-Spy-His_ST4 (HCΔ2aa); partial amino acid sequence starting from the first two amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0279] HumanCH1-EPAHIVMVDAYKPTKGAPHHHHHH

[0280] SEQ ID NO: 17 (Fab-Spy-His_ST4+1 (HCΔ1aa); partial amino acid sequence starting from the first 3 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0281] PEOPLECH1-EPKAHIVMVDAYKPTKGAPHHHHHH

[0282] SEQ ID NO: 18 (Fab-Spy-His_ST4+1 (HCΔ1aa); partial amino acid sequence starting from the first two amino acid residues of the hinge domain of human IgG1 and the third amino acid residue being replaced by "E"; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0283] HUMANCH1-EPEAHIVMVDAYKPTKGAPHHHHHH

[0284] SEQ ID NO: 19 (Fab-Spy-His_ST4+1 (HCΔ1aa); partial amino acid sequence starting from the first two amino acid residues of the hinge domain of human IgG1 with the third amino acid residue replaced by "G"; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0285] CH1-EPGAHIVMVDAYKPTKGAPHHHHHH

[0286] SEQ ID NO:20 (Fab-Spy-His_ST4+1 (HCΔ1aa); partial amino acid sequence starting from the first two amino acid residues of the hinge domain of human IgG1 and the third amino acid residue being substituted with "R"; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0287] PEOPLECH1-EPRAHIVMVDAYKPTKGAPHHHHHH

[0288] SEQ ID NO:21 (Fab-Spy-His_ST4+2(HC); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0289] Human CH1-EPKSAHIVMVDAYKPTKGAPHHHHHHH

[0290] SEQ ID NO:22 (Fab-Spy-His_ST4+2(HC); partial amino acid sequence starting from the first 3 amino acid residues of the hinge domain of human IgG1 and the 4th amino acid residue being replaced by "E"; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0291] Human CH1-EPKEAHIVMVDAYKPTKGAPHHHHHHH

[0292] SEQ ID NO:23 (Fab-Spy-His_ST4+2(HC); partial amino acid sequence starting from the first 3 amino acid residues of the hinge domain of human IgG1 with the 4th amino acid residue replaced by "K"; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0293] CH1-EPKKAHIVMVDAYKPTKGAPHHHHHH

[0294] SEQ ID NO:24 (Fab-Spy-His_ST4+2(HC); partial amino acid sequence starting from the first 3 amino acid residues of the hinge domain of human IgG1 with the 4th amino acid residue replaced by "S"; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0295] Human CH1-EPKGAHIVMVDAYKPTKGAPHHHHHHH

[0296] SEQ ID NO:25 (Fab-Spy-His_ST4+3(HC+1); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0297] Human CH1-EPKSDAHIVMVDAYKPTKGAPHHHHHHH

[0298] SEQ ID NO:26 (Fab-Spy-His_ST4+3(HC+1); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0299] Human CH1-EPKSKAHIVMVDAYKPTKGAPHHHHHHH

[0300] SEQ ID NO:27 (Fab-Spy-His_ST4+3(HC+1); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0301] CH1-EPKSPAHIVMVDAYKPTKGAPHHHHHH

[0302] SEQ ID NO:28 (Fab-Spy-His_ST4+3(HC+1); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0303] Human CH1-EPKSSAHIVMVDAYKPTKGAPHHHHHHH

[0304] SEQ ID NO:29 (Fab-Spy-His_ST4+4(HC+2); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0305] Human CH1-EPKSGGAHIVMVDAYKPTKGAPHHHHHHH

[0306] SEQ ID NO:30 (Fab-Spy-His_ST4+4(HC+2); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0307] Human CH1-EPKSGFAHIVMVDAYKPTKGAPHHHHHHH

[0308] SEQ ID NO:31 (Fab-Spy-His_ST4+5(HC+3); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0309] Human CH1-EPKSEGGAHIVMVDAYKPTKGAPHHHHHHH

[0310] SEQ ID NO:32

[0311] SEQ ID NO:32 (Fab-Spy-His_ST4+5(HC+3); partial amino acid sequence starting from the first 4 amino acid residues of the hinge domain of human IgG1; human Ig CH1 according to the IMGT definition, ending at the conserved valine at IMGT position number 121):

[0312] Human CH1-EPKSGGSAHIVMVDAYKPTKGAPHHHHHHH

[0313] SEQ ID NO:33(MBP(Δ4aa)-Spy-His_ST4):

[0314] MKKTAIAIAVALAGFATVAQAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITP DKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTAHIVMVDAYKPTKGAPHHHHHHH

[0315] SEQ ID NO:34 (scFv-F-Spy-H):

[0316] QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGQEFDYKDDDDKGGSAHIVMVDAYKPTKGAPHHHHHH

[0317] SEQ ID NO:35 (scFv(Δ6aa)-Spy-H):

[0318] QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKAHIVMVDAYKPTKGAPHHHHHH

[0319] SEQ ID NO:36 (SpyTag003)

[0320] RGVPHIVMVDAYKRYK

[0321] References

[0322] U.S. Patent No. 9,547,003

[0323] U.S. Patent Application No.: 2003 / 0198956

[0324] WO 2016 / 193746

[0325] WO 2016 / 183387

[0326] WO 2018 / 053180

[0327] IMGT definition according to: Lefranc M.-P.. De RK, Tomar NV, Immunoinformatics of C and G domains: IG, TR and IgSF, MH and MhSF Defining systems, Immunoinformatics: From biology to informatics, 2014, Volume 1184, 2nd edition Springer, NY Humana Press (pp. 59-107).

[0328] Abe, H., Rie, W., Yonemura, H., Yamada, S., Goto, M., and Kamiya, N., (2013). Split Spy0128 as a strong scaffold for protein cross-linking and immobilization. Bioconjugate Chem., 24(2), 242–250.

[0329] Alam et al., 2017. Construction of synthetic model antibodies using the SpyTag / SpyCatcher protein ligation system. Chembiochem. 18(22), 2217-2221.

[0330] Alves, NJ, Turner, KB, Daniele, MA, Oh, E., Medintz, IL, Walper, SA, Bacterial nanobioreactors - directing enzyme packaging into bacterial outer membrane vesicles. ACS Appl Mater Interfaces, 2015; 7: 24963-24972.

[0331] Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE., 2000, Protein Data Bank. Nucleic Acids Res. 28(1), 235-42.

[0332] Brannon, J. R., Burk, D. L., Leclerc, J. M., Thomassin, J. L., Portt, A., Berghuis, A. M., Gruenheid, S., Le Moual, H., 2015. Inhibition of outer membrane proteases of the omptin family by aprotinin. Infect Immun., 83: 2300–2311.

[0333] Buldun, CM, Jean, J., Bedford, MR, Howarth, M., 2018, Snoop ligase catalyzes peptide-peptide locking and enables solid-phase coupling separation. J Am Chem Soc. 140(8), 3008-3018.

[0334] Datsenko, KA and Wanner, BL, 2000, One-step inactivation of chromosomal genes in Escherichia coli K12 using PCR products. Proc Natl Acad Sci USA. 97(12), 6640–6645.

[0335] Ezkurdia I, Tress ML., 2011, Definition and prediction of protein domains. Curr Protoc Protein Sci. Chapter 2 Unit 2. 14. doi:10.1002 / 0471140864.ps0214s66.

[0336] Fierer, J. O., Veggiani, G., Howarth, M., 2014, Spy ligase peptide-peptide linked polymeric affimers for enhanced magnetic cancer cell capture. Proc Natl Acad Sci USA. 111: E1176-1181.

[0337] Geller, BL, 2005, Antimicrobial antisense. Curr Opin Mol Ther, 7: 109–113.

[0338] Keeble, A.H., Banerjee, A., Ferla, M.P., Reddington, S.C., Khairil Anuar, I.A., Howarth, M., 2017, Accelerating amidation reactions using SpyTag / SpyCatcher evolution for analyzing membrane dynamics. Ange, Chem. Int. Ed. 56: 16521-16525.

[0339] Keeble, A.H., Howarth, M., 2019. Inside information on successful covalent protein coupling with the help of SpyBank. 617: 443-461. doi.org / 10.1016 / bs.mie.2018.12.010.

[0340] Keeble,AH,Turkki,P.,Stokes,S.,Khairil Anuar,INA,Rahikainen,R., VP, Howarth, M., 2019, Towards unlimited affinity through engineering of peptide-protein interactions. Proc Natl Acad Sci USA. 116: 26526-26533.

[0341] Keiler, K. and Sauer, R., 1995, Identification of active site residues in Tsp protease. J Biol Chem, 270(48), 28864-28868.

[0342] Knappik, A., Brundiers, R., 2009, Expression and purification of recombinant antibodies, in: Walker, JM, ed.: The Protein Protocols Handbook. 3rd ed. New York: Humana Press Inc., 1929-1943.

[0343] Li et al., 2014, Structural analysis and optimization of covalent binding between SpyCatcher and peptide tags, J Mol Biol. 426(2), 309-17.

[0344] Nguyen, G.K.T., Wang, S., Qiu, Y., Hemu, X., Lian, Y., Tam, JP. 2014. Clitoria ternatea mucinase 1 is an Asx-specific ligase that enables peptide macrocyclization and synthesis. Nat Chem Biol. 10: 732-738.

[0345] Plückthun A., 1990, Antibodies from Escherichia coli. Nature 347, 497–498.

[0346] Prouty, WF, Goldberg, AL, 1972, Effects of protease inhibitors on protein degradation in Escherichia coli. J Biol Chem, 247: 3341–3352.

[0347] Qi, F. et al., 2018, Evolutionary analysis of polyproline motifs in Escherichia coli reveals their regulatory role in translation. PLoS Comput Biol. 14(2), e1005987.

[0348] Reddington, SC, Howarth, M., 2015, Secrets of covalent interactions in biomaterials and biotechnology: SpyTag and SpyCatcher. Current Opinion in Chemical Biology. 29: 94-99.

[0349] Schmohl, L., Schwarzer, D., 2014, Sortase-mediated ligation of site-specific modifications of proteins. Current Opinion in Chemical Biology. 22: 122-128.

[0350] Siegmund et al., 2016. Spontaneous formation of isopeptide bonds is a powerful tool for engineering site-specific antibody-drug conjugates. Scientific Reports. 6, 39291.

[0351] Silber, KR, Keiler, KC, Sauer, RT, 1991, Tsp: a tail-specific protease that selectively degrades proteins with a nonpolar C-terminus. Proc Natl Acad Sci USA, 89: 295-299.

[0352] Tan et al. (2016). Kinetically controlled tag-catcher interactions for directed covalent protein assembly. PLoS ONE, 11(10), e0165074.

[0353] Toplak, A., Nuljens, T., Quaedflieg, P. J. L., Wu, B., Janssen, D. B., 2016. Peptidyl ligase, an enzyme for efficient chemoenzymatic peptide synthesis and cyclization in water. Adv Synth Catal. 358: 32140-32147.

[0354] Veggiani, G. et al., 2016, Programmable multimeric proteomes constructed using dual-peptide superglue. Proc Natl Acad Sci USA 113: 1202-1207.

[0355] Waugh, DS, 2016, Crystal structure of MBP fusion protein. Protein Sci. 25: 559-571.

[0356] Wu, C., Leroux, JC, Gauthier, MA, 2012. Double disulfide bonds for orthogonal disulfide pairing and directional folding of polycyclic peptides. Nat Chem. 4: 1044-1049.

[0357] Yumura, K. et al., 2017, Construction of bispecific antibodies targeting two epitopes of a single antigen using SpyTag / SpyCatcher. J Biochem. 162(3), 203-210.

[0358] Zakeri, B. et al., 2012, Peptide tag forming a rapid covalent bond to a protein, through engineering abacterial adhesion. Proc Natl Acad Sci USA. 109: E690-697. Sequence Listing <110> BIO-RAD ABD SEROTEC GMBH <120> Periplasmic fusion protein <130> BRL.130XC1PCT <150> US 62 / 819,758 <151> 2019-03-18 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 <210> 2 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Val Pro Thr Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys 1 5 10 <210> 3 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Glu Pro Lys Ser Glu Phe Gly Ala His Ile Val Met Val Asp Ala Tyr 1 5 10 15 Lys Pro Thr Lys Gly Ala Pro His His His His His His 20 25 <210> 4 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Glu Pro Lys Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly 1 5 10 15 Ser Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ala 20 25 30 Pro His His His His His His 35 <210> 5 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Glu Pro Lys Ser Glu Phe Gly Gly Gly Ser Gly Gly Gly Ser Ala His 1 5 10 15 Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ala Pro His His 20 25 30 His His His His 35 <210> 6 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Glu Pro Lys Ser Glu Phe His His His His His Gly Ala Pro Gly 1 5 10 15 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 20 25 <210> 7 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 7 Glu Pro Lys Ser Glu Phe His His His His His Gly Ala Pro Gly 1 5 10 15 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Gly Ser 20 25 30 Asp Tyr Lys Asp Asp Asp Asp Lys 35 40 <210> 8 <211> 53 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 8 Glu Pro Lys Ser Glu Phe Gly Ala His Ile Val Met Val Asp Ala Tyr 1 5 10 15 Lys Pro Thr Lys Gly Ala Pro Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30 Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala Trp Ser His 35 40 45 Pro Gln Phe Glu Lys 50 <210> 9 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 9 Glu Pro Lys Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly 1 5 10 15 Ser Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ala 20 25 30 Pro Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly 35 40 45 Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 50 55 60 <210> 10 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Glu Pro Lys Ser Glu Phe Gly Gly Gly Ser Gly Gly Gly Ser Ala His 1 5 10 15 Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ala Pro Ser Ala 20 25 30 Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser 35 40 45 Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 50 55 60 <210> 11 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile 20 25 30 Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe 35 40 45 Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu 50 55 60 Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile 65 70 75 80 Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu 85 90 95 Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro 100 105 110 Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro 115 120 125 Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro 130 135 140 Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu 145 150 155 160 Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr 165 170 175 Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr 180 185 190 Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly 195 200 205 Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His 210 215 220 Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys 225 230 235 240 Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile 245 250 255 Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys 260 265 270 Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn 275 280 285 Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr 290 295 300 Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu 305 310 315 320 Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro 325 330 335 Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro 340 345 350 Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val 355 360 365 Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp 370 375 380 Ala Gln Thr Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Ser 385 390 395 400 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Ala Pro 405 410 415 His His His His His His 420 <210> 12 <211> 423 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile 20 25 30 Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe 35 40 45 Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu 50 55 60 Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile 65 70 75 80 Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu 85 90 95 Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro 100 105 110 Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro 115 120 125 Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro 130 135 140 Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu 145 150 155 160 Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr 165 170 175 Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr 180 185 190 Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly 195 200 205 Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His 210 215 220 Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys 225 230 235 240 Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile 245 250 255 Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys 260 265 270 Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn 275 280 285 Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr 290 295 300 Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu 305 310 315 320 Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro 325 330 335 Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro 340 345 350 Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val 355 360 365 Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp 370 375 380 Ala Gln Thr Glu Phe His His His His His His Gly Ala Pro Gly Ala 385 390 395 400 His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly Gly Ser Asp 405 410 415 Tyr Lys Asp Asp Asp Asp Lys 420 <210> 13 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 13 Glu Pro Lys Ser Glu Phe Gly Val Pro Thr Ile Val Met Val Asp Ala 1 5 10 15 Tyr Lys Arg Tyr Lys Gly Ala Pro His His His His His 20 25 30 <210> 14 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Glu Pro Lys Ser Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly 1 5 10 15 Ser Val Pro Thr Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys Gly 20 25 30 Ala Pro His His His His His 35 40 <210> 15 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 15 Glu Pro Lys Ser Glu Phe His His His His His Gly Ala Pro Gly 1 5 10 15 Val Pro Thr Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys 20 25 30 <210> 16 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 16 Glu Pro Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly 1 5 10 15 Ala Pro His His His His His 20 <210> 17 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 17 Glu Pro Lys Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 15 Gly Ala Pro His His His His His 20 25 <210> 18 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 18 Glu Pro Glu Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 15 Gly Ala Pro His His His His His 20 25 <210> 19 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 19 Glu Pro Gly Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 15 Gly Ala Pro His His His His His 20 25 <210> 20 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 20 Glu Pro Arg Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 15 Gly Ala Pro His His His His His 20 25 <210> twenty one <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty one Glu Pro Lys Ser Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr 1 5 10 15 Lys Gly Ala Pro His His His His His 20 25 <210> twenty two <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty two Glu Pro Lys Glu Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr 1 5 10 15 Lys Gly Ala Pro His His His His His 20 25 <210> twenty three <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty three Glu Pro Lys Lys Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr 1 5 10 15 Lys Gly Ala Pro His His His His His 20 25 <210> twenty four <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> twenty four Glu Pro Lys Gly Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr 1 5 10 15 Lys Gly Ala Pro His His His His His 20 25 <210> 25 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 25 Glu Pro Lys Ser Asp Ala His Ile Val Met Val Asp Ala Tyr Lys Pro 1 5 10 15 Thr Lys Gly Ala Pro His His His His His 20 25 <210> 26 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 26 Glu Pro Lys Ser Lys Ala His Ile Val Met Val Asp Ala Tyr Lys Pro 1 5 10 15 Thr Lys Gly Ala Pro His His His His His 20 25 <210> 27 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 27 Glu Pro Lys Ser Pro Ala His Ile Val Met Val Asp Ala Tyr Lys Pro 1 5 10 15 Thr Lys Gly Ala Pro His His His His His 20 25 <210> 28 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 28 Glu Pro Lys Ser Ser Ala His Ile Val Met Val Asp Ala Tyr Lys Pro 1 5 10 15 Thr Lys Gly Ala Pro His His His His His 20 25 <210> 29 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 29 Glu Pro Lys Ser Gly Gly Ala His Ile Val Met Val Asp Ala Tyr Lys 1 5 10 15 Pro Thr Lys Gly Ala Pro His His His His His 20 25 <210> 30 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 30 Glu Pro Lys Ser Gly Phe Ala His Ile Val Met Val Asp Ala Tyr Lys 1 5 10 15 Pro Thr Lys Gly Ala Pro His His His His His 20 25 <210> 31 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 31 Glu Pro Lys Ser Glu Gly Gly Ala His Ile Val Met Val Asp Ala Tyr 1 5 10 15 Lys Pro Thr Lys Gly Ala Pro His His His His His His 20 25 <210> 32 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 32 Glu Pro Lys Ser Gly Gly Ser Ala His Ile Val Met Val Asp Ala Tyr 1 5 10 15 Lys Pro Thr Lys Gly Ala Pro His His His His His His 20 25 <210> 33 <211> 409 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 33 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile 20 25 30 Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe 35 40 45 Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu 50 55 60 Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile 65 70 75 80 Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu 85 90 95 Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro 100 105 110 Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro 115 120 125 Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro 130 135 140 Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu 145 150 155 160 Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr 165 170 175 Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr 180 185 190 Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly 195 200 205 Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His 210 215 220 Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys 225 230 235 240 Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile 245 250 255 Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys 260 265 270 Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn 275 280 285 Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr 290 295 300 Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu 305 310 315 320 Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro 325 330 335 Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro 340 345 350 Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val 355 360 365 Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp 370 375 380 Ala Gln Thr Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 385 390 395 400 Gly Ala Pro His His His His His His 405 <210> 34 <211> 289 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 34 Gln Val Gln Leu Val Glu Ser Gly Gly Asn Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Gly Gly Leu Glu Trp Val 35 40 45 Ala Gly Leu Ser Ala Arg Ser Ser Leu Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ser Tyr Asp Ser Ser Gly Tyr Trp Gly His Phe Tyr Ser 100 105 110 Tyr Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Ser Ser Val Ser Ala Ala Pro Gly Gln Lys Val 145 150 155 160 Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Asn Asn Tyr Val 165 170 175 Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr 180 185 190 Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Asn Ser Ala Ser Leu Asp Ile Ser Gly Leu Gln Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Glu 225 230 235 240 Phe Leu Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Gln Glu Phe 245 250 255 Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Ser Ala His Ile Val Met 260 265 270 Val Asp Ala Tyr Lys Pro Thr Lys Gly Ala Pro His His His His His 275 280 285 His <210> 35 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Asn Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Gly Gly Leu Glu Trp Val 35 40 45 Ala Gly Leu Ser Ala Arg Ser Ser Leu Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ser Tyr Asp Ser Ser Gly Tyr Trp Gly His Phe Tyr Ser 100 105 110 Tyr Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Ser Ser Val Ser Ala Ala Pro Gly Gln Lys Val 145 150 155 160 Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Asn Asn Tyr Val 165 170 175 Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr 180 185 190 Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Asn Ser Ala Ser Leu Asp Ile Ser Gly Leu Gln Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser Glu 225 230 235 240 Phe Leu Phe Gly Thr Gly Thr Lys Ala His Ile Val Met Val Asp Ala 245 250 255 Tyr Lys Pro Thr Lys Gly Ala Pro His His His His His 260 265 270 <210> 36 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 36 Arg Gly Val Pro His Ile Val Met Val Asp Ala Tyr Lys Arg Tyr Lys 1 5 10 15

Claims

1. A method for producing a fusion protein, the method comprising: The E. coli host cells transformed with the vector containing the nucleic acid encoding the fusion protein are cultured in a culture medium under conditions effective to express the fusion protein, wherein: The fusion protein includes a binding motif linked to an antibody fragment and a signal sequence, wherein the signal sequence directs the fusion protein to be transported to the periplasmic space of E. coli, wherein the binding motif is linked to the C-terminus of the antibody fragment; (I) the binding motif consists of SEQ ID NO: 1; and Compared with wild-type cells, the Tsp protease activity of E. coli host cells is reduced or there is no Tsp protein activity due to: a) a mutation in a Tsp gene encoding a mutant Tsp protease which reduces or eliminates protease activity; or b) a mutation in the Tsp gene or in a Tsp gene regulatory sequence which reduces or eliminates the expression of the Tsp protease; or c) one or more deletions in bacterial chromosomal regions that reduce or eliminate Tsp protease activity; or d) an inhibitor or inactivator that reduces or eliminates Tsp protease activity or an inhibitor of Tsp protease expression; or (II) the binding motif consists of SEQ ID NO: 2 or SEQ ID NO: 36; and Compared with wild-type cells, the Tsp protease and ompT protease activities of E. coli host cells are reduced or absent due to: a) a mutation in a Tsp protease gene encoding a mutant Tsp protein, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protease, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates Tsp protease activity; and b) a mutation in an OmpT gene encoding a mutant OmpT protein, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protease, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates OmpT protease activity; or (III) the binding motif consists of the sequence of SEQ ID NO: 2 or SEQ ID NO: 36; and Compared with wild-type cells, the Tsp and ompT protease activities of E. coli host cells are reduced or absent due to: a) a Tsp protease inhibitor or inactivator or a Tsp expression inhibitor; and b) an ompT protease inhibitor or inactivator or an ompT expression inhibitor; and The fusion protein was recovered from the E. coli host cells.

2. The method according to claim 1, wherein the binding motif is linked to the C-terminus of the antibody fragment directly or via a linker sequence.

3. The method of claim 1, wherein the antibody fragment is an antigen-binding fragment comprising VHH, sdAb, Fab, scFv or scFab.

4. The method according to claim 1, wherein (i) The E. coli host cell in (I) is a mutant E. coli TG1 F-strain deposited on January 8, 2019 with DSM Accession No. 33004; or (ii) The E. coli host cells of (II) and (III) are mutant E. coli TG1 F-strain deposited on January 8, 2019 with DSM Accession No. 33005.

5. Escherichia coli strain TG1, TG1 F-, XL1 Blue, MC1061, SS320, BL21, JM83, JM109, HB2151, W3110 or Cmax5α, comprising: (I) a nucleic acid encoding a fusion protein, the fusion protein comprising a binding motif and a signal sequence, the signal sequence directing the fusion protein to be transported into the periplasmic space of E. coli, wherein the binding motif is directly or via a linker connected to the C-terminus of an antibody fragment selected from Fab, scFv or scFab, and the binding motif consists of SEQ ID NO: 1, wherein the E. coli strain has reduced or no Tsp protease activity compared to wild-type cells due to a mutation in a Tsp gene encoding a mutant Tsp protein, which mutation reduces or eliminates the protease activity, or a mutation in the Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates the expression of the Tsp protease, or one or more deletions in a region of the bacterial chromosome that reduces or eliminates the Tsp protease activity; or (II) a nucleic acid encoding a fusion protein, the fusion protein comprising a binding motif and a signal sequence, the signal sequence directing the fusion protein to be transported into the periplasmic space of E. coli, wherein the binding motif is directly or via a linker linked to the C-terminus of the antibody fragment, and the binding motif consists of SEQ ID NO: 2, Compared with wild-type cells, the Tsp and ompT protease activities of E. coli strains are reduced or absent due to: a) a mutation in a Tsp gene encoding a mutant Tsp protease, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protease, or one or more deletions in a region of the bacterial chromosome that reduces or eliminates Tsp protease activity; and b) a mutation in an OmpT gene encoding a mutant OmpT protease, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protease, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates OmpT protease activity; or (III) a nucleic acid encoding a fusion protein, the fusion protein comprising a binding motif and a signal sequence, the signal sequence directing the fusion protein to be transported into the periplasmic space of E. coli, wherein the binding motif is directly or via a linker linked to the C-terminus of the antibody fragment, and the binding motif consists of SEQ ID NO: 36, Compared with wild-type cells, the Tsp and ompT protease activities of E. coli strains are reduced or absent due to: a) a mutation in a Tsp gene encoding a mutant Tsp protease, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protease, or one or more deletions in a region of the bacterial chromosome that reduces or eliminates Tsp protease activity; and b) a mutation in an OmpT gene encoding a mutant OmpT protease, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protease, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates OmpT protease activity.

6. A mutant Escherichia coli strain: a) having reduced or no Tsp protease activity compared to wild-type cells due to a mutation in a Tsp gene encoding a mutant Tsp protease, which mutation reduces or eliminates the protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates the expression of the Tsp protease, or one or more deletions in the bacterial chromosome that reduce or eliminate the activity of the Tsp protein; and Expressing a fusion protein comprising a binding motif and a signal sequence, wherein the signal sequence directs the transport of the fusion protein to the periplasmic space of E. coli, wherein the binding motif is directly or via a linker connected to the C-terminus of the antibody fragment, and the binding motif consists of SEQ ID NO: 1; or b) have reduced Tsp protease activity and ompT protease activity or no Tsp and ompT protein activity compared to wild-type cells, due to: i) a mutation in a Tsp gene encoding a mutant Tsp protease, which mutation reduces or eliminates protease activity, or a mutation in a Tsp gene or a Tsp gene regulatory sequence, which mutation reduces or eliminates expression of the Tsp protease, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates Tsp protease activity; and ii) a mutation in an OmpT gene encoding a mutant OmpT protein, which mutation reduces or eliminates protease activity, or a mutation in an OmpT gene or an OmpT gene regulatory sequence, which mutation reduces or eliminates expression of the OmpT protease, or one or more deletions of a region in the bacterial chromosome that reduces or eliminates OmpT protease activity; and For expressing a fusion protein, the fusion protein comprises a binding motif and a signal sequence, the signal sequence directing the transport of the fusion protein to the periplasmic space of E. coli, wherein the binding motif is directly or via a linker connected to the C-terminus of the antibody fragment, and wherein the binding motif consists of SEQ ID NO: 2 or SEQ ID NO: 36, respectively; or (c) having DSM accession number 33004 or 33005 deposited on January 8, 2019, wherein the mutant E. coli strain expresses a fusion protein, the fusion protein comprising a binding motif and a signal sequence, the signal sequence directing the fusion protein to be transported to the periplasmic space of E. coli, wherein the binding motif is directly or via a linker connected to the C-terminus of the antibody fragment, and wherein the binding motif consists of SEQ ID NO: 1, or SEQ ID NO: 2 or SEQ ID NO:

36.

7. The E. coli strain of claim 5 or 6, wherein the antibody fragment is an antigen binding protein, including VHH, sdAb, Fab, scFv or scFab.

8. A fusion protein comprising a binding motif and a signal sequence, wherein the signal sequence directs the transport of the fusion protein to the periplasmic space of Escherichia coli, wherein the binding motif is directly or via a linker connected to a binding motif at the C-terminus of a protein domain in an antibody fragment, wherein the binding motif consists of SEQ ID NO: 1, the binding motif is resistant to proteolysis, the linker is 1-5 amino acids long, and: (i) the protein domain of the antibody fragment is a Fab heavy chain ending with the first 2 amino acids of the hinge domain after the CH1 domain according to the IMGT definition, and the binding motif is linked to the C-terminus directly or via a linker of 1, 2 or 3 amino acids; or (ii) the protein domain of the antibody fragment is a Fab light chain, ending according to the IMGT definition of an IGKC or IGLC antibody domain, and the binding motif is linked to the C-terminus directly or via a linker of 1 or 2 or 3 amino acids; or (iii) The protein domain of the antibody fragment is a scFv fragment having a FR4 domain according to the IMGT definition, which is truncated after 6 amino acids and the binding motif is linked to the C-terminus directly or via a linker of 1, 2 or 3 amino acids.

9. The fusion protein according to claim 8, wherein the protein domain is a human scFv single-chain antibody fragment with a C-terminal truncation in the FR4 region.

10. The fusion protein according to claim 8, wherein the binding motif is connected to the C-terminus of the protein domain in the antibody fragment via a linker of 1, 2 or 3 amino acids.

11. The fusion protein of claim 8, wherein: (i) the binding motif is linked to the C-terminus of the Fab at IMGT position 121 of the human heavy chain CH1 antibody domain via a linker of 2, 3, 4, or 5 amino acids; or (ii) the binding motif is linked to the C-terminus of the Fab at IMGT position 121 of the human constant light chain antibody domain via a linker of 2, 3, 4, or 5 amino acids.

12. The fusion protein of claim 8, wherein the fusion protein comprises an antibody fragment selected from maltose binding protein (MBP) or an antigen binding fragment, wherein the antigen binding fragment comprises VHH, sdAb, Fab, scFv or scFab.

13. A nucleic acid construct comprising a polynucleotide sequence encoding a fusion protein as defined in any one of claims 8 to 12.

14. A vector comprising the nucleic acid construct of claim 13.

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