Patent Description:
Today, due to the advancement of the gene recombination technique, production of useful proteins by heterologous expression has become common. For production of a useful protein by heterologous expression, factors such as selection of a promoter and a terminator, a translational enhancer, codon modification of the transgene, and intracellular transport and localization of the protein are studied for improving expression of the protein and the amount of the protein accumulated. For example, Patent Document <NUM> discloses a technique in which a bacterial toxin protein is expressed in a plant or the like, wherein the bacterial toxin protein is expressed in a state where it is linked through a peptide linker comprising prolines arranged at constant intervals (Patent Document <NUM>, <CIT>). For example, Non-patent Document <NUM> (Wu, <NUM>) discloses the production of the proline-rich peptide metchnikowin in E. coli and Non-patent Document <NUM> (UniProt entry G8B5T8) discloses the amino acid sequence of a protein from Candida parapsilosis.

There are also several techniques for improving expression of a protein of interest by linking a peptide tag thereto (Patent Document <NUM> (<CIT>), Non-patent Documents <NUM> to <NUM> (Smith, <NUM>; Marblestone, <NUM>; di Guan, <NUM>, Maria, <NUM>)). Most of these peptide tag-linking techniques improve the solubility of the protein of interest, suppress formation of inclusion bodies in the cell, and promote normal expression of the protein of interest. However, the techniques are not intended for improvement of the expression level of the protein. Moreover, most of such techniques are applied to expression systems using E.

By linking of a toxin protein using the peptide linker comprising prolines arranged at constant intervals as disclosed in Patent Document <NUM> (<CIT>), high-level accumulation of a toxin fusion protein in a plant is possible. However, although this peptide linker is useful as a linker for linking a plurality of proteins to each other, its ability as a peptide tag for the purpose of high-level expression of a single protein of interest has not been sufficiently studied, and such an ability remains to be further studied. In view of this, an object of the present invention is to provide a peptide tag for use in cases where a protein of interest is expressed in a host cell, wherein the peptide tag is linked to the protein of interest to enable achievement of an increased expression level of the protein of interest.

In an attempt to improve the performances of the linker peptides disclosed in Patent Document <NUM> (<CIT>) as peptide tags for high-level expression of protein, the present inventors first focused on the presence of proline in these peptides (PG12 and PG17), and expected that further improvement of useful properties of the peptides may be possible by substituting serine (S) and/or glycine (G) present between proline (P) and proline (P) with an amino acid(s) having different physicochemical properties. More specifically, peptide tags were prepared by substituting serine (S) and/or glycine (G) in the peptides with a basic amino acid(s) such as lysine (K) and/or arginine (R), with an acidic amino acid(s) such as aspartic acid (D) and/or glutamic acid (E), and/or with an amino acid(s) having different steric properties and/or polarity whose side chain(s) is/are uncharged such as alanine (A), threonine (T), leucine (L), methionine (M), asparagine (N), and/or glutamine (Q), and each peptide tag was fused with a protein of interest to attempt improvement of expression of the protein of interest. As a result, it was found that, by the addition of the peptide prepared by the substitution of S and/or G present between P and P with K, L, N, Q, and/or R in PG12 or PG17 to the protein of interest, the expression level of the protein of interest can be improved. The present invention was accomplished based on such findings.

The present invention as also characterized in the claims relates to a tagged protein comprising a peptide tag bound to a protein of interest, the peptide tag having the following sequence:
Xm(PYn)qPZr.

Further the present invention relates to the use of the peptide tag as defined herein above and/or below for increasing and/or for improving the expression level of the protein of interest in a host cell. Accordingly, the present invention provides for the use of a peptide tag for increasing and/or for improving the expression level of the protein of interest in a host cell, the peptide tag having the following sequence:
Xm(PYn)qPZr.

By using the peptide tag disclosed herein and comprised in the tagged protein of the present invention, the expression level of a protein of interest can be improved. Thus, the present invention is useful for production of a protein using a cell such as yeast, E. coli, or Brevibacillus. In particular, since effects of tags on the expression level have so far been unclear in E. coli and Brevibacillus, achievement of improvement of expression in these cells using the tag is industrially very useful. Since the peptide tag of the present invention may have a length of as small as <NUM> to <NUM> amino acids, it is less likely to affect the structure or function of the protein of interest to which the peptide tag is added. Thus, it is highly likely that cleavage treatment after the expression can be omitted. If removal of the peptide tag is required, a protease recognition sequence may be inserted therefor.

The present invention also relates to a DNA encoding the herein above and/or below detailed tagged protein. Accordingly, the present invention relates to a DNA encoding a tagged protein comprising a peptide tag bound to a protein of interest, the peptide tag having the following sequence:
Xm(PYn)qPZr.

Further, the present invention relates to a recombinant vector comprising the herein above and/or below detailed DNA. Accordingly, the present invention relates to a recombinant vector comprising a DNA encoding a tagged protein comprising a peptide tag bound to a protein of interest, the peptide tag having the following sequence:
Xm(PYn)qPZr.

The present invention also relates to a transformant prepared by transformation with the herein above and/or below detailed DNA or the herein above and/or below detailed recombinant vector. Accordingly, the present invention relates to a transformant prepared by transformation with a DNA encoding a tagged protein comprising a peptide tag bound to a protein of interest or a recombinant vector comprising said DNA, the peptide tag having the following sequence:
Xm(PYn)qPZr.

Further, the present invention relates to a method for producing a tagged protein, comprising culturing the herein above and/or below detailed transformant to allow accumulation of the tagged protein, and collecting the tagged protein. Accordingly, the present invention relates to a method for producing a tagged protein, comprising culturing a transformant prepared by transformation with a DNA encoding a tagged protein comprising a peptide tag bound to a protein of interest or a recombinant vector comprising said DNA to allow accumulation of the tagged protein, and collecting the tagged protein, the peptide tag having the following sequence:
Xm(PYn)qPZr.

The peptide (also referred to as peptide tag) of the present invention has the following sequence. Xm(PYn)qPZr.

Xm means m-consecutive "X"s, wherein the m "X"s may be either the same amino acid residues or different amino acid residues selected from the group consisting of R, G, S, K, T, L, N, Q, and H. m is an integer of <NUM> to <NUM>, preferably an integer of <NUM> to <NUM>, more preferably an integer of <NUM> to <NUM>.

(PYn)q means q-consecutive "PYn"s, that is, since n is <NUM>, <NUM>, or <NUM>, it means q-consecutive "PY"(s), "PYY"(s), and/or "PYYY"(s) (wherein P represents proline). The total number of the consecutive "PY"(s), "PYY"(s), and/or "PYYY"(s) is q.

Here, the "Y"s may be either the same amino acid residues or different amino acid residues selected from the group consisting of R, G, S, K, T, L, N, and Q, with the proviso that at least one of the "Y"s included in the q consecutive "PYn"s is K, L, N, Q, H, or R. More preferably, at least two "Y"s included in the q-consecutive "PYn"s are K, L, N, Q, H, or R. q is an integer of <NUM> to <NUM>, preferably an integer of <NUM> to <NUM>, more preferably an integer of <NUM> to <NUM>, still more preferably an integer of <NUM> to <NUM>.

PZr means r-consecutive "Z"s after "P", wherein the r "Z"s may be either the same amino acid residues or different amino acid residues selected from the group consisting of R, G, S, K, T, L, N, and Q. r is an integer of <NUM> to <NUM>, preferably an integer of <NUM> to <NUM>, more preferably an integer of <NUM> to <NUM>.

The peptide of the present invention has a length of <NUM> to <NUM> amino acids, preferably <NUM> to <NUM> amino acids, more preferably <NUM> to <NUM> amino acids, still more preferably <NUM> to <NUM> amino acids, still more preferably <NUM> to <NUM> amino acids, especially preferably <NUM> to <NUM> amino acids.

In the peptide of the present invention, the total content of glycine and serine with respect to the total amino acids is less than <NUM>%, preferably less than <NUM>%.

In one mode of the peptide of the present invention, the peptide has the same amino acid sequence as PG12 or PG17 except that one or several (for example, <NUM> to <NUM>, preferably <NUM> to <NUM>) amino acids other than P are substituted with K, L, N, and/or Q. In another mode, the peptide has the same amino acid sequence as PG12 or PG17 except that one or several (for example, <NUM> to <NUM>, preferably <NUM> to <NUM>) amino acids other than P or R are substituted with R. Here, the amino acid(s) substituted in PG12 or PG17 is/are more preferably an amino acid(s) between P and P.

The peptide of the present invention is a peptide comprising the amino acid sequence represented by SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

In the tagged protein of the present invention, the peptide tag of the present invention is bound to a protein of interest (the tagged protein is also referred to as a fusion protein of the tag, a useful protein, and the protein of interest). The peptide tag may be bound to the N-terminus of the protein of interest; the peptide tag may be bound to the C-terminus of the protein of interest; or the peptide tag may be bound to each of both the N-terminus and the C-terminus of the protein of interest. Accordingly, the peptide tag may be bound to the N-terminus, the C-terminus or to each of both the N-terminus and the C-terminus of the protein of interest. The peptide tag(s) may be directly bound to the N-terminus and/or the C-terminus of the protein of interest, or may be bound thereto through a sequence(s) of one to several amino acids (for example, <NUM> to <NUM> amino acid(s)). The sequence of the one to several amino acids may be an arbitrary sequence as long as the sequence does not adversely affects the function and the expression level of the tagged protein. In cases where the sequence is a protease recognition sequence, the peptide tag can be cleaved off from the useful protein after the expression and purification. Accordingly, the peptide tag may be linked to the protein of interest through a protease recognition sequence. Examples of the protease recognition sequence include the factor Xa recognition sequence (IEGR; SEQ ID NO:<NUM>). The tagged protein of the present invention may also include another tag sequence necessary for detection or purification, such as a His tag, HN tag (for example, SEQ ID NO:<NUM>), or FLAG tag.

Examples of the useful protein contained in the tagged protein of the present invention include, but are not limited to, growth factors, hormones, cytokines, blood proteins, enzymes, antigens, antibodies, transcription factors, receptors, fluorescent proteins, and partial peptides thereof. Accordingly, the protein of interest may be selected from the group consisting of human growth hormone, interferon β, xylanase, esterase, and green fluorescent protein (GFP).

Examples of the enzymes include lipase, protease, steroid-synthesizing enzyme, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase, and chitinase.

Examples of the growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).

Examples of the hormones include insulin, glucagon, somatostatin, growth hormones (for example, SEQ ID NO: <NUM>), parathyroid hormone, prolactin, leptin, and calcitonin.

Examples of the cytokines include interleukins, interferons (IFNα, IFNβ (for example, SEQ ID NO:<NUM>), IFNγ), and tumor necrosis factor (TNF).

Examples of the blood proteins include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

Examples of the antibodies include complete antibodies, Fab, F(ab'), F(ab')<NUM>, Fc, Fc fusion proteins, heavy chain (H-chain), light chain (L-chain), single-chain Fv (scFv), sc(Fv)<NUM>, disulfide-linked Fv (sdFv), and diabodies.

The antigen proteins to be used as vaccines are not limited as long as the immune response can be induced, and may be appropriately selected depending on the expected target of the immune response. Examples of the antigen proteins include proteins derived from pathogenic bacteria and proteins derived from pathogenic viruses.

To the tagged protein of the present invention, a secretion signal peptide that functions in a host cell may be added for secretory production. Accordingly, the tagged protein may further comprise a secretion signal. Examples of the secretion signal peptide include invertase secretion signal (for example, SEQ ID NO:<NUM>), P3 secretion signal, and α-factor secretion signal (SEQ ID NO: <NUM>) in cases where yeast is used as the host; PelB secretion signal in cases where E. coli is used as the host; and P22 secretion signal in cases where Brevibacillus is used as the host. In cases where a plant is used as the host, examples of the secretion signal peptide include those derived from plants belonging to the families Solanaceae, Rosaceae, Brassicaceae, and Asteraceae, preferably those derived from plants belonging to genera such as Nicotiana, Arabidopsis, Fragaria, and Lactuca, more preferably those derived from Nicotiana tabacum, Arabidopsis thaliana, Fragaria × ananassa, Lactuca sativa, and the like.

For allowing expression in a particular cellular compartment, a transport signal peptide such as an endoplasmic reticulum retention signal peptide or a vacuole transport signal peptide may be added to the tagged protein of the present invention.

The tagged protein of the present invention may be chemically synthesized, or may be produced by genetic engineering. A method for its production by genetic engineering will be described later.

The DNA of the present invention is characterized in that it comprises a DNA encoding the tagged protein of the present invention. That is, the DNA of the present invention comprises a DNA encoding the useful protein and a DNA encoding the peptide tag. The DNA encoding the useful protein and the DNA encoding the peptide tag are linked to each other in the same reading frame.

The DNA encoding the useful protein may be obtained by, for example, a common genetic engineering method based on a known nucleotide sequence.

Preferably, in the DNA encoding the tagged protein of the present invention, a codon(s) corresponding to an amino acid(s) constituting the tagged protein is/are modified as appropriate such that the translation level of the hybrid protein increases depending on the host cell in which the protein is to be produced. For the method of the codon modification, one may refer to, for example, the method of Kang et al. Examples of the method also include methods in which codons frequently used in the host cell are selected, methods in which codons with high GC contents are selected, and methods in which codons frequently used in house-keeping genes of the host cell are selected.

For improving expression in the host cell, the DNA of the present invention may comprise an enhancer sequence or the like that functions in the host cell. Examples of the enhancer include the Kozak sequence, and the <NUM>'-untranslated region of an alcohol dehydrogenase gene derived from a plant.

The DNA of the present invention can be prepared by a common genetic engineering technique. For example, a DNA encoding the peptide tag of the present invention, a DNA encoding the useful protein, and the like may be linked to each other using PCR, DNA ligase, and/or the like, to construct the DNA of the present invention.

The recombinant vector of the present invention may be a vector in which the DNA encoding the tagged protein is inserted such that expression of the protein is possible in the host cell to which the vector is introduced. The vector is not limited as long as it can replicate in the host cell. Examples of the vector include plasmid DNAs and viral DNAs. The vector preferably contains a selection marker such as a drug resistance gene. Specific examples of the plasmid vectors include the pTrcHis2 vector, pUC119, pBR322, pBluescript II KS+, pYES2, pAUR123, pQE-Tri, pET, pGEM-3Z, pGEX, pMAL, pRI909, pRI910, pBI221, pBI121, pBI101, pIG121Hm, pTrc99A, pKK223, pA1-<NUM>, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, p3×FLAG-CMV-<NUM>, pCAT3, pcDNA3. <NUM>, and pCMV.

The promoter used in the vector may be appropriately selected depending on the host cell to which the vector is introduced. In cases of expression in yeast, examples of the promoter include the GAL1 promoter, PGK1 promoter, TEF1 promoter, ADH1 promoter, TPI1 promoter, and PYK1 promoter. In cases of expression in a plant, examples of the promoter include the cauliflower mosaic virus <NUM> promoter, rice actin promoter, maize ubiquitin promoter, and lettuce ubiquitin promoter. In cases of expression in E. coli, examples of the promoter include the T7 promoter. In cases of expression in Brevibacillus, examples of the promoter include the P2 promoter and the P22 promoter. The promoter may be an inducible promoter. Examples of the inducible promoter include lac, tac, and trc, which are inducible with IPTG; trp, which is inducible with IAA; ara, which is inducible with L-arabinose; Pzt-<NUM>, which is inducible with tetracycline; the PL promoter, which is inducible by heat (<NUM>); and the promoter of the cspA gene, which is a cold shock gene.

When necessary, a terminator sequence may also be included depending on the host cell.

The recombinant vector of the present invention may be prepared by, for example, cleaving a DNA construct with an appropriate restriction enzyme, or adding a restriction site by PCR, and then inserting the resulting DNA into a restriction site or a multicloning site of a vector.

The transformant of the present invention is characterized in that it is transformed with the DNA or a recombinant vector comprising the DNA. The host cell used for the transformation may be either a eukaryotic cell or a prokaryotic cell. A eukaryotic cell is preferred.

Preferred examples of the eukaryotic cell include yeast cells, mammalian cells, plant cells, and insect cells. Examples of the yeast include Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe, and Pichia pastoris. Further, a microorganism such as Aspergillus may be used. Examples of the prokaryotic cell include Escherichia coli, Lactobacillus, Bacillus, Brevibacillus, Agrobacterium tumefaciens, and actinomycetes. Examples of the plant cells include cells of plants belonging to Astaraceae such as Lactuca; Solanaceae; Brassicaceae; Rosaceae; or Chenopodiaceae.

The transformant to be used in the present invention can be prepared by introducing the recombinant vector of the present invention into host cells by a common genetic engineering technique. Examples of the method used include the electroporation method (<NPL>), the protoplast method (<NPL>)), the polyethylene glycol method (<NPL>), the introduction method utilizing Agrobacterium (<NPL>, <NPL>), the particle gun method (<NPL>), and the polycation method (<NPL>. The gene expression may be transient expression, or may be stable expression based on incorporation into the chromosome.

After the introduction of the recombinant vector of the present invention into the host cells, a transformant can be selected based on a phenotype of the selection marker. By culturing the selected transformant, the tagged protein can be produced. The medium and the conditions used for the culture may be appropriately selected depending on the species of the transformant.

In cases where the host cell is a plant cell, a plant body can be regenerated by culturing a selected plant cell by a conventional method, and the tagged protein can be accumulated in the plant cell or outside the cell membrane of the plant cell.

The protein comprising the peptide tag of the present invention accumulated in the medium or cells can be separated and purified according to a method well known to those skilled in the art. For example, the separation and purification can be carried out by an appropriate known method such as salting-out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion-exchange column chromatography, affinity chromatography, high/medium-pressure liquid chromatography, reversed-phase chromatography, or hydrophobic chromatography, or by combination of any of these.

Examples of the present invention are described below.

As proteins to which a peptide tag is to be added, <NUM>) human growth hormone (hGH, SEQ ID NO:<NUM>) and <NUM>) human interferon β-1b (IFNβ, SEQ ID NO:<NUM>) were used. An artificial synthetic DNA encoding hGH (SEQ ID NO:<NUM>) was inserted into the EcoRV recognition site of a pUC19-modified plasmid pTRU5 (Fasmac), to obtain plasmid <NUM>. An artificial synthetic DNA encoding IFNβ (SEQ ID NO:<NUM>) was inserted into the EcoRV recognition site of a pUC19-modified plasmid pTRU5, to obtain plasmid <NUM>.

An artificial synthetic DNA (SEQ ID NO:<NUM>) prepared by adding a DNA sequence encoding a yeast invertase SUC2 signal peptide (SUC2SP, SEQ ID NO:<NUM>; <NPL>) and a <NUM> × HN tag for detection and purification (SEQ ID NO:<NUM>) to the <NUM>'-end of a multicloning site composed of the NotI, SalI, SfiI, XhoI, and AscI recognition sequences was inserted into the HindIII-XbaI site of pYES2 (Invitrogen) such that the SUC2SP and the <NUM> × HN tag were added to the N-terminus of the expressed protein, to prepare plasmid <NUM> for expression in yeast.

By the following procedure, plasmids for expression of hGH or IFNβ having various tags (Table <NUM>) at the N- or C-terminus, or at both the N- and C-termini, in yeast were constructed (<FIG>).

First, for the addition of the various tags to the N- or C-terminus of hGH or IFNβ, or to both the N- and C-termini, PCR was carried out using the combinations of a template plasmid, a forward primer, and a reverse primer shown in Table <NUM>. To the <NUM>'-end of each primer, a sequence homologous to plasmid <NUM> was added. For the PCR, KOD-PLUS-Ver. <NUM> (Toyobo Co. ) was used. A reaction liquid in an amount of <NUM>µl was prepared such that it contained <NUM> pg/µl template plasmid, <NUM> forward primer, <NUM> reverse primer, <NUM> dNTPs, <NUM> × Buffer for KOD-Plus-Ver. <NUM>, <NUM> MgSO<NUM>, and <NUM> U/µl KOD-PLUS-Ver. The reaction liquid was heated at <NUM> for <NUM> minutes, and this was followed by <NUM> cycles of treatment each composed of heating at <NUM> for <NUM> seconds, at <NUM> for <NUM> seconds, and then at <NUM> for <NUM> seconds. Finally, the reaction liquid was heated at <NUM> for <NUM> minutes. The resulting amplification fragment was purified with a QIAquick PCR Purification Kit (QIAGEN). Plasmid <NUM> was digested with NotI and AscI, and then separated by electrophoresis using <NUM>% SeaKem GTG Agarose (Lonza), followed by extraction from the gel using a QIAquick Gel Extraction Kit (QIAGEN). With the extracted plasmid <NUM> in an amount corresponding to about <NUM> ng, <NUM>µl of the purified PCR product was mixed, and the liquid volume was adjusted to <NUM>µl. The resulting mixture was mixed with <NUM>µl of <NUM> × Enzyme Mix attached to a Gene Art Seamless PLUS Cloning and Assembly Kit (Applied Biosystem), and then left to stand at room temperature for <NUM> minutes, followed by being left to stand on ice for <NUM> minutes. With the competent cells DH10B T1 SA attached to the kit, <NUM>µl of the reaction liquid was mixed, and the resulting mixture was left to stand on ice for <NUM> minutes. The mixture was then warmed at <NUM> for <NUM> minutes, and left to stand on ice for <NUM> minutes, followed by addition of <NUM>µl of SOC thereto and shaking at <NUM> at <NUM> rpm for <NUM> hour. Subsequently, <NUM>µl of the shaken product was applied to <NUM> × YT agar medium (<NUM>/l Bacto tryptone, <NUM>/l Bacto Yeast Extract, <NUM>/l NaCl, <NUM>/l Bacto Agar) supplemented with <NUM>/l ampicillin, and static culture was carried out at <NUM> overnight, to obtain transformed colonies. A colony was transferred to <NUM> × YT liquid medium (<NUM>/l Bacto tryptone, <NUM>/l Bacto Yeast Extract, and <NUM>/l NaCl) supplemented with <NUM>/l ampicillin, and shake culture was carried out at <NUM> at <NUM> rpm overnight, followed by extraction of plasmid. After confirmation of the nucleotide sequence, the plasmid was used for transformation of yeast.

Yeast (Saccharomyces cerevisiae INVSc1, Invitrogen) was subjected to shake culture in YPD medium (<NUM>% yeast extract, <NUM>% peptone, <NUM>% dextrose (D-glucose)) at <NUM> at <NUM> rpm overnight. The resulting culture was diluted such that the turbidity at a wavelength of <NUM> (OD<NUM>) in <NUM> of YPD became <NUM> to <NUM>. Shake culture was then carried out at <NUM> at <NUM> rpm until OD<NUM> reached <NUM> to <NUM>. After performing centrifugation at <NUM> × g at room temperature for <NUM> minutes, the cells were pelletized, and the supernatant was discarded. The resulting pellet was suspended in <NUM> of Solution I (S. EasyComp Transformation Kit, Invitrogen). Further, after performing centrifugation at <NUM> × g at room temperature for <NUM> minutes, the cells were pelletized, and the supernatant was discarded. The resulting pellet was suspended in <NUM> of Solution II (S. EasyComp Transformation Kit, Invitrogen), and aliquoted in <NUM>-µl volumes to provide competent cells. The competent cells were stored in a deep freezer at -<NUM> until use.

The competent cells obtained were thawed and allowed to warm to room temperature. After adding <NUM>µg of the plasmid for expression of the peptide-tagged protein prepared as described above to the competent cells, <NUM>µl of Solution III (room temperature) was added to the resulting mixture, followed by vortexing the mixture and then leaving the mixture to stand at <NUM> for <NUM> hour (while vortexing the mixture at <NUM>-minute intervals). Thereafter, <NUM>µl of the mixture after being left was applied to SC-Ura medium (<NUM>/L yeast nitrogen base, <NUM>/L adenine, <NUM>/L arginine, <NUM>/L cysteine, <NUM>/L leucine, <NUM>/L lysine, <NUM>/L threonine, <NUM>/L tryptophan, <NUM>/L aspartic acid, <NUM>/L histidine, <NUM>/L isoleucine, <NUM>/L methionine, <NUM>/L phenylalanine, <NUM>/L proline, <NUM>/L serine, <NUM>/L tyrosine, <NUM>/L valine) supplemented with <NUM>% glucose and <NUM>% Bacto Agar, and static culture was carried out at <NUM> for <NUM> to <NUM> days to obtain transformed colonies.

A single colony after the transformation was smeared on a plate medium (SC-Ura, <NUM>% dextrose), and then left to stand in an incubator at <NUM> for <NUM> hours to perform culture. Subsequently, cells were scraped with a <NUM>-µl sterile disposable loop from the plate medium after the culture, and then inoculated into <NUM> of a preculture medium (SC-Ura, <NUM>% galactose) placed in a sterile <NUM>-ml polystyrene tube. Shake culture was carried out at <NUM> at <NUM> rpm for <NUM> hours. After completion of the culture, the turbidity was measured at <NUM> using a spectrophotometer. The culture in the amount required for the turbidity to become <NUM> by resuspension in <NUM> of a medium was taken into a sterile <NUM>-ml Eppendorf tube, and then centrifugation was carried out at <NUM> × g at <NUM> for <NUM> minutes. After removing the supernatant, the precipitate was suspended in <NUM> of an induction medium (SC-Ura, <NUM>% galactose, <NUM>% raffinose), and the resulting suspension was combined with <NUM> of the induction medium preliminarily placed in a sterile <NUM>-ml polystyrene tube, followed by performing shake culture at <NUM> at <NUM> rpm for <NUM> hours. After completion of the culture, <NUM>µl of the culture liquid was taken into a <NUM>-ml Eppendorf tube, and centrifugation was carried out at <NUM> × g at <NUM> for <NUM> minutes. After removing the supernatant, the cells were frozen in liquid nitrogen, and then stored in a deep freezer at -<NUM>.

Using the transgenic yeast cells stored at -<NUM> after the freezing in liquid nitrogen, protein extraction was carried out according to the method by Akira Hosomi et al. To the stored sample, <NUM>µl of distilled water was added, and the resulting mixture was stirred using a vortex mixer. Thereafter, <NUM>µl of <NUM> N NaOH was added to the mixture, and the mixture was stirred again using a vortex mixer, followed by being left to stand on ice for <NUM> minutes. Subsequently, centrifugation was carried out at <NUM> at <NUM>,<NUM> for <NUM> minutes, and the supernatant was discarded, followed by collecting the precipitate. To the precipitate, <NUM>µl of a sample buffer (EZ Apply, manufactured by ATTO) was added, and the resulting mixture was stirred using a vortex mixer, followed by heating in boiling water for <NUM> minutes to perform SDS treatment of the sample.

An artificial synthetic DNA (SEQ ID NO:<NUM>) was prepared and inserted into the XbaI-BlpI site of pET-15b (Novagen) to prepare plasmid <NUM> for expression in E. The artificial synthetic DNA (SEQ ID NO:<NUM>) is a DNA prepared from the gene expression cassette between XbaI-BlpI in pET-22b(+) (Novagen) by replacing the region from immediately after the E. coli PelB signal peptide (PelBSP) to the stop codon with a <NUM> × HN tag for detection/purification (SEQ ID NO:<NUM>) followed by a multicloning site composed of the NotI, SalI, SfiI, XhoI, and AscI recognition sequences.

By the following procedure, plasmids for expression of hGH or IFNβ in E. coli, having various tags at the N- or C-terminus, or at both the N- and C-termini, were constructed (<FIG>).

First, for the addition of the various tags to the N- or C-terminus of hGH or IFNβ, or to both the N- and C-termini, PCR was carried out using the combinations of a template plasmid, a forward primer, and a reverse primer shown in Table <NUM>. To the <NUM>'-end of each primer, a sequence homologous to plasmid <NUM> was added. For the PCR, KOD-PLUS-Ver. <NUM> (Toyobo Co. ) was used. A reaction liquid in an amount of <NUM>µl was prepared such that it contained <NUM> pg/µl template plasmid, <NUM> forward primer, <NUM> reverse primer, <NUM> dNTPs, <NUM> × Buffer for KOD-Plus-Ver. <NUM>, <NUM> MgSO<NUM>, and <NUM> U/µl KOD-PLUS-Ver. The reaction liquid was heated at <NUM> for <NUM> minutes, and this was followed by <NUM> cycles of treatment each composed of heating at <NUM> for <NUM> seconds, at <NUM> for <NUM> seconds, and then at <NUM> for <NUM> seconds. Finally, the reaction liquid was heated at <NUM> for <NUM> minutes. The resulting amplification fragment was purified with a QIAquick PCR Purification Kit (QIAGEN). Plasmid <NUM> was digested with NotI and AscI, and then separated by electrophoresis using <NUM>% SeaKem GTG Agarose (Lonza), followed by extraction from the gel using a QIAquick Gel Extraction Kit (QIAGEN). With the extracted plasmid <NUM> in an amount corresponding to about <NUM> ng, <NUM>µl of the purified PCR product was mixed, and the liquid volume was adjusted to <NUM>µl. The resulting mixture was mixed with <NUM>µl of <NUM> × Enzyme Mix attached to a Gene Art Seamless PLUS Cloning and Assembly Kit (Applied Biosystem), and then left to stand at room temperature for <NUM> minutes, followed by being left to stand on ice for <NUM> minutes. With the E. coli competent cells DH10B T1 SA attached to the kit, <NUM>µl of the reaction liquid was mixed, and the resulting mixture was left to stand on ice for <NUM> minutes. The mixture was then warmed at <NUM> for <NUM> minutes, and left to stand on ice for <NUM> minutes, followed by addition of <NUM>µl of SOC thereto and shaking at <NUM> at <NUM> rpm for <NUM> hour. Subsequently, <NUM>µl of the shaken product was applied to <NUM> × YT agar medium (<NUM>/l Bacto tryptone, <NUM>/l Bacto Yeast Extract, <NUM>/l NaCl, and <NUM>/l Bacto Agar) supplemented with <NUM>/l ampicillin, and static culture was carried out at <NUM> overnight to obtain transformed colonies. A colony was transferred to <NUM> × YT liquid medium (<NUM>/l Bacto tryptone, <NUM>/l Bacto Yeast Extract, <NUM>/l NaCl) supplemented with <NUM>/l ampicillin, and shake culture was carried out at <NUM> at <NUM> rpm overnight, followed by extraction of plasmid. After confirmation of the nucleotide sequence, the plasmid was used for transformation of E. coli for expression of protein.

A glycerol stock of E. coli BL21 (DE3) (Novagen) was inoculated into <NUM> of SOB medium (<NUM>/l Bacto tryptone, <NUM>/l Bacto Yeast Extract, <NUM> NaCl, <NUM> KCl, <NUM> MgSO<NUM>, <NUM> MgClz) placed in a sterile <NUM>-ml polystyrene tube, and shake culture was carried out at <NUM> at <NUM> rpm overnight. To <NUM> of SOB medium placed in a sterile Erlenmeyer flask, <NUM> of the resulting culture liquid was inoculated, and shake culture was carried out at <NUM> at <NUM> rpm. When the turbidity at a wavelength of <NUM> (OD600) reached <NUM> to <NUM>, the culture liquid was cooled with ice for <NUM> to <NUM> minutes to stop the culture. The culture liquid was transferred to <NUM>-ml conical tubes, and centrifugation was carried out at <NUM> × g at <NUM> for <NUM> minutes (× <NUM> tubes). After discarding the supernatant, ice-cold <NUM> TB (<NUM> PIPES-KOH, pH <NUM>, <NUM> CaCl<NUM>, <NUM> KCl, <NUM> MnCh) was added to the pellets, and the pellets were gently suspended (× <NUM> tubes). The suspension contained in the two tubes was combined into one tube, and centrifugation was carried out at <NUM> × g at <NUM> for <NUM> minutes. After discarding the supernatant, <NUM> of ice-cold TB was added to the pellet, and the pellet was gently suspended. After addition of <NUM>µl of DMSO thereto, the pellet was gently suspended under ice-cooling. The resulting suspension was aliquoted in <NUM>-µl volumes into <NUM>-ml microtubes to provide competent cells. After freezing the competent cells with liquid nitrogen, the cells were stored at -<NUM> until use.

The obtained competent cells were thawed on ice, and <NUM> ng of the plasmid for expression of the peptide-tagged protein for E. coli prepared as described above was added to the cells, followed by gently stirring the resulting mixture and leaving the mixture to stand on ice for <NUM> minutes. The cells were then treated (heat-shocked) at <NUM> for <NUM> to <NUM> seconds, and left to stand on ice for <NUM> minutes. After adding <NUM>µl of SOC, the tube was kept in a horizontal position, and shaken at <NUM> at <NUM> rpm for <NUM> hour. Subsequently, <NUM>µl of the shaken product was applied to <NUM> × YT agar medium supplemented with <NUM>/l ampicillin, and static culture was carried out at <NUM> overnight to obtain transformed colonies.

A single colony after the transformation was smeared on a plate medium (<NUM> × YT, <NUM> ppm Ampicillin), and then left to stand in an incubator at <NUM> overnight to perform culture. Subsequently, bacterial cells were scraped with a sterile disposable loop from the plate medium after the culture, and then inoculated into <NUM> of a preculture medium (LB, <NUM> ppm Ampicillin) placed in a sterile <NUM>-ml polystyrene tube. Shake culture was carried out at <NUM> at <NUM> rpm until the OD<NUM> value reached <NUM> to <NUM>. The culture in the amount required for the OD<NUM> value to become <NUM> by addition of <NUM> of LB medium (<NUM> ppm Ampicillin) to the precipitate obtained after removal of the centrifuge supernatant from the culture was taken into a <NUM>-ml Eppendorf tube, and left to stand at <NUM> (in a refrigerator) overnight. On the next day, the sample was centrifuged at <NUM> rpm at <NUM> for <NUM> minutes, and then the supernatant was removed, followed by adding <NUM> of fresh LB medium (<NUM> ppm Ampicillin) to the sample and suspending the precipitate. Further, <NUM>µl out of <NUM> of the sample was inoculated into <NUM> of LB medium (<NUM> ppm Ampicillin) such that the OD<NUM> value became <NUM>, and then shake culture was carried out at <NUM> at <NUM> rpm until the OD<NUM> value reached <NUM> to <NUM>. Subsequently, <NUM>µl (final concentration, <NUM>) of <NUM> IPTG (inducer) was added to the culture, and shake culture was carried out at <NUM> at <NUM> rpm for <NUM> hours. After completion of the culture, the test tube containing the sample was cooled on ice for <NUM> minutes to stop the growth of E. coli, and <NUM>µl of the culture liquid was taken into another <NUM>-ml Eppendorf tube, followed by performing centrifugation at <NUM> rpm at <NUM> for <NUM> minutes. Subsequently, the supernatant was removed, and then the bacterial cells were frozen with liquid nitrogen, followed by cryopreservation at - <NUM>.

To the cryopreserved sample, <NUM>µl of a sample buffer (EZ Apply, manufactured by ATTO) was added, and the resulting mixture was stirred using a vortex mixer, followed by heating the mixture in boiling water for <NUM> minutes to perform SDS treatment of the sample.

Plasmid construction and transformation of Brevibacillus were carried out using a Brevibacillus Expression System -BIC System- (Takara Bio Inc.

First, for addition of various tags to the N- or C-terminus of hGH, or to both the N- and C-termini, PCR was carried out using the combinations of a template plasmid, a forward primer, and a reverse primer shown in Table <NUM>. To the <NUM>'-end of each primer, a sequence homologous to the insertion site in pBIC3 was added. For the PCR, KOD-PLUS-Ver. <NUM> (Toyobo Co. ) was used. A reaction liquid in an amount of <NUM>µl was prepared such that it contained <NUM> pg/µl template plasmid, <NUM> forward primer, <NUM> reverse primer, <NUM> dNTPs, <NUM> × Buffer for KOD-Plus-Ver. <NUM>, <NUM> MgSO<NUM>, and <NUM> U/µl KOD-PLUS-Ver. The reaction liquid was heated at <NUM> for <NUM> minutes, and this was followed by <NUM> cycles of treatment each composed of heating at <NUM> for <NUM> seconds, at <NUM> for <NUM> seconds, and then at <NUM> for <NUM> seconds. Finally, the reaction liquid was heated at <NUM> for <NUM> minutes. The resulting amplification fragment was purified with a QIAquick PCR Purification Kit (QIAGEN).

Plasmid construction by homologous recombination, and transformation therewith were carried out as follows. After mixing <NUM> ng of pBIC3, which is a plasmid for expression in Brevibacillus (attached to the kit), with the purified PCR product at a molar ratio of about <NUM>:<NUM>, the volume of the resulting mixture was adjusted to <NUM>µl with sterile water. Brevibacillus choshinensis SP3 competent cells (Takara Bio Inc. ) were left to stand on a heat block at <NUM> for <NUM> seconds to allow rapid thawing, and then centrifuged (<NUM>,<NUM> rpm, room temperature, <NUM> minute). After removing the supernatant, the whole amount of a mixture of <NUM>µl of the above DNA solution and <NUM>µl of Solution A (attached to the kit) was added, and the pellet of the competent cells was completely suspended by vortexing, followed by leaving the resulting suspension to stand for <NUM> minutes. After addition of <NUM>µl of Solution B (PEG solution), the suspension was mixed by vortexing for <NUM> seconds, and then centrifugation was carried out (<NUM> rpm, room temperature, <NUM> minutes), followed by removing the supernatant. After carrying out centrifugation (<NUM> rpm, room temperature, <NUM> seconds) again, the supernatant was completely removed. To the resulting pellet, <NUM> of MT medium as added, and the pellet was completely suspended using a micropipette, followed by shake culture at <NUM> at <NUM> rpm for <NUM> hour. The culture liquid was plated on an MTNm plate (<NUM>/L glucose, <NUM>/L Phytone peptone, <NUM>/L Ehrlich bonito extract, <NUM>/L powdered yeast extract S, <NUM>/L FeSO<NUM>·<NUM><NUM>O, <NUM>/L MnSO<NUM>·<NUM><NUM>O, <NUM>/L ZnSO<NUM>·<NUM><NUM>O, <NUM> MgCl<NUM>, <NUM>% Bacto Agar, <NUM>µg/mL neomycin, pH <NUM>), and static culture was carried out at <NUM> overnight. For the resulting clones, expression of the protein of interest was confirmed by Western analysis of their colonies. For each of the clones for which the expression could be confirmed, its colony was inoculated into TMNm medium (<NUM>/L glucose, <NUM>/L Phytone peptone, <NUM>/L Ehrlich bonito extract, <NUM>/L powdered yeast extract S, <NUM>/L FeSO<NUM>·<NUM><NUM>O, <NUM>/L MnSO<NUM>·<NUM><NUM>O, <NUM>/L ZnSO<NUM>·<NUM><NUM>O, and <NUM>µg/mL neomycin, pH <NUM>), and culture was carried out at <NUM> at <NUM> rpm overnight, followed by extraction of plasmid and confirmation of the nucleotide sequence.

An artificial synthetic DNA (SEQ ID NO: <NUM>) encoding xylanase derived from Bacillus subtilis (XynA, SEQ ID NO: <NUM>) was inserted into the EcoRV recognition site of a pUC19-modified plasmid pUCFa (Fasmac), to obtain plasmid <NUM>. An artificial synthetic DNA (SEQ ID NO: <NUM>) encoding esterase derived from Bacillus subtilis (EstA, SEQ ID NO: <NUM>) was inserted into the EcoRV recognition site of the pUC19-modified plasmid pUCFa (Fasmac), to obtain plasmid <NUM>.

An artificial synthetic DNA (SEQ ID NO: <NUM>) encoding an HA tag (SEQ ID NO:<NUM>) and a <NUM> × His tag (SEQ ID NO: <NUM>) for detection and purification, and a stop codon, was inserted to the NcoI-HindIII site of pNCMO2 (Takara Bio Inc. ) such that the HA tag and the <NUM> × His tag were added to the C-terminus of the expressed protein, to prepare plasmid <NUM> for expression in Brevibacillus.

By the following procedure, plasmids for expression of xylanase or esterase in Brevibacillus, having a PX12-<NUM> tag(s) at the N- or C-terminus, or at both the N- and C-termini, were constructed (<FIG> and <FIG>).

First, for addition of various tags to the N- or C-terminus of xylanase or esterase, or to both the N- and C-termini, PCR was carried out using the combinations of a template plasmid, a forward primer, and a reverse primer shown in Table <NUM>. To the <NUM>'-end of each primer, a sequence homologous to plasmid <NUM> was added. In designing of the forward primer, the two amino acid residues AD were added such that they follow a signal peptide. For the PCR, KOD-PLUS-Ver. <NUM> (Toyobo Co. ) was used. A reaction liquid in an amount of <NUM>µl was prepared such that it contained <NUM> pg/µl template plasmid, <NUM> forward primer, <NUM> reverse primer, <NUM> dNTPs, <NUM> × Buffer for KOD-Plus-Ver. <NUM>, <NUM> MgSO<NUM>, and <NUM> U/µl KOD-PLUS-Ver. The reaction liquid was heated at <NUM> for <NUM> minutes, and this was followed by <NUM> cycles of treatment each composed of heating at <NUM> for <NUM> seconds, at <NUM> for <NUM> seconds, and then at <NUM> for <NUM> seconds. Finally, the reaction liquid was heated at <NUM> for <NUM> minutes. The resulting amplification fragment was purified with a QIAquick PCR Purification Kit. Plasmid <NUM> was digested with NcoI and NdeI, and then separated by electrophoresis using <NUM>% SeaKem GTG Agarose, followed by extraction from the gel using a QIAquick Gel Extraction Kit. With the extracted plasmid <NUM> in an amount corresponding to about <NUM> ng, <NUM>µl of the purified PCR product was mixed, and the liquid volume was adjusted to <NUM>µl. The resulting mixture was mixed with <NUM>µl of <NUM> × Enzyme Mix attached to a Gene Art Seamless PLUS Cloning and Assembly Kit, and then left to stand at room temperature for <NUM> minutes, followed by being left to stand on ice for <NUM> minutes. With competent cells DH10B T1 SA, <NUM>µl of the reaction liquid was mixed, and the resulting mixture was left to stand on ice for <NUM> minutes. The mixture was then warmed at <NUM> for <NUM> minutes, and left to stand on ice for <NUM> minutes, followed by addition of <NUM>µl of SOC thereto and shaking at <NUM> at <NUM> rpm for <NUM> hour. Subsequently, <NUM>µl of the shaken product was applied to <NUM> × YT agar medium supplemented with <NUM>/l ampicillin, and static culture was carried out at <NUM> overnight, to obtain transformed colonies. A colony was transferred to <NUM> × YT liquid medium supplemented with <NUM>/l ampicillin, and shake culture was carried out at <NUM> at <NUM> rpm overnight, followed by extraction of plasmid. After confirmation of the nucleotide sequence, the plasmid was used for transformation of Brevibacillus.

Brevibacillus choshinensis SP3 competent cells were left to stand on a heat block at <NUM> for <NUM> seconds to allow rapid thawing, and then centrifuged (<NUM>,<NUM> rpm, room temperature, <NUM> minute). After removing the supernatant, the whole amount of a mixture of <NUM>µl of the above plasmid solution and <NUM>µl of Solution A was added, and the pellet of the competent cells was completely suspended by vortexing, followed by leaving the resulting suspension to stand for <NUM> minutes. After addition of <NUM>µl of Solution B, the suspension was mixed by vortexing for <NUM> seconds, and then centrifugation was carried out (<NUM> rpm, room temperature, <NUM> minutes), followed by removing the supernatant. After carrying out centrifugation (<NUM> rpm, room temperature, <NUM> seconds) again, the supernatant was completely removed. To the resulting pellet, <NUM> of MT medium as added, and the pellet was completely suspended using a micropipette, followed by shake culture at <NUM> at <NUM> rpm for <NUM> hour. The culture liquid was plated on an MTNm plate, and static culture was carried out at <NUM> overnight, to obtain transformed Brevibacillus.

A single colony of the transformed Brevibacillus was smeared on an MTNm plate, and left to stand at <NUM> overnight to perform culture. Subsequently, bacterial cells were scraped with a <NUM>-µl sterile disposable loop from the plate medium after the culture, and then inoculated into <NUM> of TMNm medium placed in a sterile <NUM>-ml polystyrene tube. Preculture was carried out at <NUM> at <NUM> rpm overnight. In cases of hGH expression, <NUM>µl of the preculture liquid was inoculated into <NUM> of TMNm medium, and shake culture was carried out at <NUM> at <NUM> rpm, followed by sampling of the culture liquid containing bacterial cells <NUM> hours later. In cases of xylanase or esterase expression, <NUM>µl of the preculture liquid was inoculated into <NUM> of TMNm medium, and shake culture was carried out at <NUM> at <NUM> rpm, followed by sampling of the culture liquid containing bacterial cells <NUM> hours later. The culture liquid was aliquoted in <NUM>-µl volumes, and centrifugation (<NUM>,<NUM> × g, <NUM>, <NUM> minutes) was carried out to separate the bacterial cells from the culture supernatant, followed by storing <NUM>µl of the culture supernatant and the whole amount of the bacterial cells at -<NUM>.

To <NUM>µl of the cryopreserved culture supernatant, <NUM>µl of <NUM> × sample buffer (EZ Apply, manufactured by ATTO) was added, and the resulting mixture was stirred using a vortex mixer, followed by heating in boiling water for <NUM> minutes to perform SDS treatment. To the bacterial cells, <NUM>µl of <NUM> × sample buffer (<NUM>-fold dilution of EZ Apply) was added, and SDS treatment was carried out by the same process.

An artificial synthetic DNA (SEQ ID NO: <NUM>) in which a sequence encoding the extracellular secretion signal peptide for factor α derived from Saccharomyces cerevisiae (AFSP, SEQ ID NO:<NUM>) and a stop codon sequence are placed downstream of a Kozak sequence was inserted into the BsaI-BsaI site of pJ902-<NUM> (Invivogen), to prepare plasmid <NUM> for expression in Pichia yeast.

By the following procedure, plasmids for expression of hGH in Pichia yeast, having various tags at the N- or C-terminus, or at both the N- and C-termini, were constructed (<FIG>).

First, for the addition of a tag(s) to the N- or C-terminus of hGH, or to both the N- and C-termini, PCR was carried out using the combinations of a template plasmid, a forward primer, and a reverse primer shown in Table <NUM>. To the <NUM>'-end of each primer, a sequence homologous to plasmid <NUM> was added. For the PCR, KOD-PLUS-Ver. <NUM> (Toyobo Co. ) was used. A reaction liquid in an amount of <NUM>µl was prepared such that it contained <NUM> pg/µl template plasmid, <NUM> forward primer, <NUM> reverse primer, <NUM> dNTPs, <NUM> × Buffer for KOD-Plus-Ver. <NUM>, <NUM> MgSO<NUM>, and <NUM> U/µl KOD-PLUS-Ver. The reaction liquid was heated at <NUM> for <NUM> minutes, and this was followed by <NUM> cycles of treatment each composed of heating at <NUM> for <NUM> seconds, at <NUM> for <NUM> seconds, and then at <NUM> for <NUM> seconds. Finally, the reaction liquid was heated at <NUM> for <NUM> minutes. The resulting amplification fragment was purified with a QIAquick PCR Purification Kit. Plasmid <NUM> was digested with XhoI and NotI, and then separated by electrophoresis using <NUM>% SeaKem GTG Agarose, followed by extraction from the gel using a QIAquick Gel Extraction Kit. With the extracted plasmid <NUM> in an amount corresponding to about <NUM> ng, <NUM>µl of the purified PCR product was mixed, and the liquid volume was adjusted to <NUM>µl. The resulting mixture was mixed with <NUM>µl of <NUM> × Enzyme Mix attached to a Gene Art Seamless PLUS Cloning and Assembly Kit, and then left to stand at room temperature for <NUM> minutes, followed being left to stand on ice for <NUM> minutes. With competent cells DH10B T1 SA, <NUM>µl of the reaction liquid was mixed, and the resulting mixture was left to stand on ice for <NUM> minutes. The mixture was then warmed at <NUM> for <NUM> minutes, and left to stand on ice for <NUM> minutes, followed by addition of <NUM>µl of SOC thereto and shaking at <NUM> at <NUM> rpm for <NUM> hour. Subsequently, <NUM>µl of the shaken product was applied to <NUM> × YT agar medium supplemented with <NUM>/l ampicillin, and static culture was carried out at <NUM> overnight, to obtain transformed colonies. A colony was transferred to <NUM> × YT liquid medium supplemented with <NUM>/l ampicillin, and shake culture was carried out at <NUM> at <NUM> rpm overnight, followed by extraction of plasmid and confirmation of the nucleotide sequence. The plasmid was linearized by digestion with SacI, and protein was removed by phenol-chloroform extraction. After ethanol precipitation and drying, the plasmid was dissolved in TE buffer for use in transformation of Pichia yeast.

Pichia yeast (Pichia pastoris PPS-<NUM>) was subjected to shake culture in YPD medium at <NUM> at <NUM> rpm overnight. To <NUM> of YPD medium placed in an Erlenmeyer flask, the culture liquid was added such that the OD<NUM> became <NUM> to <NUM>. Shake culture was carried out at <NUM> at <NUM> rpm until OD<NUM> reached <NUM> to <NUM>. Centrifugation was carried out at <NUM> × g at room temperature for <NUM> minutes, and the supernatant was discarded to obtain a cell pellet. To the pellet, <NUM> of ice-cold BEDS solution (<NUM> bicine, <NUM>% (v/v) Ethylene glycol, <NUM>% (v/v) Dimethyl sulfoxide, <NUM> Sorbitol) and <NUM> of ice-cold <NUM> Dithiothreitol were added, and the resulting mixture was suspended, followed by incubation at <NUM> at <NUM> rpm for <NUM> minutes. Centrifugation was carried out at <NUM> × g at room temperature for <NUM> minutes, and the supernatant was discarded to obtain a cell pellet. To the pellet, <NUM> of BEDS solution was added, and the resulting mixture was suspended, followed by aliquoting the suspension in <NUM>-µl volumes to provide competent cells. The competent cells were stored in a deep freezer at -<NUM> until use.

The plasmid solution after the linearization in an amount corresponding to about <NUM> ng of the plasmid was mixed with competent cells thawed on ice, and the resulting mixture was placed in an electroporation cuvette (interelectrode distance, <NUM>; BIO-RAD). The cuvette was then left to stand on ice for <NUM> minutes. The cuvette was set in an electroporation device (MicroPulser, BIO-RAD), and electroporation was carried out under programmed conditions (Pic, <NUM>µF, <NUM> Q, <NUM> kV, <NUM> pulse). Immediately thereafter, <NUM> of YPD medium supplemented with <NUM> sorbitol was added to the mixture, and the whole mixture was transferred to a microtube, followed by shaking at <NUM> at <NUM> rpm for <NUM> hour. Thereafter, <NUM>µl of the mixture was applied to YPD plate medium supplemented with <NUM> sorbitol, <NUM>% Bacto Agar, and <NUM>/L Zeocine (Invivogen), and static culture was carried out at <NUM> for <NUM> to <NUM> days, to obtain transformed colonies.

A single colony after the transformation was applied to YPD plate medium supplemented with <NUM> Sorbitol, <NUM>% Bacto Agar, and <NUM>/L Zeocine, and static culture was carried out at <NUM> for <NUM> hours. Subsequently, cells were scraped with a <NUM>-µl sterile disposable loop from the plate medium after the culture, and then inoculated into <NUM> of BMGY medium (<NUM>% Bacto Yeast Extract, <NUM>% Bacto peptone, <NUM> potassium phosphate buffer (pH <NUM>), <NUM>% Yeast nitrogen base with ammonium sulfate without amino acids, <NUM>/L Biotin, <NUM>% Glycerol) placed in a sterile <NUM>-ml tube, followed by performing shake culture at <NUM> at <NUM> rpm until OD<NUM> reached <NUM> to <NUM>. The culture in the amount required for OD<NUM> to become <NUM> by resuspension in <NUM> of a medium was taken into a sterile <NUM>-ml Eppendorf tube, and then centrifugation was carried out at <NUM> × g at <NUM> for <NUM> minutes. After removing the supernatant, the precipitate was suspended in <NUM> of BMMY medium (<NUM>% Bacto Yeast Extract, <NUM>% Bacto peptone, <NUM> potassium phosphate buffer (pH <NUM>), <NUM>% Yeast nitrogen base with ammonium sulfate without amino acids, <NUM>/L Biotin, <NUM>% Methanol), and the whole amount of the resulting suspension was mixed with <NUM> of BMMY medium preliminarily provided in a sterile <NUM>-ml tube, followed by performing shake culture at <NUM> at <NUM> rpm for <NUM> hours. After completion of the culture, <NUM>µl of the culture liquid was taken into a microtube, and centrifugation was carried out at <NUM>,<NUM> × g at <NUM> for <NUM> minutes, followed by taking <NUM>µl of the supernatant into another tube to obtain a culture supernatant. The remaining supernatant was removed to obtain a yeast cell pellet. The culture supernatant and the yeast cell pellet were frozen in liquid nitrogen, and then stored in a deep freezer at -<NUM>.

To <NUM>µl of the cryopreserved culture supernatant, <NUM>µl of <NUM> × sample buffer (EZ Apply, manufactured by ATTO) was added, and the resulting mixture was stirred using a vortex mixer, followed by heating in boiling water for <NUM> minutes to perform SDS treatment.

The yeast cell pellet was suspended in <NUM>µl of an ice-cold suspension buffer (<NUM> × PBS (BIO-RAD), <NUM>× cOmplete-EDTA free (Roche)), and the whole amount of the resulting suspension was added to <NUM>µl of glass beads (diameter, <NUM>; acid-treated; Sigma) placed in a microtube, followed by homogenization by shaking using TissueLyzer II (QIAGEN) at <NUM> for <NUM> minutes. After taking <NUM>µl of the homogenate, <NUM>µl of <NUM> × sample buffer (EZ Apply, manufactured by ATTO) was added thereto. The resulting mixture was stirred using a vortex mixer, and then heated in boiling water for <NUM> minutes to perform SDS treatment.

As reference materials for protein quantification, standard samples of hGH and IFNβ were used. For quantification of xylanase, an HA sequence was added to Stx2eB to provide a standard sample. Each standard sample was serially <NUM>-fold diluted with a sample buffer (EZ Apply, manufactured by ATTO) to prepare a dilution series to be used as standards.

For electrophoresis (SDS-PAGE) of protein, an electrophoresis tank (Criterion cell, BIO RAD) and Criterion TGX-gel (BIO RAD) were used. In the electrophoresis tank, an electrophoresis buffer (Tris/Glycine/SDS Buffer, BIO RAD) was placed, and <NUM>µl of each SDS-treated sample was applied to each well, followed by performing electrophoresis at a constant voltage of <NUM> V for <NUM> minutes.

The gel after the electrophoresis was subjected to blotting by Trans-Blot Turbo (BIO RAD) using a Trans-Blot Transfer Pack (BIO RAD).

The membrane after the blotting was immersed in a blocking solution (TBS system, pH <NUM>; Nacalai Tesque, Inc. ), and shaken at room temperature for <NUM> hour or left to stand at <NUM> for <NUM> hours. Thereafter, the membrane was subjected to three times of washing by shaking in TBS-T (<NUM> sodium chloride, <NUM> potassium chloride, <NUM>% polyoxyethylene sorbitan monolaurate, <NUM> Tris-HCl, pH <NUM>) at room temperature for <NUM> minutes. For detection of hGH, an antiserum Rabbit-monoclonal Anti-Growth Hormone antibody [EPR11047(B)] (abcam) was used after <NUM>-fold dilution with TBS-T. For detection of IFNP, an antiserum Mouse-monoclonal Anti-Human IFNβ antibody (R&D Systems) was used after <NUM>,<NUM>-fold dilution with TBS-T. For detection of xylanase and esterase, an antiserum Rat-monoclonal Anti-HA antibody (Roche) was used after <NUM>,<NUM>-fold dilution with TBS-T. The membrane was immersed in the antibody dilution, and shaken at room temperature for <NUM> hour to allow antigen-antibody reaction, followed by three times of washing by shaking in TBS-T at room temperature for <NUM> minutes. As a secondary antibody, an Anti-Rabbit IgG, AP-linked Antibody (Cell Signaling TECHNOLOGY) diluted <NUM>-fold with TBS-T was used for detection of hGH, or an Anti-Mouse IgG, AP-linked Antibody (Cell Signaling TECHNOLOGY) was used for detection of IFNβ. For detection of xylanase and esterase, an Anti-Rat IgG, AP-linked Antibody (EDM Millipore Corp. ) diluted <NUM>-fold with TBS-T was used.

The membrane was immersed in the secondary antibody dilution, and shaken at room temperature for <NUM> hour to allow antigen-antibody reaction, followed by three times of washing by shaking in TBS-T at room temperature for <NUM> minutes. Chromogenic reaction with alkaline phosphatase was carried out by immersing the membrane in a coloring solution (<NUM> sodium chloride, <NUM> magnesium chloride, <NUM>/ml nitroblue tetrazolium, <NUM>/ml <NUM>-bromo-<NUM>-chloro-<NUM>-indolyl-phosphate, <NUM> Tris-HCl, pH <NUM>), and shaking the membrane at room temperature for <NUM> minutes. The membrane was washed with distilled water, and then dried at normal temperature.

From the membrane after the coloring, an image was obtained using a scanner (PM-A900, Epson) at a resolution of <NUM> dpi, and quantification of hGH or IFNβ protein was carried out using image analysis software (CS Analyzer ver. <NUM>, Atto Corporation).

As the protein to which the peptide tag was added, green fluorescent protein derived from Aequorea victoria (GFP; amino acid sequence, SEQ ID NO: <NUM>; DNA nucleotide sequence, SEQ ID NO: <NUM>) was used. PCR reaction was carried out using combinations of a forward primer (pENTR1A-<NUM> (SEQ ID NO: <NUM>), for addition of no tag; pENTR1A-<NUM> (SEQ ID NO: <NUM>), for PG12 tag; pENTR1A-<NUM> (SEQ ID NO: <NUM>), for PX12-<NUM> tag; pENTR1A-<NUM> (SEQ ID NO: <NUM>), for PX12-20v7 tag) and a reverse primer (pENTR1A-Flag-GFP (SEQ ID NO:<NUM>)) such that various peptide tags were added to the N-terminal side of the GFP protein and a Flag tag was added to the C-terminal side, to prepare DNA fragments for cloning. Each prepared DNA fragment was cloned into pENTR 1A (ThermoFisher Scientific) to construct a plasmid having the DNA fragment encoding each peptide tag-GFP-Flag tag. Based on this plasmid, LR reaction was used to insert the DNA encoding the peptide tag-GFP-Flag tag into pFastbac (ThermoFisher Scientific), which is a donor vector. The donor vector was introduced into E. coli DH10bac (ThermoFisher Scientific) to allow its transposition into the lacZ region of a bacmid vector, to prepare a recombinant bacmid. The recombinant bacmid DNA containing the DNA encoding the peptide tag-GFP-Flag tag was introduced into BmN cells derived from silkworm, to prepare a baculovirus. The operation of adding a solution of the obtained baculovirus to a medium for BmN cells to prepare a baculovirus solution again was repeated three times, to prepare a baculovirus solution with a sufficiently increased baculovirus concentration. To <NUM> of IPL41-<NUM>% FCS medium containing <NUM> × <NUM><NUM> BmN cells, <NUM>µl of the baculovirus solution was added, and, <NUM> hours later, the cells were detached by pipetting, followed by collecting the culture liquid and counting the cell number. Thereafter, the culture supernatant was separated from the cell fraction by centrifugation operation. The collected cell fraction was suspended in <NUM>µl of the solution of <NUM> Tris-HCl (pH7. <NUM>), <NUM> NaCl, and <NUM> MgCh. The resulting suspension was then subjected to ultrasonic treatment and centrifugation operation, followed by collecting the supernatant. The collected cell homogenate supernatant was added to the culture supernatant to provide an analysis sample. SDS-PAGE was carried out for <NUM>µl of the analysis sample containing the peptide tag-GFP-Flag tag, and the peptide tag-GFP-Flag tag protein was detected using ImmunoStar Zeta (Wako Pure Chemical Industries, Ltd. ) with an anti-Flag antibody (Sigma Aldrich) as a primary antibody and an anti-mouse IgG HRP-labeled antibody (GE healthcare) as a secondary antibody. By comparison of the values calculated by dividing the band intensity of each peptide tag-GFP-Flag tag protein by the number of cells, the effect of each peptide tag on improvement of the protein expression level was determined.

As the protein to which the peptide tag was added, green fluorescent protein derived from Aequorea victoria (GFP; amino acid sequence, SEQ ID NO: <NUM>; DNA nucleotide sequence, SEQ ID NO: <NUM>) was used. PCR reaction was carried out using combinations of a forward primer (pENTR1A-<NUM> (SEQ ID NO: <NUM>), for addition of no tag; pENTR1A-<NUM> (SEQ ID NO: <NUM>), for PG12 tag; pENTR1A-<NUM> (SEQ ID NO: <NUM>), for PX12-<NUM> tag) and a reverse primer (pENTR1A-Flag-GFP (SEQ ID NO: <NUM>)) such that various peptide tags were added to the N-terminal side of the GFP protein and a Flag tag was added to the C-terminal side, to prepare DNA fragments for cloning. Each prepared DNA fragment was cloned into pENTR 1A (ThermoFisher Scientific) to construct a plasmid having the DNA fragment encoding each peptide tag-GFP-Flag tag. Based on this plasmid, LR reaction was used to insert the DNA encoding the peptide tag-GFP-Flag tag into pAd/CMV/v5-DEST adenovirus vector (ThermoFisher Scientific). Each resulting vector was introduced into HEK293A cells to prepare an adenovirus solution. The operation of inoculating the obtained adenovirus solution to HEK293A cells to prepare an adenovirus solution again was repeated four times, to prepare an adenovirus solution with a sufficiently increased adenovirus concentration. To <NUM> × <NUM><NUM> A549 cells in <NUM> of DMEM high glucose-<NUM>% FCS medium, <NUM>µl of this adenovirus solution was added, and, <NUM> hours later, the cells were detached by trypsin treatment (<NUM>, <NUM> minutes), followed by collecting the culture liquid and counting the cell number. Thereafter, the culture supernatant was separated from the cell fraction by centrifugation operation. The collected cell fraction was suspended in <NUM>µl of the solution of <NUM> Tris-HCl (pH7. <NUM>), <NUM> NaCl, and <NUM> MgCl<NUM>. The resulting suspension was then subjected to ultrasonic treatment and centrifugation operation, followed by collecting the supernatant and adding the supernatant to the culture supernatant. The fluorescence intensity was measured at an excitation wavelength of <NUM> and a measurement wavelength of <NUM>. By comparison of the values calculated by dividing the fluorescence intensity of each peptide tag-GFP-Flag tag protein by the number of cells, the effect of each peptide tag on improvement of the protein expression level was determined.

The prepared recombinant yeast was cultured under predetermined conditions, and hGH or IFNβ was extracted under predetermined conditions, followed by measuring the expression level of the protein of interest by Western analysis. As a result, as shown in <FIG>, the protein expression level was higher in the cases where PX12-<NUM> (SEQ ID NO:<NUM>), which was prepared by changing three Ss in the PG12 sequence (SEQ ID NO:<NUM>) to Ks, was added to IFNβ, than in the cases where PG12 was added to IFNβ. The effect of the PX12-<NUM> tag was higher in the case of addition to the C-terminus than in the case of addition to the N-terminus, and even higher in the case of addition to both the N-terminus and the C-terminus.

As shown in <FIG>, as a result of preparation of peptide tags by various modifications of the sequence of PG12 or PG17, and investigation of the influences of such modifications on expression of hGH, it was found that not only K, but also L, N, Q, and R are effective for increasing the expression when they are used as amino acids for substitution of S and/or G in PG12. Further, addition of a protease recognition sequence to each tag having a modified amino acid sequence still allowed maintenance of the high-expression effect.

The prepared recombinant E. coli was cultured under predetermined conditions, and hGH or IFNβ was extracted under predetermined conditions, followed by measuring the expression level of the protein of interest by Western analysis. As a result, as shown in <FIG>, the protein expression level was higher in the cases where PX12-<NUM> (SEQ ID NO:<NUM>) or PX12-20v7 (SEQ ID NO:<NUM>) was added to IFNβ than in the cases where PG12 was added to IFNβ. The effect of PX12-<NUM> or PX12-20v7 was even higher in the case of addition to both the N-terminus and the C-terminus than in the case of addition to only the N-terminus. As shown in <FIG>, the effect of PX12-<NUM> was also exerted on hGH.

The prepared recombinant Brevibacillus was cultured under predetermined conditions, and hGH was extracted under predetermined conditions, followed by measuring the expression level of the protein of interest by Western analysis. As a result, as shown in <FIG>, the expression level of hGH increased in the cases where PX12-<NUM> (SEQ ID NO:<NUM>), PX12-<NUM> (SEQ ID NO:<NUM>), or PX12-20v7 (SEQ ID NO:<NUM>) was added to hGH.

The prepared recombinant Brevibacillus was cultured under predetermined conditions, and xylanase was extracted under predetermined conditions, followed by measuring the expression level of the protein of interest by Western analysis. As a result, as shown in <FIG>, the expression level of xylanase increased in the cases where PX12-<NUM> (SEQ ID NO:<NUM>) was added to the C-terminus or to both the N-terminus and the C-terminus.

The prepared recombinant Brevibacillus was cultured under predetermined conditions, and esterase was extracted under predetermined conditions, followed by measuring the expression level of the protein of interest by Western analysis. As a result, as shown in <FIG>, the expression level of esterase increased in the case where PX12-<NUM> (SEQ ID NO:<NUM>) was added to the N-terminus.

The prepared recombinant Pichia yeast was cultured under predetermined conditions, and hGH was extracted under predetermined conditions, followed by measuring the expression level of the protein of interest by Western analysis. As a result, as shown in <FIG>, the expression level of hGH increased in the cases where PX12-<NUM> (SEQ ID NO:<NUM>) or PX12-20v7 (SEQ ID NO:<NUM>) was added to hGH.

The prepared recombinant insect cells or mammalian cells were cultured under predetermined conditions, and fluorescence of GFP was measured under predetermined conditions.

As a result, as shown in <FIG>, when GFPs having various peptide tags were expressed in insect cells, the proteins having the PX12-<NUM> tag or the PX12-20v7 tag showed higher expression levels than the non-tagged protein.

As a result, as shown in <FIG>, when GFPs having various peptide tags were expressed in mammalian cells, the protein having the PX12-<NUM> tag showed a higher expression level than the non-tagged protein.

Claim 1:
A tagged protein comprising a peptide tag bound to a protein of interest, the peptide tag having the following sequence:
Xm(PYn)qPZr
wherein X, Y, and Z each represent an amino acid residue independently selected from the group consisting of arginine (R), glycine (G), serine (S),
lysine (K), threonine (T), leucine (L), asparagine (N), glutamine (Q), and histidine (H),
with the proviso that at least one Y represents K, L, N, Q, H, or R; and
wherein m represents an integer of <NUM> to <NUM>; n represents <NUM>, <NUM>, or <NUM>; q represents an integer of <NUM> to <NUM>; and r represents an integer of <NUM> to <NUM>;
wherein the peptide tag has a length of <NUM> to <NUM> amino acids; and, wherein the content of G and S is less than <NUM>%; and wherein the peptide tag comprises the amino acid sequence of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.