Recombinant protein

Abstract

To improve, in one aspect, the expression of heterologous proteins in Escherichia coli and other gram-negative bacteria, in particular for immunoglobulin-binding proteins derived from the B and C domains of Staphylococcus aureus Protein A.SOLUTION: One aspect of the present application is to provide a recombinant protein comprising a functional polypeptide and, linked to the N-terminus of the functional polypeptide, an N-terminal spacer having a length such that the number of amino acid residues between a signal peptide cleaving site and an N-terminus proximal structural unit of the functional polypeptide is 14-24.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] FIELD OF THE PRESENT ART The present invention relates to recombinant proteins, in particular recombinant proteins expressed in Gram-negative bacteria such as Escherichia coli (E. coli). The present invention also relates to nucleic acids, vectors, and Gram-negative bacteria for the expression of recombinant proteins, as well as separation matrices having covalently linked recombinant protein ligands and methods for separating immunoglobulins on such matrices. [Background technology]

[0002] 2. Background of the Invention Expression of heterologous proteins in E. coli is commonly used for recombinant proteins at laboratory and commercial scale. Expression in E. coli using secretion generally means transport of the produced protein across the inner membrane that separates the cytoplasm and the periplasm. Secretion into the periplasm frequently also results in leakage of proteins into the extracellular medium (Mergulhao et al., Biotech Adv 23, 177-202, 2005). Secretion has many advantages over cytoplasmic expression, such as facilitating correct protein folding, correct N-terminal processing, simplified downstream processing, and preventing aggregation into inclusion bodies. However, not all proteins are successfully expressed in soluble form in the periplasm. Some of the possible problems are lack of secretion and no or incorrect processing of the signal peptide. A particular example of a protein with problems regarding insufficient secretion is in the expression of immunoglobulin conjugates based on the native or mutated Fc-binding domains B or C of protein A of Staphylococcus aureus (L Abrahmsen et al., EMBO J 4(13B), pp. 3901-3906, 1985). Such immunoglobulin conjugates are commonly used as ligands in the affinity chromatographic separation of monoclonal antibodies, a major category of modern pharmaceuticals.

[0003] Protein expression depends on a promoter sequence that initiates the transcription of messenger ribonucleotide acid (mRNA), followed by a ribosome binding site (RBS) that attracts the translation machinery, followed by a signal peptide sequence that facilitates transport of the protein into the periplasm. The mature protein is often cloned after the signal peptide, from which it is cleaved by a signal peptidase as it crosses the membrane. However, a problem with cloning constructs after a signal peptide is that restriction enzymes often require specific sequences to cut the DNA, which leaves a cloning trail behind the signal peptide sequence. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US 8,329,860 [Patent Document 2] US 8,754,196 [Patent Document 3] US 9,040,661 [Patent Document 4] US 9,403,883 [Patent Document 5] JP 2006304633A [Patent Document 6] US 8,674,073 [Patent Document 7] US 2010 / 0221844 [Patent Document 8] US 2012 / 0208234 [Patent Document 9] US 9,051,375 [Patent Document 10] US 2014 / 0031522 [Patent Document 11] US 2014 / 0107315 [Patent Document 12] US 2013 / 0096276

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Non-licensed literature

[0005] [Non-licensed document 1] Mergulhaoら, Biotech Adv 23, pages 177~202, 2005

Non-licensed Document 2

[0006] Thus, there is a need to improve the expression of heterologous proteins in E. coli and other Gram-negative bacteria, particularly for immunoglobulin-binding proteins derived from the B and C domains of Staphylococcus aureus protein A. [Means for solving the problem]

[0007] SUMMARY OF THE PRESENT APPLICATION One aspect of the present invention is to provide a functional protein that is easily expressed and secreted in Gram-negative bacteria such as E. coli, which is achieved with a recombinant protein comprising a functional polypeptide and an N-terminal spacer linked to the N-terminus of said functional polypeptide, the N-terminal spacer having a length such that the distance between the signal peptide cleavage site and the N-terminal proximal structural unit of the functional polypeptide is 14 to 24 amino acid residues.

[0008] One advantage is that the expression level is improved by the introduction of an N-terminal spacer, and a further advantage is that the selectivity of signal peptide cleavage is improved.

[0009] A second aspect of the present invention provides a nucleic acid molecule encoding a recombinant protein, which is accomplished with a nucleic acid molecule comprising, in a 5' to 3' direction, the following elements, said elements being operably linked: a) an inducible or constitutive promoter DNA sequence; b) a DNA sequence encoding a signal peptide; c) a DNA sequence encoding an N-terminal spacer; and d) A DNA sequence encoding a functional or immunoglobulin-binding polypeptide.

[0010] A third aspect of the present invention is to provide a cloning vector for expressing and secreting a recombinant protein according to any one of the preceding claims into the bacterial periplasm of Gram-negative cells, which is achieved with a cloning vector comprising the above-mentioned nucleic acid molecule.

[0011] A fourth aspect of the present invention provides a Gram-negative bacterium transformed with the cloning vector.

[0012] A fifth aspect of the present invention is to provide a method for expressing and secreting a recombinant protein in a gram-negative bacterium, which is achieved by a method comprising the steps of providing a gram-negative bacterium and culturing the gram-negative bacterium.

[0013] A sixth aspect of the present invention is to provide a separation matrix comprising a recombinant protein covalently linked to a support.

[0014] A seventh aspect of the present invention is a method for producing a composition comprising the steps of: i) providing a separation matrix as described above, wherein the recombinant protein comprises an immunoglobulin-binding polypeptide; ii) contacting the separation matrix with a liquid sample containing immunoglobulins to bind the immunoglobulins; iii) optionally washing the separation matrix with a washing solution; iv) contacting the separation matrix with an elution liquid to elute the immunoglobulins The present invention provides a method for isolating immunoglobulins, comprising:

[0015] Further suitable embodiments of the invention are set forth in the dependent claims. drawing [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows an example of a cloning site sequence with a spacer sequence inserted, with restriction enzyme cleavage sites marked above the sequence. [Diagram 2] FIG. 1 shows integrated peak areas at 237 nm from the eluate of an IgG Sepharose 6FF Tricorn 10 column. [Diagram 3] UV measurements at 210 nm, integrated peak areas. a) pGE120 OmpA-AQGT (reference), 52% correct processed signal peptide. b) pGE144 OmpA-DsbA8AA, 96% correct signal peptide cleavage. c) pGE140 DsbA-DsbA8AA, 97% correct signal peptide cleavage. [Figure 4]FIG. 1 shows protein expression measured in heat-treated fermentation broth for Zvar26 with and without the DsbA 8AA N-terminal spacer using concentration analysis with a standard curve. Arrows indicate the time points of induction. [Diagram 5] UV measurements at 210 nm, integrated peak areas. a) pGE0002 OmpA-AQGT-Zvar26 (reference). b) pGE0180 OmpA-DsbA8AA- Zvar26. [Figure 6] FIG. 1 is a schematic diagram of a construct having a signal peptide, an N-terminal spacer, and a functional polypeptide. [Figure 7] Figure 1 shows a summary of expression results from shake flask cultures. Error bars corresponding to one standard deviation are included where applicable. Three replicates were performed for construct pGE180. Two replicates were performed for samples DsbA7 and DsbA4. Single replicates were performed for all other constructs. [Figure 8] FIG. 1 shows examples of relevant total ion chromatogram (TIC) peaks for a) a construct with correct signal peptide cleavage (in this case pGE0180) and b) a construct with incorrect signal peptide cleavage (in this case pGE0002). [Figure 9] Figure 1 shows an example of deconvoluted TIC peaks after 24 hours of incubation in 1M NaOH. The image on the left shows the typical pattern seen in pGE0180 (cluster of peaks between 500 and 800 m / z). The image on the right shows the same area in DsbA8_noGT, with no peaks present except in the background. [Figure 10] Figure 1 shows the integrated extracted ion chromatogram (XIC) areas of peptide peaks corresponding to truncated N-terminal sequences of different lengths after 0 hours (top left), 4 hours (top right), and 24 hours (bottom left). Areas are shown in arbitrary units (AU). [Figure 11]Figure 1 shows a zoomed-in view of the relevant peptide regions of the most promising candidates, with areas shown in arbitrary units (AU). [Figure 12] FIG. 1 shows the degradation of the most prominent peptide in the N-terminal region of the construct exhibiting the lowest alkaline stability. Areas are shown in arbitrary units (AU). [Figure 13] Figure 1 shows measured protein concentration, estimated protein concentration from the extinction coefficient at 280 nm, and OD600 at the end of fed-batch culture. One replicate run was performed for pGE180, all other constructs were cultured only once. Error bars corresponding to the 95% confidence interval are included where data were available. [Figure 14] FIG. 13 shows a comparison between the dynamic binding capacities of candidate constructs (DsbA8_noGT and DsbA8_DT), a reference sample containing Zvar2, and previous data from other immobilizations using base matrices with similar dry weight, porosity, and ligand density. [Figure 15] FIG. 1 shows the alignment of Fc binding domains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] definition The terms "antibody" and "immunoglobulin" are used interchangeably herein and are understood to also include fragments of antibodies, fusion proteins comprising antibodies or antibody fragments, and conjugates comprising antibodies or antibody fragments.

[0018] The terms "Fc binding polypeptide" and "Fc binding protein" refer to a polypeptide or protein, respectively, capable of binding to the crystallizable portion (Fc) of an antibody, and include, for example, Staphylococcus aureus protein A and Staphylococcus (Streptococcus) protein G, or any fragment or fusion protein thereof which maintains said binding properties.

[0019] The terms "Fab-binding polypeptide" and "Fab-binding protein" refer to a polypeptide or protein, respectively, capable of binding to the antigen-binding portion (Fab) of an antibody and include, for example, Peptostreptococcus magnus protein L, Streptococcus protein G, native Staphylococcus aureus protein A, or any fragment or fusion protein thereof which maintains said binding properties.

[0020] As used herein, the term "linker" refers to an element that connects two polypeptide units, monomers, or domains together as a multimer.

[0021] The term "% identity" for comparison of amino acid sequences is determined by standard alignment algorithms such as, for example, the basic local alignment tool (BLAST™) described in Altschul et al. (1990) J. Mol. Biol., 215: 403-410. Web-based software for this is freely available from the US National Library of Medicine at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. In this case, the algorithm "blastp (protein-protein BLAST)" is used to determine the alignment, in particular the % identity, of a query sequence with a subject sequence.

[0022] The abbreviation "DsbA" herein refers to E. coli, thiol:disulfide exchange protein, UniProt P0AEG4.

[0023] The abbreviation "OmpA" herein means Escherichia coli, outer membrane protein A, UniProt P0A910.

[0024] The abbreviation "PrA" herein means Staphylococcus aureus Protein A, UniProt P38507.

[0025] The abbreviation "GIII" herein refers to Gene 3 from bacteriophage M13, UniProt P69168.

[0026] The term "signal peptide" as used herein refers to a short (usually 16-30 amino acids long) peptide present at the N-terminus of most newly synthesized proteins that are committed to the secretory pathway. It may also be called a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide. Signal peptides are usually cleaved off from proteins by a signal peptidase enzyme.

[0027] The term "signal peptide cleavage site" as used herein refers to the dipeptide between which a signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases, the dipeptide is Ala-Ala. Signal peptide cleavage sites can be calculated with algorithms such as SignalP 4.1 (Center for Biological Sequence Analysis, Technical University of Denmark), available online at http: / / www.cbs.dtu.dk / services / SignalP / .

[0028] As used herein, the term "heterologous expression" refers to the expression of a gene or part of a gene in a host organism that does not naturally possess this gene or gene fragment. "Secreted" refers to crossing the inner membrane of gram-negative bacteria such as E. coli.

[0029] The cytoplasm (cell cytoplasm) is the space inside the inner cell membrane in gram-negative bacteria that contains the genetic material. As used herein, the term "cytoplasmic expression" refers to protein expression within the cytoplasm.

[0030] The periplasm is a thickened gel-like matrix in the space between the inner cytoplasmic membrane and the outer bacterial membrane, called the periplasmic space in gram-negative bacteria.

[0031] The term "promoter" as used herein refers to a region of DNA that initiates transcription (writing into mRNA) of a particular gene. A promoter is usually located near the transcription start site of a gene, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). A promoter can be inducible, meaning that expression of a gene operably linked to the promoter can be turned on by the presence of an inducer. Alternatively, a promoter can be constitutive, i.e., not controlled by any inducer.

[0032] The abbreviation "RBS" herein means ribosome binding site, or ribosome binding site, which is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for recruitment of ribosomes during the initiation of protein translation.

[0033] The abbreviation "RhaBAD" herein refers to the E. coli rhamnose operon promoter (also called the rhamnose promoter) of genes RhaB, RhaA, and RhaD, which is a promoter widely used in molecular biology.

[0034] The abbreviation "T5" herein refers to the bacteriophage T5 promoter for E. coli RNA polymerase with an embedded lac operator, a segment of DNA to which transcription factors bind and repress, thereby controlling gene expression.

[0035] The abbreviation "pD861-SR" herein refers to a plasmid for E. coli protein expression with a rhamnose promoter (RhaBAD) and a strong ribosome binding site (SR).

[0036] The abbreviation "pJ401" herein refers to a plasmid for E. coli protein expression having a bacteriophage T5 promoter and two embedded opposed lac operators on each side of the promoter.

[0037] The abbreviation "OptEc" herein means optimized for E. coli expression, i.e., the codon triplets have been selected to be compatible with the E. coli translational machinery.

[0038] The abbreviation "FspI" used herein refers to the cyanobacterium F ischerella s By this reference is meant the DNA restriction enzyme from the Streptococcus pecies (ATCC 29114), which cuts to produce blunt ends at the sequence TGCGCA.

[0039] The abbreviation "KpnI" used herein refers to Klebsiella pneumoniae ( K Lebsiella p.n. "GGTACC" refers to the DNA restriction enzyme from E. eumoniae OK8 (ATCC 49790), which cuts with an overhang at the sequence GGTACC.

[0040] The abbreviation "SRP" herein refers to the signal recognition particle pathway, a universally conserved pathway for targeting polypeptides for secretion via the cotranslational pathway.

[0041] The abbreviation "Sec" as used herein refers to secretion or type II secretion pathway, the system responsible for the secretion of proteins across the cell membrane.

[0042] As used herein, the term "E. coli K12-017" refers to the E. coli expression strain as described in Olsson MO and Isaksson LA, Molec. Gen. Genet. 169, 251-257 (1979).

[0043] The term "functional polypeptide" as used herein refers to a polypeptide that has technically useful properties, such as a) highly specific binding to a target species (use as an affinity binder, in particular as a ligand for affinity chromatography), b) therapeutic properties (use as a pharmaceutical), c) enzymatic properties (use as a biocatalyst), and d) signal-emitting properties (use as a reporter protein, such as a fluorescent reporter protein).

[0044] As used herein, the terms "comprises," "comprising," "containing," "having," and the like, may have the meaning given them in United States patent law and may mean "includes," "including," and the like; similarly, "consisting essentially of" or "consisting essentially of" may have the meaning given them in United States patent law and the terms are open-ended, permitting the presence of things other than those recited, so long as the presence of things other than those recited does not change the basic or novel characteristics of what is recited, but excluding prior art embodiments.

[0045] Detailed Description of the Embodiments In one aspect, as illustrated in Figures 1 to 3, the present invention discloses a recombinant protein comprising a functional polypeptide and an N-terminal spacer linked to the N-terminus of the functional polypeptide, the N-terminal spacer having a length such that the number of amino acid residues between the signal peptide cleavage site and the N-terminal proximal structural unit of the functional polypeptide is 14 to 24.

[0046] The functional polypeptide may be an immunoglobulin-binding polypeptide. Such a polypeptide may, for example, comprise one or more immunoglobulin-binding domains derived from a bacterial protein selected from the group consisting of Staphylococcus aureus protein A, Peptostreptococcus magnus protein L, and Streptococcus spp. protein G, for example, the group consisting of Staphylococcus aureus protein A and Peptostreptococcus magnus protein L. The immunoglobulin-binding domain may, for example, have at least 80%, for example at least 90% or 95% sequence identity with domains E, D, A, B, or C of Staphylococcus aureus protein A, with protein Z (variant of domain B of Staphylococcus aureus protein A), Zvar, or Zvar2 (alkali-stabilized mutant of protein Z), or with domains 1, 2, 3, 4, or 5 of Peptostreptococcus magnus protein L. In this context, an immunoglobulin-containing domain may be defined by or have at least 80%, such as at least 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11. SEQ ID NOs: 1-7 (Staphylococcus aureus Protein A Domains E, D, A, B, and C, Protein Z, and Zvar) are listed in Figure 15, and SEQ ID NOs: 8-11 are specified below.

[0047] Sequence number 8 - Zvar2 [ka] SEQ ID NO:9 - Staphylococcus aureus protein A truncated domain C [ka] SEQ ID NO:10 - A shortened version of Zvar [ka] SEQ ID NO:11 - A shortened version of Zvar2 [ka]

[0048] SEQ ID NOs:1-11 can all be characterized as Fc-binding domains derived from Staphylococcus aureus protein A. Such domains can be further alkali-stabilized by mutation of the native domain, as has been done in Zvar and Zvar2. Further examples of such alkaline stabilizing domains can be SEQ ID NOs: 48-93 (listed in Example 6), other examples are, for example, US 8,329,860, US 8,754,196, US 9,040,661, US 9,403,883, JP 2006304633A, US 8,674,073, US 2010 / 0221844, US 2012 / 0208234, US 9,051,375, US 2014 / 0031522, US 2014 / 0107315, US 2013 / 0096276, US 2013 / 0274451, US 2005 / 0143566, US 2016 / 0159855, US No. 2016 / 0168209, US 2016 / 0237124, WO 2014 / 146350, WO 2016 / 079033, WO 2016 / 079034, WO 2016 / 152946, PCT EP2017 / 061162, PCT EP2017 / 061164, PCT EP2017 / 061160, PCT EP2017 / 061158, PCT EP2017 / 061159, US 14 / 961164, US 15 / 348699, and US 15 / 282367, all of which are incorporated by reference in their entireties. Specifically, the alkali-stabilized Fc-binding domain may have at least 80%, for example at least 90% or at least 95%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-11, 48-64, and 74-93.

[0049] The immunoglobulin-binding polypeptide may suitably be a multimer of immunoglobulin-binding domains, e.g. Fc-binding domains, as discussed above. The multimer may be, for example, a dimer, trimer, tetramer, pentamer, hexamer, or heptamer, such as a dimer, tetramer, or hexamer. Suitably, the multimer may comprise at least four domains. The domains may be directly linked to each other (e.g., in the case of SEQ ID NOs: 1-8, 48-64, and 74-93), but they may also be linked to each other via a linker, typically comprising 1 to 25 (e.g., 3 to 20) amino acid residues (e.g., in the case of SEQ ID NOs: 9-11). Examples of suitable linkers are: [ka] Some specific examples of multimers include SEQ ID NO: 12 (tetramer), SEQ ID NO: 13 (hexamer), and SEQ ID NOs: 65-73 (dimers).

[0050] SEQ ID NO:12 - Zvar tetramer containing a C-terminal cysteine [ka] SEQ ID NO:13 - Zvar2 hexamer containing a C-terminal cysteine [ka]

[0051] The recombinant protein may include a coupling moiety at or proximal to the C-terminus, such as the C-terminus, or the N-terminus. This coupling moiety may be used for specific coupling to a support as discussed below, and may include a cysteine, allowing coupling via a thioether bond. Alternatively, or in addition, the coupling moiety may include one or more lysines, such as a cluster of 2-8 lysines.

[0052] A functional or immunoglobulin-binding polypeptide may have secondary and tertiary structure and may suitably comprise one or more structural units, as exemplified by an alpha helix, a beta sheet, and / or a beta barrel. In particular, a polypeptide may comprise multiple alpha helices, such as at least three alpha helices. The Fc-binding domains from Staphylococcus aureus Protein A (e.g., SEQ ID NOs: 1-11, 48-64, and 74-93) each comprise three alpha helices, and thus the number of alpha helices in a multimer as discussed above may be three times the number of domains in the multimer. The first alpha helix in the domains as exemplified by SEQ ID NOs: 1-11, 48-64, and 74-93 begins at position 9 (using the position nomenclature of FIG. 15), which is glutamine in the case of SEQ ID NOs: 1-7 and 9-10, and alanine in the case of SEQ ID NOs: 8, 11, 48-64, and 74-93.

[0053] The N-terminal spacer suitably has a length such that the number of amino acid residues between the signal peptide cleavage site and the N-terminal proximal structural unit of the functional or immunoglobulin-binding polypeptide is 14-24. As discussed above, this structural unit may suitably be an α-helix (or alternatively a β-sheet or β-barrel). The number of amino acid residues between the N-terminus of the functional or immunoglobulin-binding polypeptide and the N-terminal proximal structural unit may vary, for example between 0 (SEQ ID NO: 9-11) and 11 (SEQ ID NO: 2), and therefore the length of the N-terminal spacer may vary, for example between 3 and 24 amino acid residues, such as between 8 and 24 amino acid residues or between 14 and 24 amino acid residues. For example, when the N-terminal spacer is linked to an Fc binding domain having at least 90% identity with SEQ ID NO: 1-8, 48-64, or 74-93, the N-terminal spacer may have a length of, for example, 8 to 12 amino acid residues, and when the N-terminal spacer is linked to an Fc binding domain having at least 90% identity with SEQ ID NO: 9-11, the N-terminal spacer may have a length of, for example, 16 to 20 amino acid residues. The N-terminal spacer may be composed of, for example, an amino acid residue selected from the group consisting of alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine. For improved alkaline stability, it may be advantageous to exclude asparagine. In this case, the N-terminal spacer may consist of amino acid residues selected from the group consisting of alanine, aspartic acid, glutamine, glutamic acid, glycine, histidine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine. It may be advantageous if the N-terminal spacer does not contain any cluster of arginine or lysine and / or if it contains at most two amino acid residues selected from the group consisting of lysine and arginine. In some embodiments, the two N-terminal spacer amino acid residues may be AQ (alanine followed by glutamine).

[0054] In particular, the N-terminal spacer may comprise, essentially comprise, or have an amino acid sequence having at least 80% sequence identity with, or defined by, an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-18, 29-30, 33-40, 43-45, and 47. The N-terminal spacer may further comprise [ka] The N-terminal spacer may comprise, essentially comprise or have an amino acid sequence having at least 80% sequence identity with or defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-11. The latter sequences are particularly advantageous in combination with immunoglobulin-binding polypeptides derived from SEQ ID NOs: 9-11. With regard to alkaline stability, it may be further advantageous to use an N-terminal spacer that comprises, essentially comprises or has an amino acid sequence having at least 80% sequence identity with or defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, 38, 40, 43, 44, and 47. The N-terminal spacer may also be selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 43, 44, and 47 for alkaline stability. [ka] The immunoglobulin-binding polypeptide may comprise, essentially comprise or have at least 80% sequence identity with or defined by an amino acid sequence selected from the group consisting of: The latter sequences are particularly advantageous in combination with immunoglobulin-binding polypeptides derived from SEQ ID NOs: 9-11.

[0055] The alkaline stability of a recombinant protein can be assessed by coupling it to an SPR chip, for example a Biacore CM5 sensor chip as described for example in WO2016079033, using, for example, NHS or maleimide coupling chemistry, and measuring the immunoglobulin binding capacity of the chip, typically with polyclonal human IgG, before and after incubation in an alkaline solution at a specified temperature, for example 22+ / -2°C. The incubation can be carried out, for example, in 0.5M NaOH for a number of 10 minute cycles, for example 100 cycles, 200 cycles, or 300 cycles. The IgG capacity of the matrix after 100 cycles of 10 minute incubation in 0.5M NaOH at 22+ / -2°C can be at least 55%, for example at least 60%, at least 80%, or at least 90% of the IgG capacity before incubation. Alternatively, the remaining IgG capacity after 100 cycles for a particular variant, measured as above, can be compared to the remaining IgG capacity for the parent recombinant protein, in which case the remaining IgG capacity for the variant may be at least 105%, e.g., at least 110%, at least 125%, at least 150%, or at least 200% of that of the parent recombinant protein.

[0056] In a second aspect, the present invention discloses a nucleic acid molecule encoding a recombinant protein as disclosed above, the nucleic acid molecule comprising, in a 5' to 3' direction, the following operably linked elements: a) Inducible or constitutive promoter DNA sequences, such as the RhaBAD or T5 promoter sequences. RhaBAD is inducible with rhamnose and T5 is inducible with isopropyl β-D-1-thiogalactopyranoside (IPTG). An example of a constitutive promoter is the spa promoter (the naturally occurring Staphylococcus aureus protein A promoter); b) a DNA sequence encoding a signal peptide, such as the OmpA (SEQ ID NO: 14) or DsbA (SEQ ID NO: 15) signal peptide, or a signal peptide having at least 80% sequence identity to any of these; c) a DNA sequence encoding an N-terminal spacer as discussed above; and d) A DNA sequence encoding a functional or immunoglobulin-binding polypeptide as discussed above. The nucleic acid molecule may further comprise a ribosome binding site (RBS) and an origin of replication. It may also suitably contain an antibiotic resistance marker.

[0057] In a third aspect, the present invention discloses a cloning vector, e.g., a plasmid, that expresses and secretes a recombinant protein as disclosed above into the bacterial periplasm of a gram-negative cell, e.g., E. coli. The cloning vector comprises a nucleic acid molecule as discussed above.

[0058] In a fourth aspect, the present invention discloses a Gram-negative bacterium transformed with the cloning vector disclosed above. The bacterium can be identified as Escherichia coli, in particular the K12 strain of Escherichia coli, such as E. coli K12-017. Other examples of Gram-negative bacteria include the genus Pseudomonas, such as Pseudomonas fluorescens. Transformation can be accomplished, for example, by the heat shock method, although other methods such as electroporation are also possible.

[0059] In a fifth aspect, the present invention discloses a method for expressing and secreting the recombinant proteins discussed above in Gram-negative bacteria, the method comprising: i) providing a Gram-negative bacterium as disclosed above; and ii) Culturing Gram-negative bacteria In the event that the nucleic acid molecule as discussed above comprises an inducible promoter, the method may further comprise the step of inducing recombinant protein expression in the Gram-negative bacterium.

[0060] In a sixth aspect, the present invention discloses a separation matrix comprising a recombinant protein as disclosed above, covalently linked to a support.

[0061] As will be appreciated by those of skill in the art, the expressed recombinant protein should be purified to an appropriate extent before immobilization to a support. Such purification methods are well known in the art, and immobilization of protein-based ligands to a support is readily accomplished using standard methods. Suitable methods and supports are discussed in more detail below.

[0062] The alkaline stability of a matrix can be assessed by measuring its immunoglobulin binding capacity, typically with polyclonal human IgG, before and after incubation in alkaline solution at a specified temperature, e.g., 22+ / -2°C. The incubation can be carried out, for example, in 0.5 M NaOH for a number of 15 minute cycles, e.g., 100, 200, or 300 cycles, corresponding to a total incubation time of 25, 50, or 75 hours. The IgG capacity of the matrix after 96-100 cycles of 15 minute incubation or a total incubation time of 24 or 25 hours in 0.5 M NaOH at 22+ / -2°C can be at least 80%, e.g., at least 85%, at least 90%, or at least 95%, of the IgG capacity before incubation.

[0063] The solid support of the matrix according to the invention can be of any suitable known type. Conventional affinity separation matrices are often organic and based on polymers exposing hydrophilic surfaces to the aqueous medium used, i.e. exposing hydroxyl (-OH), carboxyl (-COOH), carboxamide (-CONH2, optionally in N-substituted), amino (-NH2, optionally in substituted), oligo- or polyethyleneoxy groups on their outer surface and, if present, also on the inner surface. The solid support can suitably be porous. Porosity can be expressed as a Kav or Kd value (fraction of pore volume available for a probe molecule of a certain size) measured by inverse size exclusion chromatography, for example according to the method described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pages 6-13. By definition, both Kd and Kav values ​​are always in the range of 0 to 1. The Kav value may advantageously be between 0.6 and 0.95, for example between 0.7 and 0.90 or between 0.6 and 0.8, measured with dextran of molecular weight 110 kDa as the probe molecule. The advantage of this is that the support has a large percentage of pores that can accommodate both the recombinant protein of the invention and the immunoglobulins that bind to the recombinant protein, and provide mass transport of the immunoglobulins to and from the binding site.

[0064] The recombinant protein can be attached to the support by conventional coupling techniques, for example, exploiting thiol, amino, and / or carboxyl groups present on its ligand. Bisepoxide, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS), etc. are well-known coupling reagents. Between the support and the recombinant protein, molecules known as spacers can be introduced, which improve the availability of the recombinant protein and facilitate the chemical coupling of the recombinant protein to the support. Depending on the nature of the recombinant protein and the coupling conditions, the coupling can be a multipoint coupling (e.g., via multiple lysines) or a single point coupling (e.g., via a single cysteine). Alternatively, the recombinant protein can be attached to the support by non-covalent bonds, such as physical or biospecific adsorption.

[0065] In some embodiments, the matrix comprises 5-25 mg / ml, e.g., 5-20 mg / ml, 5-15 mg / ml, 5-11 mg / ml, or 6-11 mg / ml, of recombinant protein coupled to the support. The amount of coupled protein can be adjusted by the concentration of protein used in the coupling process, by the activation and coupling conditions used, and / or by the pore structure of the support used. As a general rule, the absolute binding capacity of the matrix increases with the amount of coupled protein, at least up to the point where the pores become significantly constricted by the coupled protein. The relative binding capacity per mg of coupled protein decreases at high coupling levels, resulting in a cost-effective optimum within the ranges specified above.

[0066] In certain embodiments, the protein is coupled to the support via a thioether bond. Methods for performing such coupling are well known in the art and are readily performed by those skilled in the art using standard techniques and equipment. Thioether bonds are flexible and stable, and are generally suitable for use in affinity chromatography. In particular, when the thioether bond is via a terminal or near-terminal cysteine ​​residue on the recombinant protein, the mobility of the coupled recombinant protein is enhanced, which results in improved binding capacity and binding kinetics. In some embodiments, the recombinant protein is coupled via a C-terminal cysteine ​​provided on the protein as described above. This allows for efficient coupling of the cysteine ​​thiol to electrophilic groups on the support, such as epoxide groups, halohydrin groups, etc., resulting in thioether cross-link coupling. Alternatively, the recombinant protein can be covalently linked to the support via one or more amide bonds. This can be achieved, for example, through a reaction between one or more lysines in the protein and one or more activated carboxyl groups on the support. Activation can be done, for example, with the commonly known N-hydroxysuccinimide (NHS) reagent. Yet another alternative is for the protein to be linked via one or more secondary amine linkages. Such linkages can be formed from lysines in the protein and any hydroxyl groups on the support that have been activated, for example, using tresyl chloride or tosyl chloride chemistry, or by a reductive amination reaction between lysines and aldehydes on the support. The aldehydes can be formed, for example, from vicinal diols on the support by periodate oxidation.

[0067] In certain embodiments, the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc. Polysaccharides are hydrophilic in nature, with a low degree of non-specific interactions, they offer a high content of reactive (activatable) hydroxyl groups, and they are generally stable to the alkaline cleaning solutions used in bioprocessing.

[0068] In some embodiments, the support comprises agar or agarose. The supports used in the present invention can be readily prepared by standard methods such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), pp. 393-398 (1964)). Alternatively, the base matrix is ​​a commercially available product, such as cross-linked agarose beads sold under the name SEPHAROSE™ FF (GE Healthcare). In certain embodiments, particularly those advantageous for large-scale separations, the support is adapted to increase its rigidity using the methods described in US6602990 or US7396467 (incorporated herein by reference in their entirety), thus making the matrix more suitable for high flow rates.

[0069] In certain embodiments, the support, such as a polysaccharide or agarose support, is crosslinked, for example with a hydroxyalkyl ether crosslink. The crosslinking reagent that produces such crosslinks can be, for example, epihalohydrins such as epichlorohydrin, diepoxides such as butanediol diglycidyl ether, allyl halides or allylation reagents such as allyl glycidyl ether. Crosslinking is beneficial to the rigidity of the support and improves its chemical stability. Hydroxyalkyl ether crosslinks are alkaline stable and do not cause significant nonspecific adsorption.

[0070] Alternatively, the solid support is based on synthetic polymers such as polyvinyl alcohol, polyhydroxyalkylacrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides, etc. In the case of hydrophobic polymers such as matrices based on divinyl and monovinyl substituted benzenes, the surface of the matrix is ​​often hydrophilized to expose hydrophilic groups as defined above to the surrounding aqueous liquid. Such polymers are easily produced by standard methods, see for example "Styrene based polymer supports developed by suspension polymerization", R Arshady: Chimica e L'Industria 70(9), pp. 70-75 (1988). Alternatively, commercial products such as SOURCE™ (GE Healthcare) are used. In another alternative, the solid support according to the invention comprises an inorganic support, such as silica, zirconium oxide, etc.

[0071] In certain embodiments, the solid support takes another form, such as a surface, a chip, a capillary, or a filter (eg, a membrane or depth filter matrix).

[0072] In a seventh aspect, the present invention provides a method for producing a composition comprising the steps of: i) providing a separation matrix as discussed above, wherein the recombinant protein comprises an immunoglobulin-binding polypeptide, e.g., comprising one or more Fc-binding domains derived from Staphylococcus aureus protein A; ii) contacting the liquid sample containing immunoglobulins with a separation matrix to bind the immunoglobulins; iii) optionally washing the separation matrix with a washing solution; iv) contacting the separation matrix with an elution liquid to elute the immunoglobulins; v) optionally cleaning the separation matrix with a cleaning solution Alkaline cleaning solutions are commonly used in bioprocessing, and the cleaning solution may comprise at least 0.1 M NaOH or KOH, for example at least 0.5 M NaOH or KOH, or 0.5-2.5 M NaOH or KOH, provided that the recombinant protein is alkaline stable. EXAMPLES

[0073] Working Example First, two different signal peptides were tested with four different N-terminal spacers to find the best cleavage and protein expression. This experiment (Example 1) was performed with Zvar2 monomer using the RhaBAD promoter system. In Example 2, the most promising signal peptide and N-terminal start were cloned in Zvar2 hexamer. This construct was tested in fed-batch fermentation with and without N-terminal start sequences. This study was performed using the T5 promoter expression system. In Example 3, all native domains of Staphylococcus aureus protein A, Zvar monomer (Zvar1), Zvar2 monomer (Zvar21), and Zvar tetramer (Zvar4), were tested with the selected signal peptide and N-terminal start to see if the start sequence also had an effect on closely related domains. Example 3 was also performed using the T5 expression system. Example 4 was performed with a set of different N-terminal spacers. Example 5 is a scale-up of fermentation using two selected spacers, and Example 6 is a scale-up of fermentation using two selected spacers in combination with different mutants of immunoglobulin binding proteins. [ka] This was a study using an N-terminal spacer.

[0074] Materials and Methods Constructs Genes as listed in Table 1 were synthesized by a synthetic gene contract manufacturer (ATUM, CA, USA). Double-stranded dideoxyribonucleic acid (dsDNA) was synthesized based on the amino acid (AA) sequence and optimized for expression in E. coli by the manufacturer's proprietary algorithm. The dsDNA was inserted into the expression vector pD861-SR or pJ401 with the signal peptide from either DsbA or OmpA (Table 2).

[0075] [Table 1]

[0076] [Table 2]

[0077] N-Terminal Cloning All plasmids were transformed into E. coli K12-017 using chemically competent cells. To modify the vector, a short spacer sequence of 8AA was inserted between the FspI and KpnI cleavage sites. The 8AA sequence was inserted into OmpA( [ka] , SEQ ID NO: 16), DsbA( [ka] , SEQ ID NO: 17), SpA( [ka] 18) or the flexible linker structure AA 236-243 ( [ka] , SEQ ID NO: 19), with modifications in OmpA to accommodate a restriction site, and mutations in OmpA and SpA to mutate an asparagine to a glutamine ( FIG. 1 ). Additionally, a variety of different sequences were cloned into the plasmid pGE0002 (Table 1 ).

[0078] Digestion of plasmids The plasmid was cut with the restriction enzymes FspI and KpnI (New England Biolabs (NEB), MA, USA) and 6 μl NEB buffer 2.1 was mixed with 6 μg plasmid and 2 μl FspI to a total of 58 μl. The solution was incubated at 37° C. for 1 h before adding KpnI. Incubation was continued for 2 h, after which 1 μl calf intestinal phosphatase (CIP) was added followed by further incubation for 30 min. Excised bases were removed from the digested plasmid using a QIAquick PCR purification kit (Qiagen, Hilden, Germany).

[0079] Hybridization Oligonucleotides were ordered from Integrated DNA technologies (IDT, IA, USA). All oligonucleotides were modified with a 5' phosphate group by the manufacturer. Two complementary oligonucleotide pairs were mixed in ligation buffer and heated to 95°C for 4 min, then cooled to room temperature. Hybridized fragments were ligated into the FspI and KpnI cut plasmid by using T4 DNA ligase (NEB, MA, USA). A complete list of oligonucleotides used can be found in Table 3.

[0080] [Table 3]

[0081] Cloned constructs The ligated plasmids were transformed into chemically competent E. coli K12-017 cells. The cells were thawed on ice for 30 min and 100 μl competent cells were added to the 10 μl ligation reaction. The cells were incubated on ice for 20 min, then heat shocked at 42° C. for 1 min, then incubated on ice for 5 min. Then 900 μl SOC medium (NEB, MA, USA) was added. The transformation reactions were incubated at 37° C. for 60 min in a rotary shaking incubator and 100 μl of each reaction was spread on Luria agar plates containing the appropriate selection antibiotic. Positive clones were screened by polymerase chain reaction (PCR) to select clones with the correct insert. Selected clones were grown overnight (o / n) in 10 ml Luria Broth (LB) containing the appropriate antibiotics, and then plasmids were prepared using the Qiagen Plasmid Miniprep kit (Qiagen, Hilden, Germany). Plasmids were sent to GATC Biotech (Cologne, Germany) for sequence verification.

[0082] [Table 4A]

[0083] [Table 4B]

[0084] Example 1 Protein expression and purification in shake flasks E. coli strain K12-017 transformed with the recombinant plasmid pGE0138-145 was grown in 100 ml Terrific Broth (TB) medium (containing 12 g tryptophan, 24 g yeast extract, 5 g glycerol (85%), 2.31 g KH2PO4, 12.54 g K2HPO4, and 50 mg kanamycin sulfate per liter) at 30°C for 4 hours. The culture was grown at OD 600nmWhen the β-amino acid concentration reached 1-2, the culture was induced with 4 mM L-rhamnose (Sigma Aldrich, MO, USA). The culture was further incubated at 30°C for 17-20 h. The culture solution was subjected to low-speed centrifugation (4,000 rpm) in a swing-out rotor for 20 min, and the wet cell pellet was collected. The bacterial cell pellet was suspended in 20 ml of 25 mM phosphate buffer solution (pH 7.4), and the cells were lysed by heat treatment at 85°C for 10 min in a heat block. Then, high-speed centrifugation (13,000 rpm) was performed for 10 min to separate the supernatant. The supernatant was then filtered through a 0.2 μm syringe filter to remove any remaining particles, and then it was applied to an IgG Sepharose 6FF (GE Healthcare Bio-Sciences, Uppsala, Sweden) affinity chromatography column. The column was equilibrated with loading buffer (25 mM phosphate pH 7, 250 mM NaCl) before loading the sample. After loading, the column was washed with 5 column volumes (CV) of loading buffer and 1 CV of low-salt wash buffer (50 mM acetate pH 6) and then eluted with 50 mM acetic acid, pH 2.8. The absorbance at 237 nm was measured in-line using an AKTA explorer 100 chromatography system (GE Healthcare Bio-Sciences, Uppsala, Sweden) and peak integration of the eluted peaks was performed in the system software (Unicorn 5.1).

[0085] Shake flask results for two signal peptides and four N-terminal starts In the first work of optimizing the expression system, two different signal peptides were tested that utilize different secretory pathways in cells. DsbA utilizes the signal recognition particle (SRP) pathway, and OmpA utilizes the Sec pathway. Results from protein expression using DsbA and OmpA signal peptides showed that the OmpA signal peptide resulted in higher protein expression compared to the DsbA signal peptide; 2-5 fold. Furthermore, the first N-terminal AA after the cleavage site of the signal peptide had a significant effect on expression levels. In this study, the DsbA start AA resulted in the highest expression levels with either signal peptide. The lowest expression levels were seen for the Flex8AA start sequence with either signal peptide.

[0086] Liquid chromatography coupled with mass spectrometry (LC / MS) results The eluate from IgG Sepharose 6FF was analyzed using LC / MS (Waters, PA, USA). The results showed that the eluate from the reference construct pGE0120 with AQGT as the N-terminal start sequence had a variety of different signal peptide cleavage sites (Figure 3, a). The integrated area of ​​the peak with the correct mass, where the signal peptide was correctly processed, was 52% of the total area. Other peaks corresponded to 6 AA extra, 9 AA extra, intact signal peptide (21 AA extra), and 7 AA deletion from the main construct. After the addition of 8-AA from DsbA, the signal peptide was correctly cleaved up to 96% (Figure 3, b). Similar results were seen for the DsbA signal peptide and the DsbA 8AA N-terminus, with 97% correctly cleaved signal peptide.

[0087] Example 2 Protein expression in fed-batch fermenters Six 1 L working volume fermenters, GRETA (Belach Bioteknik, Skogás, Sweden) were used. The starting volume was set at 750 ml and the final volume was approximately 1 L. Aeration was set at 1 L / min and temperature, pH and antifoam were automatically controlled. pH was maintained at the set point by addition of 25% ammonia and 2 M phosphoric acid. Antifoam control was automatic when a lot of foaming occurred, with the addition of Breox FMT 30 (BASF, Ludwigshafen, Germany). pH and dissolved oxygen (DO) were controlled with Broadley James (CA, USA) probes. DO was kept constant at 30% by increasing the stirrer speed from 300 rpm to 1500 rpm. When the stirrer reached maximum speed, the DO second set point was 20% and was kept constant by adding pure oxygen mixed into the air stream. Terrific Broth (TB) supplemented with 50 mg / L kanamycin or neomycin was used for shake flask precultures, which were initiated by addition of 100 μl cell suspension to 100 mL TB supplemented with 50 μg / mL kanamycin or neomycin and incubated at 37° C. for 17 h. The main fermentation medium was inoculated with 10 ml preculture. After the first batch glucose was consumed, a glucose feed at 60% (w / v) (VWR, PA, USA) was fed to the fermenter according to a preset profile. The total duration of the main fermentation was 26 h.

[0088] Concentration analysis using a standard curve The fermentation samples were heat treated at 85°C for 5 min in 1.5 ml tubes. Then, high speed centrifugation (13500 rpm, tabletop centrifuge) was performed for 5 min to separate the supernatants. The supernatants were filtered through a 0.2 μm syringe filter to remove any remaining particles, after which they were applied to an IgG Sepharose 6FF HiTrap™ column (GE Healthcare Bio-Sciences, Uppsala, Sweden). The column was equilibrated with loading buffer (phosphate buffered saline (Medicago, Uppsala, Sweden)) and then 50 μl sample was loaded. After loading, the column was washed with 5 CV of loading buffer and then eluted with 200 mM phosphate buffer, pH 2.9. The absorbance at 237 nm was measured in-line and peak integration of the eluted peaks was performed in the chromatography system software. The protein concentration of the samples was determined by a standard curve using purified proteins of known concentrations.

[0089] Fermentation and protein expression results Plasmid pGE0002, containing the Zvar2 hexamer (Zvar26), was digested with FspI and KpnI restriction enzymes. The N-terminal start from DsbA was ligated into the digested vector, and the resulting plasmid was designated pGE0180. These two vectors were transformed into E. coli K12-017, and the resulting constructs were expressed in fed-batch fermentation to see the difference in protein expression and signal peptide cleavage patterns. Protein concentration analysis using a standard curve showed an increase in protein expression from 3.5 g / L to 16.8 g / L, i.e., a more than four-fold increase in protein expression (Figure 4).

[0090] LC / MS results The eluate from IgG Sepharose was analyzed using LC / MS (Waters, PA, USA). The results showed that the eluate from the reference construct pGE0002 showed a variety of different signal peptide sites, including the cleavage sites with the extra 6AA and 9AA, previously seen in experiment 1 (Figure 5, a). However, when the 8-AA from DsbA was added to the N-terminus, the signal peptide was correctly cleaved, resulting in a clear single peak of the correct mass (Figure 5, b).

[0091] Example 3 Protein expression in fed-batch fermenters and concentration analysis using standard curves were performed as in Example 2.

[0092] Protein preparation The filtered sample (see above) was applied to an IgG Sepharose 6FF Tricorn 10 column (GE Healthcare Bio-Sciences, Uppsala, Sweden) with a CV of 10.5 ml. The column was equilibrated with high salt loading buffer (50 mM phosphate, pH 7.0, 500 mm NaCl) and then loaded with 0.5–5 mL sample based on the concentration calculation (see above). After loading, the column was washed with 5 CV of loading buffer and then eluted with 100 mM acetic acid. The eluted sample was collected and concentrated to approximately 1 mg / ml using a Vivaspin 5, 3000 Da cutoff (GE Healthcare Bio-Sciences, Uppsala, Sweden). Protein quantity was estimated by absorbance measurements at 280 nm using the specific extinction coefficient calculated based on Pace et al., Protein Science 4, 2411-2423, (1995) and the Beer-Lambert law. Sample concentrations were back-calculated using the total protein mass in the elution pool and the volume of the injected sample. The concentration was confirmed by amino acid analysis. Homogeneity and molecular weight were analyzed using mass spectrometry on a Waters Q-Tof (PA, USA).

[0093] Protein expression results of additional constructs To determine the effect of N-terminal start sequences on other domains from Staphylococcus aureus protein A as well as the alkali-stabilizing domains Zvar monomer (Zvar1) and tetramer (Zvar4), new plasmids with additional N-termini were constructed and expressed in fed-batch fermentation using plasmid pJ401. Results showed that the E domain did not result in measurable expression without an insert. However, with the 8AA DsbA start, protein expression reached 2.9 g / L. Both the D and A domains showed low expression, which was not improved by adding an N-terminal start. Both the C and B domains (which have the same AA sequence at the start) showed very similar expression, and with the 8AA DsbA start, there was a clear increase in expression. However, the greatest increase in expression was seen in the Zvar monomer and Zvar2 monomer (Zvar1 and Zvar21), with over 4-fold and 10-fold protein expression, respectively. Also, the Zvar tetramer (Zvar4) showed a significant increase in protein expression with the 8AA DsbA initiation, although the increase was not as dramatic as for the monomer.

[0094] [Table 5]

[0095] In initial experiments with N-terminal spacers, two different signal peptides from two secretory pathways were tested, and it was shown in these experiments that the OmpA signal peptide, which utilizes the Sec-dependent pathway, resulted in the highest expression and signal peptide cleavage. Other signal peptides that use this pathway are e.g. PhoA (Escherichia coli alkaline phosphatase), MalE (Escherichia coli maltose binding protein), and PelB (Erwinia carotovora pectate lyase B). In contrast, the reference construct ( [ka] The constructs with the DsbA signal peptide (with the start part) resulted in higher protein expression and signal peptide cleavage with the DsbA signal peptide, indicating that it is the combination of the signal peptide and the N-terminus. Furthermore, four different N-termini were tested and all performed better compared to the reference construct with the OmpA signal peptide. Surprisingly, when the signal peptide cleavage worked optimally, protein expression also increased, indicating that signal peptidase cleavage may be the rate-limiting enzyme in the transport of proteins across the inner membrane. Without wishing to be bound by theory, the hypothesis is that when secretion is not working optimally, the protein becomes "stuck" in the pores of the inner membrane, stopping further expression. In cultures with high protein expression, more than 50% of the protein was found outside the cells in the fermentation broth, indicating periplasmic leakage into the extracellular space. When other domains from Staphylococcus aureus protein A were tested, it was found that the E domain did not result in measurable protein expression, which was unexpected considering that it is the first domain after the signal peptide in native Staphylococcus aureus protein A. However, one important difference is that the first AA after the cleavage site is not included in the cloning sequence. [ka] from [ka] to , and it is this substitution / insertion that may negatively affect signal peptide function. Another difference is that the signal peptide is replaced from the native SpA to OmpA, which in combination with the N-terminal start may not be optimal. When the 8AA DsbA N-terminus is added to the E domain in combination with the OmpA signal peptide, protein transport and expression function properly. Furthermore, the A and D domains, which are very closely related to the N-terminal proximal structural unit, are poorly expressed and are not improved by additional AA, whereas the identical B and C domains in this unit are well expressed with the AQGT start, and expression is further increased when the 8AA DsbA is added to the N-terminus. Zvar as monomers and tetramers also increases expression with additional AA. However, the most dramatic increase is seen in the Zvar2 monomer, where protein expression goes from 0.68 g / L to 7.28 g / L. Without wishing to be bound by theory, a hypothesis for the difference between Zvar and Zvar2 is that Zvar2 has two mutations at positions Q9 and N11 in the first α-helix, which, as calculated with algorithms such as GORIV (available online at https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_gor4.html (PRABI, Rhone-Alpes Bioinformatics Center)), are predicted to increase the α-helical structure at the start of the protein and therefore increase the steric hindrance for the signal peptidase to cleave the signal peptide of the nascent protein.

[0096] Example 4 N-Terminal Spacer Variants Each N-terminal insert was designed to examine the effect of the insert sequence on one or more of the following parameters: protein yield, signal peptide cleavage, and alkaline stability. The designs can be divided into four separate categories as follows: 1. Changing the length of an existing insert without modification. 2. To remove the site (glycine-threonine) suspected to be truncated under alkaline conditions and at the same time change the length of the remaining insert. 3. Substitution of glycines at known sites of truncation with various other residues. 4. Replacing the entire sequence.

[0097] The purpose of category 1 was primarily to examine the effect of insert length on signal peptide cleavage and protein yield, and did not explicitly address the suspected truncation site in alkaline conditions. Categories 2 and 3 were mostly aimed at addressing the alkaline stability issue, but they also provide some data on the other two issues. Category 4 is a broader category, providing a broader set of sequences to provide data in all three areas of interest. The nucleotide sequences were codon-optimized for E. coli, but some degeneracy was added in the more highly repeated sequences. The inserts in categories 1 and 4 were designed to be complementary to the FspI digestion site at the 5' end and to the KpnI digestion site at the 3' end. Because the glycine-threonine truncation site was part of the KpnI restriction site, instead the inserts in categories 2 and 3 were designed to be complementary to the adjacent AccI restriction site at the 3' end, while retaining the 5' complementarity with FspI. The amino acid sequences of all the inserts are shown in table 6. An example of a general construct is shown in FIG.

[0098] [Table 6]

[0099] Constructs were assembled by ligating the different hybridized oligonucleotides into either pGE0002 or pGE0180 plasmids that had been digested with the relevant restriction enzymes to remove the first N-terminal insert. After ligation, the plasmids were transformed into E. coli K12-017 cells. Positive colonies were screened using colony PCR and electrophoresis. Two or three colonies from each construct were selected and sent to GATC biotech (Cologne, Germany) for sequence verification.

[0100] One sequence-verified clone for each construct was selected for protein expression in shake flask cultures. Figure 7 shows a summary of the results from the quantification of protein yield in the different shake flask cultures, performed on IgG Sepharose 6FF. For each construct, the left bar is the protein concentration after linear correction using 5 g / L and 10 g / L standard solutions as reference. The right bar represents this concentration as a function of the OD at the end of the culture. 600nm This value is intended to give an idea of ​​whether low protein concentrations are due to an actual low rate of protein production per cell or simply due to low culture density. Note that the concentrations were measured after pelleting the cells and resuspending them in phosphate buffered saline (Medicago, Uppsala, Sweden) and as such are not directly comparable to concentrations from samples taken from, for example, multifermenters.

[0101] Protein solutions for each construct resulting from shake flask cultures were purified using IgG Sepharose 6FF (GE Healthcare, Uppsala, Sweden). Purified protein samples were examined for signal peptide cleavage using LC / MS analysis (Waters, PA, USA). Only proteins with correctly cleaved signal peptides have one major mass (e.g., total ion counts (TIC) shown in Figure 8a), whereas proteins with incorrectly cleaved signal peptides give rise to one or more secondary peaks (as shown in Figure 8b). Additional minor peaks to the left and right of the main peak are likely due to leakage from the cytoplasm during the heat treatment step when the protein is extracted from the cell culture. Peaks C1-C5, N1, and N2 are likely various partially digested versions of the protein from the cytoplasm. The small peak to the right of the main peak is the protein with an uncleaved signal peptide, which is reasonable to see if leakage from the cytoplasm has occurred.

[0102] In many cases, the peaks were difficult to conclusively interpret due to sample purity and poor separation during the liquid chromatography step, however, it could be seen that the following constructs produced the correct signal peptide cleavage: pGE0180, DsbA7, DsbA6, DsbA8_noGT, DsbA8_DT, DsbA8_ET, DsbA8_AT, DsbA7_EDT, DsbA12, DsbA16, H8, H6, and SPA.

[0103] After signal peptide cleavage investigation, protein solutions of constructs that gave correct signal peptide cleavage were further purified using a Capto Q ImpRes anion exchange column (GE Healthcare, Uppsala, Sweden). Purified samples were subjected to treatment with 1 M sodium hydroxide (NaOH) for 0, 4, and 24 h. A Vivaspin column (GE Healthcare, Uppsala, Sweden) was used to separate the major protein from any potentially truncated smaller peptides. Both peptide samples and the major protein were examined using LC / MS (Waters, PA, USA).

[0104] Peptides appear in the mass spectrum with a +2 charge, i.e. the value at mass over charge (m / z) corresponds to half their molecular weight. In the cases examined here, the peptides all had molecular weights between about 800 Da and 1600 Da. As such, a cluster of related peaks appeared between 400 m / z and 800 m / z, as seen for pGE0180 on the left in Figure 9.

[0105] If peptides were present, the MassLynx search function was used on the TIC to extract peaks of different peptide masses as extracted ion chromatograms (XICs). The XIC peaks were integrated and the integrated areas were then used to compare the amounts of different peptides in the samples.

[0106] Figure 10 shows a summary of the results from the initial alkaline stability study performed on unwashed Vivaspin columns. Despite significant background noise overlay, clear patterns are visible after 24 hours of incubation.

[0107] Figure 11 shows a different view of the same data for the most promising construct (including pGE0180 as a reference), while Figure 12 shows the degradation of the most severely cleaved construct. The sequence around the cleavage site is included to show what residues may be vulnerable. Notably, for the low peak areas (approximately 5000 area units), background noise has a significant effect.

[0108] A second run on an isopropanol-washed spin column gave results almost similar to the first run, but with reduced peak areas. A notable exception was DsbA6, which appeared to undergo some truncation. This was not directly detectable from peptide analysis, but became evident when the residue was examined; DsbA6, much like pGE0180, showed a distinct truncated peak, whereas DsbA8_noGT and DsbA_DT did not.

[0109] Example 5 Interesting constructs from the shake flask studies and subsequent analysis of purified proteins were selected for larger scale cultivation in multiple fermentors (Belach Bioteknik, Skogas, Sweden). At the end of the fermentation, the OD 600nm and protein concentration was measured (see Figure 13).

[0110] Because different proteins have slightly varying extinction coefficients, it stands to reason that they might differ in their absorbance at 237 nm (where protein concentration was estimated) as well. Therefore, the ratio of the pGE0180 extinction coefficient (0.294) to the calculated extinction coefficients for each of the other proteins was used to estimate a more "true" value.

[0111] Fed-batch cultures of constructs DsbA8_noGT and DsbA8_DT were heat treated to extract periplasmic proteins and centrifuged and diafiltered to remove cell debris. The filtrate was adjusted to remove some contaminants by precipitation and the solution was prepared for purification on a Capto S ImpAct (GE Healthcare, Uppsala, Sweden) using a pH gradient. The eluate was reduced to remove dimers, desalted to remove DTT and to obtain the correct buffer for anion exchange purification using a salt gradient. The final purified solution was concentrated by membrane ultrafiltration to reach the concentration required for immobilization at the desired ligand density. The final product was analyzed for purity by SEC and for molecular weight by LC / MS with satisfactory results.

[0112] Purified protein (>90% purity) was coupled to highly cross-linked agarose beads aiming at a ligand density similar to previous experiments where Zvar2 was immobilized on a base matrix of similar nature and analyzed for dynamic binding capacity. The coupled gel was packed into a Tricorn 5 / 100 column (GE Healthcare, Uppsala, Sweden). Packing of the gel bed was assessed by the asymmetry of the acetone peak. The packed column was used to measure the dynamic binding capacity of the gel with IgG. One measurement was performed per construct. The results are summarized in table 7. Figure 14 shows a comparison of the measurements performed in this example with those performed in the previous experiment described above.

[0113] [Table 7]

[0114] Example 6 An N-terminal spacer AQYEDGKQYTGT and a C-terminal cysteine ​​were introduced into several additional mutants of Zvar2 monomers and dimers (listed below) that use the OmpA signal peptide.

[0115] Plasmids were transformed into chemically competent E. coli K12-017 cells. The cells were thawed on ice for 30 min and 50 μl competent cells were added to 20 ng plasmid. The cells were incubated on ice for 20 min, then heat shocked at 42° C. for 1 min, then incubated on ice for 5 min, and 400 μl SOC medium (NEB, MA, USA) was added. The transformation reactions were incubated at 37° C. for 60 min in a rotary shaking incubator and 200 μl of each reaction was spread on Luria agar plates containing the appropriate selection antibiotic.

[0116] E. coli strain K12-017 transformed with the recombinant plasmids (Table 8) was grown in 100 ml Terrific Broth (TB) medium (containing 12 g tryptone, 24 g enzyme extract, 5 g glycerol (85%), 2.31 g KH2PO4, 12.54 g K2HPO4, and 50 mg kanamycin sulfate per liter) at 37°C. The cultures were grown at OD 600nmWhen the β-actin concentration reached 1, it was induced with 1 mM IPTG (Sigma Aldrich, MO, USA). The culture was further incubated at 30°C for 17-20 h. The culture was subjected to low-speed centrifugation (4,000 rpm) in a swing-out rotor for 20 min, and the wet cell pellet was collected. The bacterial cell pellet was suspended in 10 ml of 25 mM phosphate buffer solution (pH 7.4), and the cells were lysed by heat treatment at 85°C for 15 min in a heat block. Then, high-speed centrifugation (10,000xg) was performed for 10 min to separate the supernatant. The supernatant was then filtered through a 0.2 μm syringe filter to remove any remaining particles, and then it was applied to XK 16-6 IgG Sepharose 6FF (GE Healthcare Bio-Sciences, Uppsala, Sweden). The column was equilibrated with loading buffer (25 mM phosphate pH 7, 250 mM NaCl) before loading the sample. After loading, the column was washed with 5 column volumes (CV) of loading buffer and 1 CV of low-salt wash buffer (50 mM acetate pH 6) and then eluted with 50 mM acetate, pH 2.8. The absorbance at 237 nm was measured inline using an AKTA explorer 100 (GE Healthcare Bio-Sciences, Uppsala, Sweden) and peak integration of the eluted peaks was performed in the system software (Unicorn 5.1).

[0117] The IgG binding fraction was eluted and collected in a pool, and the pool volume and protein concentration were noted (see table 8). The amount of expressed IgG binding protein may in some cases be higher than the amount recovered due to loss of protein secreted into the medium and / or overloading of the IgG column.

[0118] [Table 8A]

[0119] [Table 8B]

[0120] Zvar2(A29G) monomer (SEQ ID NO:48) [ka] Zvar2 (A29S) monomer (SEQ ID NO:49) [ka] Zvar2 (A29Y) monomer (SEQ ID NO:50) [ka] Zvar2 (A29Q) monomer (SEQ ID NO:51) [ka] Zvar2 (A29T) monomer (SEQ ID NO:52) [ka] Zvar2 (A29N) monomer (SEQ ID NO:53) [ka] Zvar2 (A29F) monomer (SEQ ID NO:54) [ka] Zvar2 (A29L) monomer (SEQ ID NO:55) [ka] Zvar2 (A29W) monomer (SEQ ID NO:56) [ka] Zvar2 (A29I) monomer (SEQ ID NO:57) [ka] Zvar2 (A29M) monomer (SEQ ID NO:58) [ka] Zvar2 (A29V) monomer (SEQ ID NO:59) [ka] Zvar2 (A29D) monomer (SEQ ID NO:60) [ka] Zvar2 (A29E) monomer (SEQ ID NO:61) [ka] Zvar2 (A29H) monomer (SEQ ID NO:62) [ka] Zvar2 (A29R) monomer (SEQ ID NO:63) [ka] Zvar2 (A29K) monomer (SEQ ID NO:64) [ka] Zvar2 (Δ235,236,237) dimer (SEQ ID NO:65) [ka] Zvar2 (Δ233, 234, 235) duplex (SEQ ID NO: 66) [ka] Zvar2 (Δ5-1) dimer (SEQ ID NO:67) [ka] Zvar2 (Δ5-2) dimer (SEQ ID NO:68) [ka] Zvar2 (D3C-term) duplex (SEQ ID NO: 69) [ka] Zvar2 (D3N-term) duplex (SEQ ID NO: 70) [ka] Zvar2 (D8N-term) duplex (SEQ ID NO: 71) [ka] Zvar2 (linker+8) duplex (SEQ ID NO:72) [ka] Zvar2 (linker+4) duplex (SEQ ID NO:73) [ka] Zvar2 (ΔQ9) monomer (SEQ ID NO:74) [ka] Zvar2 (ΔQ40) monomer (SEQ ID NO:75) [ka] Zvar2 (ΔA42) monomer (SEQ ID NO:76) [ka] Zvar2 (ΔN43) monomer (SEQ ID NO:77) [ka] Zvar2 (ΔL44) monomer (SEQ ID NO:78) [ka] Zvar2 (E11N, A12F) monomer (SEQ ID NO:79) [ka] Zvar2 (E11N, A12Y) monomer (SEQ ID NO:80) [ka] Zvar2 (E11N, A12K) monomer (SEQ ID NO:81) [ka] Zvar2 (E11N, A12R) monomer (SEQ ID NO:82) [ka] Zvar2 (L22F) monomer (SEQ ID NO:83) [ka] Zvar2 (A43N, I44F) monomer (SEQ ID NO:84) [ka] Zvar2 (A43N, I44Y) monomer (SEQ ID NO:85) [ka] Zvar2 (A43N, I44W) monomer (SEQ ID NO:86) [ka] Zvar2 (A43N, I44R) monomer (SEQ ID NO:87) [ka] Zvar2 (A43N, I44K) monomer (SEQ ID NO:88) [ka] Zvar2 (D53F) monomer (SEQ ID NO:89) [ka] Zvar2 (D53Y) monomer (SEQ ID NO:90) [ka] Zvar2 (D53W) monomer (SEQ ID NO:91) [ka] Zvar2 (D53K) monomer (SEQ ID NO:92) [ka] Zvar2 (D53R) monomer (SEQ ID NO:93) [ka]

[0121] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any device or system, and practicing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that have insubstantial differences from the literal language of the claims. Any patents or patent applications mentioned herein are incorporated by reference in their entirety as if they were individually incorporated.

Claims

1. A recombinant protein comprising a functional polypeptide and an N-terminal spacer linked to the N-terminus of the functional polypeptide, the N-terminal spacer having a length such that the number of amino acid residues between the signal peptide cleavage site and the N-terminal proximal structural unit of the functional polypeptide is 14 to 24.

2. A recombinant protein comprising an immunoglobulin-binding polypeptide and an N-terminal spacer linked to the N-terminus of the immunoglobulin-binding polypeptide, the N-terminal spacer having a length such that the number of amino acid residues between the signal peptide cleavage site and the N-terminal proximal structural unit of the functional polypeptide is 14 to 24.

3. 3. The recombinant protein of claim 1 or 2, wherein the N-terminal proximal structural unit is an alpha helix.

4. The recombinant protein of claim 3, wherein the functional or immunoglobulin-binding polypeptide comprises at least three alpha helices.

5. 5. The recombinant protein according to claim 1, wherein the N-terminal spacer comprises at most two amino acid residues selected from the group consisting of lysine and arginine.

6. 6. The recombinant protein according to any one of claims 1 to 5, wherein the N-terminal spacer consists of 8 to 24 amino acid residues.

7. 1. A recombinant protein comprising a functional or immunoglobulin-binding polypeptide and an N-terminal spacer linked to the N-terminus of said functional or immunoglobulin-binding polypeptide, said N-terminal spacer comprising an amino acid sequence having at least 80% sequence identity to, or defined by, an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-18, 29-30, 33-40, 43-45, and 47.

8. 8. The recombinant protein according to any one of claims 1 to 7, wherein the N-terminal spacer has at least 80% sequence identity with or is defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-18, 29-30, 33-40, 43-45, and 47.

9. 9. The recombinant protein according to any one of claims 1 to 8, wherein the functional or immunoglobulin-binding polypeptide comprises one or more Fc-binding domains derived from Staphylococcus aureus protein A.

10. The recombinant protein of claim 9, wherein the Fc binding domain is an alkali-stabilized Fc binding domain.

11. 11. The recombinant protein of claim 10, wherein the alkali-stabilized Fc binding domain has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11 and 48-64.

12. 12. A recombinant protein according to any one of claims 9 to 11, wherein the functional or immunoglobulin binding polypeptide comprises a multimer of at least four Fc binding domains.

13. 13. The recombinant protein of any one of claims 1 to 12, wherein the functional or immunoglobulin-binding polypeptide comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 and 48-93.

14. 14. A recombinant protein according to any one of claims 1 to 13, further comprising a coupling moiety at or adjacent to the C-terminus.

15. 15. The recombinant protein of claim 14, wherein the coupling moiety comprises a cysteine ​​residue and / or multiple lysine residues.

16. 16. The recombinant protein of claim 1, wherein the N-terminal spacer is alkaline stable.

17. 17. The recombinant protein of any one of claims 1 to 16, wherein the N-terminal spacer consists of an amino acid residue selected from the group consisting of alanine, aspartic acid, glutamine, glutamic acid, glycine, histidine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

18. 18. A nucleic acid molecule encoding a recombinant protein according to any one of claims 1 to 17, comprising the following elements in the 5' to 3' direction, said elements being operably linked: a) an inducible or constitutive promoter DNA sequence; b) a DNA sequence encoding a signal peptide; c) a DNA sequence encoding an N-terminal spacer; and d) A DNA sequence encoding a functional or immunoglobulin-binding polypeptide.

19. 19. The nucleic acid molecule of claim 18, wherein the signal peptide has at least 80% sequence identity with or comprises an amino acid sequence defined by an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15.

20. 19. The nucleic acid molecule of claim 18, wherein the signal peptide comprises an amino acid sequence having at least 80% sequence identity to or defined by SEQ ID NO:

14.

21. A cloning vector for expressing and secreting into the bacterial periplasm of a gram-negative cell a recombinant protein as defined in any one of claims 1 to 17 and any one of claims 18 to 20, comprising a nucleic acid molecule as defined in any one of claims 18 to 20.

22. A Gram-negative bacterium transformed with the cloning vector of claim 21.

23. 23. The gram-negative bacterium of claim 22, identified as Escherichia coli.

24. 24. The gram-negative bacterium of claim 23, further characterized as Escherichia coli K12.

25. 25. The Gram-negative bacterium of claim 23 or 24, further characterized as Escherichia coli K12-017.

26. A method for expressing and secreting a recombinant protein as defined in any one of claims 1 to 17 and any one of claims 18 to 25 in a Gram-negative bacterium, comprising the steps of: i) providing a gram-negative bacterium according to any one of claims 22 to 25; and ii) culturing the gram-negative bacteria.

27. 18. A separation matrix comprising a recombinant protein according to any one of claims 1 to 17, covalently linked to a support.

28. 28. The separation matrix of claim 27, wherein the support comprises porous particles.

29. 30. The separation matrix of claim 28, wherein the porous particles comprise cross-linked polysaccharides.

30. 30. The separation matrix of any one of claims 27 to 29, wherein the recombinant protein is covalently linked to the support via a thioether bond.

31. 30. The separation matrix of any one of claims 27 to 29, wherein the recombinant protein is covalently linked to the support via one or more amide bonds.

32. 32. The separation matrix according to any one of claims 27 to 31, wherein the recombinant protein comprises an immunoglobulin-binding polypeptide and is alkaline stable, such that the IgG capacity of the matrix after a 24 hour incubation time in 0.5 M NaOH at 22 + / - 2°C is at least 80% of the IgG capacity before incubation.

33. 1. A method for isolating immunoglobulins, comprising the steps of: i) providing a separation matrix according to any one of claims 27 to 32, wherein the recombinant protein comprises an immunoglobulin-binding polypeptide; ii) contacting the separation matrix with a liquid sample containing immunoglobulins to bind the immunoglobulins; iii) optionally washing the separation matrix with a washing solution; iv) contacting the separation matrix with an elution liquid to elute the immunoglobulins.

34. 34. The method of claim 33, wherein in step a) the recombinant protein comprises one or more Fc binding domains derived from Staphylococcus aureus protein A, and the method further comprises, after step iv), a step v) of cleaning the separation matrix with a cleaning solution.

35. 35. The method of claim 34, wherein the cleaning liquid comprises at least 0.1 M NaOH or KOH, such as at least 0.5 M NaOH or KOH, or 0.5 to 2.5 M NaOH or KOH.

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