Additive composition for inducing protein expression and method for producing protein

Abstract

An object of the present invention is to provide a novel additive composition for inducing protein expression that can replace IPTG.SOLUTION: An additive composition for inducing expression of a target protein in a bacterium containing an expression cassette A in which a polynucleotide encoding a T7RNA polymerase is located on the 3 ' terminal side of a lac promoter and an expression cassette B in which a polynucleotide encoding the target protein is located on the 3 ' terminal side of a T7 promoter, the additive composition comprising a peptone as an active ingredient and being free of isopropyl-β-thiogalactopyranoside and lactose and being used by being added to a medium at a timing when expression of the target protein is desired to be induced during culturing of the bacterium.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an additive composition for inducing protein expression and a method for producing a protein. [Background technology]

[0002] The pET system is the most common system for protein production in E. coli and is known as the gold standard for protein expression (see Non-Patent Documents 1 and 2). This system uses lactose or its analog, isopropyl-β-thiogalactopyranoside (IPTG), as an inducer for protein expression in E. coli. Specifically, IPTG drives the lacUV5 promoter to produce T7 RNA polymerase, which then transcribes a target gene with an upstream T7lac promoter.

[0003] However, when the pET system is used, weak protein expression occurs (leakage) even without adding IPTG as an inducer, making it difficult to control the expression of the target gene (see Non-Patent Documents 3 and 4), and IPTG is cytotoxic (see Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Studier, FW et al., Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol., 189, 113-130 (1986). [Non-patent document 2] Alan H.Rosenberg et al., Gene, 56 (1987) 125-135. [Non-patent document 3] Kelly, K. et al., Regulation of sCD4-183 gene expression from phage-T7-based vectors in Escherichia coli, Gene, 156, 33-36 (1995). [Non-patent document 4] Studier, F. W: Protein Expr. Purif., 41, 207-234 (2005). [Non-patent document 5] Dvorak et al. Microb Cell Fact (2015) 14:201. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide a new additive composition for inducing protein expression that can replace IPTG. [Means for solving the problem]

[0006] The present inventors have found that, when using the pET system in LB medium, protein expression occurs without the addition of IPTG, and conversely, the addition of IPTG does not result in induced protein expression. Therefore, we attempted to control the pET inducible expression system so that expression is suppressed in the absence of IPTG and increased by the addition of an inducer. During this process, the inventors discovered that certain peptones and yeast extracts contain inducing substances, leading to the completion of the present invention.

[0007] That is, the present disclosure is as follows. [1] An additive composition for inducing protein expression, which is used to induce expression of a target protein in a bacterium containing an expression cassette A in which a polynucleotide encoding T7 RNA polymerase is located on the 3'-end of a lac promoter, and an expression cassette B in which a polynucleotide encoding the target protein is located on the 3'-end of a T7 promoter, the additive composition for inducing protein expression containing peptone as an active ingredient but not containing isopropyl-β-thiogalactopyranoside, and which is used by adding to a culture medium at the time when it is desired to induce expression of the target protein during the cultivation process of the bacterium. [2] The additive composition for inducing protein expression according to [1] above, wherein the bacterium is Escherichia coli or Bacillus subtilis. [3] The additive composition for inducing protein expression according to [1] above, wherein the bacterium is Escherichia coli BL21(DE3) strain. [4] The protein expression induction composition according to any one of [1] to [3] above, wherein the expression cassette B is incorporated into a pET vector, a pCold vector, a pQE vector, or a pGEX vector. [5] The additive composition for inducing protein expression according to any one of [1] to [4] above, wherein a pET vector in which a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 is located on the 3'-end of the T7lac promoter is introduced into an Escherichia coli BL21(DE3) strain, and the transformant is cultured in a predetermined medium at 37°C and 150 rpm for 12 hours with shaking, and then peptone is added and the culture is further cultured at 37°C and 150 rpm for 6 hours with shaking, and the amount of luciferase expressed is 0.2 or more relative to the amount of luciferase expressed when 1 mM IPTG is administered instead of the peptone under the same conditions. [6] The additive composition for inducing protein expression according to any one of [1] to [5] above, further comprising a yeast extract. [7] The additive composition for inducing protein expression according to [6] above, characterized in that a transformant obtained by introducing a pET vector, in which a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 is located at the 3' end of the T7lac promoter, into an Escherichia coli BL21(DE3) strain is cultured in a specified medium at 37°C and 150 rpm for 12 hours with shaking, and then yeast extract is added and the culture is further cultured at 37°C and 150 rpm for 6 hours with shaking, whereby the expression level of luciferase used is 0.2 or more relative to the expression level of luciferase when 1 mM IPTG is administered instead of the yeast extract under the same conditions. [8] A method for producing a target protein by inducing expression of the target protein in a bacterium containing an expression cassette A in which a polynucleotide encoding T7RNA polymerase is located on the 3'-end of a lac promoter, and an expression cassette B in which a polynucleotide encoding the target protein is located on the 3'-end of a T7lac promoter, the method comprising the steps of culturing the bacterium in a medium and adding to the medium an additive composition for inducing protein expression described in any one of [1] to [7] above at a time when it is desired to induce the expression of the target protein. [9] The method according to [8] above, which does not include a step of adding isopropyl-β-thiogalactopyranoside.

[10] The step of adding the additive composition for inducing protein expression according to any one of [1] to [7] to a medium at a timing when the expression of the target protein is to be induced comprises culturing the bacteria in the medium and increasing the OD of the medium. 600 The method according to [8] or [9] above, wherein the additive composition for inducing protein expression is added to the medium at a stage where the ratio of the total number of cells in the medium is 0.3 to 1.2.

[11] A low-induction medium, wherein the medium mainly contains yeast extract, peptone, or a mixture thereof, The yeast extract, peptone, or mixture thereof The method according to any one of [8] to

[10] above, wherein a transformant obtained by introducing a pET vector, in which a polynucleotide encoding luciferase set forth in SEQ ID NO: 1 is located at the 3' end of the T7lac promoter, into Escherichia coli BL21(DE3) strain is cultured in a predetermined medium with shaking at 37°C and 150 rpm for 12 hours, and then the yeast extract, peptone, or a mixture thereof is added and cultured further with shaking at 37°C and 150 rpm for 6 hours, and the expression level of luciferase is 0.1 or less as a relative value to the expression level of luciferase when 1 mM IPTG is administered instead of the yeast extract, peptone, or mixture thereof under the same conditions. [Effects of the Invention]

[0008] The present disclosure makes it possible to induce protein expression in bacteria such as E. coli without using IPTG. [Brief explanation of the drawings]

[0009] [Figure 1A] FIG. 1A is a photograph of a colony of an E. coli BL21 or E. coli BL21(DE3) transformant harboring pET21a-T7lacp-eEmRFP in the absence of IPTG addition in an example. [Figure 1B] FIG. 1B is a photograph of an E. coli BL21(DE3) transformant carrying pET21a-T7lacp-eEmRFP (hereinafter also referred to as "E. coli BL21(DE3) / pET21a-T7lacp-eEmRFP transformant") cultured for 24 hours after addition of IPTG in the example. [Figure 1C] FIG. 1C shows the results of SDS-PAGE evaluation of Escherichia coli BL21(DE3) / pET21a-T7lacp-eEmRFP transformants cultured with and without the addition of IPTG in the example. [Figure 1D] FIG. 1D is a diagram showing the pET21a-T7lacp-Lux vector used in the Examples. [Figure 1E]Figure 1E shows the results of measuring the luciferase activity (RUL / OD / mL × 10) of T7lacp-Lux when Escherichia coli BL21(DE3) carrying pET21a-T7lacp-Lux (hereinafter also referred to as "E. coli BL21(DE3) / pET21a-T7lacp-Lux transformant") was cultured without adding IPTG in the examples. [Figure 2A] FIG. 2A shows the results of examining the growth of Escherichia coli BL21(DE3) in the presence of yeast extract alone or peptone alone in an example. [Figure 2B] FIG. 2B shows the results of measuring luciferase activity in the culture medium of Escherichia coli BL21(DE3) / pET21a-T7lacp-Lux transformants cultured in the yeast extracts and peptones that had high OD600 values ​​at 4% based on the results of FIG. 2A in the Examples. [Figure 3A] FIG. 3A shows the results of examining luciferase activity in an example in which an Escherichia coli BL21(DE3) / pET21a-T7lacp-Lux transformant was cultured in a medium containing a mixture of yeast extract and peptone. [Figure 3B] FIG. 3B shows the results of examining eEmRFP expression after culturing in three types of LB media in an example. [Figure 4] FIG. 4 shows the results of examining luciferase activity in an example in which My and Nc, which have a high expression-inducing effect, were mixed and the pET21a-T7lacp-Lux transformant was cultured. [Figure 5A] Figure 5A shows the results of examining luciferase activity after culturing pET21a-T7lacp-Lux transformants in low-induction medium at 37°C for 12 hours with shaking until the OD reached 0.3-1.2, and then adding inducers such as 1 mM IPTG, 0.9% lactose, 0.2% My / 1.6% Nc, or 0.2% By / 1.6% Dc. [Figure 5B] Figure 5B shows the results of SDS-PAGE analysis of the induction of eEmRFP or SpCas9 expression by IPTG or My / Nc to examine the expression profiles of other proteins in the examples. [Figure 6] FIG. 6 shows the results of inducing expression using only yeast extract or only peptone in an example. DETAILED DESCRIPTION OF THE INVENTION

[0010] The contents of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

[0011] The additive composition for inducing protein expression in this specification is an additive composition for inducing protein expression that is used to induce expression of a target protein in bacteria containing expression cassette A, in which a polynucleotide encoding T7 RNA polymerase is located on the 3'-end of a lac promoter, and expression cassette B, in which a polynucleotide encoding the target protein is located on the 3'-end of a T7 promoter, and that contains peptone as an active ingredient but does not contain isopropyl-β-thiogalactopyranoside, and that is used by adding to a culture medium at a timing when it is desired to induce expression of the target protein during the cultivation of the bacteria, and hereinafter this additive composition will also be simply referred to as "the present additive composition for inducing protein expression."

[0012] Furthermore, the method for producing a target protein in this specification is a method for producing the target protein by inducing expression of the target protein in a bacterium containing expression cassette A, in which a polynucleotide encoding T7 RNA polymerase is located at the 3' end of a lac promoter, and expression cassette B, in which a polynucleotide encoding the target protein is located at the 3' end of a T7 promoter, the method comprising the steps of culturing the bacterium in a medium and adding the present additive composition for inducing protein expression to the medium at a time when it is desired to induce expression of the target protein, and hereinafter this method will also be referred to simply as the "present method for producing the target protein."

[0013] 1. Additive composition for inducing protein expression [Expression cassette A / B] The expression cassette A used in this specification is not particularly limited as long as it is an expression cassette A in which a polynucleotide encoding T7 RNA polymerase is located on the 3' end of the lac promoter, and by including expression cassette A, T7 RNA polymerase is expressed in bacteria when the present additive composition for inducing protein expression is added.

[0014] The lac promoter is a promoter that is induced by lactose or IPTG, and specifically, the lac promoter set forth in SEQ ID NO: 2, the lacUV5 lac promoter set forth in SEQ ID NO: 3, or the L8-UV5 lac promoter set forth in SEQ ID NO: 4 can be used. In addition, the tac promoter set forth in SEQ ID NO: 5, which is a hybrid promoter of the trp promoter and the L8-UV5 lac promoter, or the trc promoter set forth in SEQ ID NO: 6 can be used.

[0015] T7 RNA polymerase is an RNA polymerase derived from T7 phage, which specifically binds to the T7 promoter sequence and enables expression of a gene located on the 3' end of the T7 promoter. An example of a polynucleotide encoding T7 RNA polymerase is a polynucleotide consisting of the nucleic acid sequence set forth in SEQ ID NO:7.

[0016] The expression cassette A may contain a lac operator located on the 3'-end of the lac promoter and on the 5'-end of the polynucleotide encoding T7 RNA polymerase, the expression of which is controlled by lacI, a lac repressor expressed by the lacI gene.

[0017] The polynucleotide encoding T7 RNA polymerase being located on the 3' end of the lac promoter means that the polynucleotide encoding T7 RNA polymerase is operably linked to the 3' end of the lac promoter and is linked so that it is expressed under the control of the lac promoter.

[0018] The expression cassette B in this specification is not particularly limited as long as it is an expression cassette B in which a polynucleotide encoding a target protein is located on the 3' end of the T7 promoter. By including the expression cassette B, when the present additive composition for inducing protein expression is added, the target gene is expressed in bacteria.

[0019] The T7 promoter is a promoter that induces expression by T7 RNA polymerase, and expresses a target gene when T7 RNA polymerase binds to it. Examples of T7 promoters include the T7lac promoter shown in SEQ ID NO: 8, the T7 promoter shown in SEQ ID NO: 9, and the consensus φ(phi)10 (T7) promoter (T7p) shown in SEQ ID NO: 10. CONS ) can be mentioned.

[0020] The target protein is not particularly limited, and examples thereof include proteins that are used for pharmaceutical or industrial purposes and can be expressed in bacteria, such as hormones such as insulin, growth hormone, and natriuretic peptides, hormones such as parathyroid hormone and leptin, interferons such as interferon α, interferon β, and interferon γ, cytokines such as G-CSF, interleukin 2, and bFGF, and antibodies such as humanized anti-VEGF antibody fragments and Fc-TPOR agonist peptide fusion proteins.

[0021] The expression cassette B may contain a lac operator located at the 3' end of the T7 promoter and at the 5' end of the polynucleotide encoding the target protein, whose expression is controlled by lacI, a lac repressor expressed by the lacI gene.

[0022] The phrase "the polynucleotide encoding the target protein is located on the 3' end of the T7 promoter" means that the polynucleotide encoding the target protein is operably linked to the 3' end of the T7 promoter so that the polynucleotide encoding the target protein is expressed under the control of the T7 promoter.

[0023] The expression cassettes A and / or B may each be incorporated into the genome of the bacterium, or may each be contained in a plasmid vector incorporated into the bacterium. Examples of the plasmid vector include pET vector, pCold vector, pQE vector, and pGEX vector. Furthermore, when the expression cassettes A and / or B contain the lac operator, the plasmid vector may contain a polynucleotide encoding the lacl gene.

[0024] The expression cassette A and / or B may contain a drug resistance gene, and examples of the drug resistance gene include ampicillin, kanamycin, chloramphenicol, and the like.

[0025] [Bacteria] The bacteria are not particularly limited, and may be either gram-negative or gram-positive bacteria, with gram-negative bacteria being preferred, including Escherichia coli and Bacillus subtilis. Examples of E. coli include BL21, JM109, and DH5α strains, as well as CD41(DE3), CD43(DE3), CD44(DE3), and CD45(DE3) (Miroux, B. et al., J. Mol. Biol., 260, 289-298 (1996); Kwon, S. K. et al., Sci. Rep., 5, 16076 (2015); Schlegel, S. et al., Cell Rep., 10, 1758-1766 (2015); Angius, F. et al., Sci. Rep., 8, 8572 (2018)) and Mutant56(DE3) (Baumgarten, T. et al., Sci. Rep., 7, 45089). (2017)) and Lemo21(DE3) (Wagner, S. et al., Proc. Natl. Acad. Sci. USA, 105, 14371-14376 (2008)), and their mutant strains available as pET systems. Alternatively, bacteria lysogenized with bacteriophage λDE3, in which expression cassette A has been integrated into the λDE3 gene, may be used. Specific examples include E. coli BL21(DE3) strain, Origami TM 2(DE3), Rosetta-gami TM Examples include bacteriophage λDE3 lysogenized bacteria, and Rosetta-gami B (DE3). By incorporating expression cassette B into the bacteria, bacteria containing expression cassettes A and B can be prepared.

[0026] Furthermore, when the expression cassette A and / or B contains the lac operator, the genome of the bacterium may contain a polynucleotide encoding the lacl gene.

[0027] [peptone] The term "peptone" as used herein is not particularly limited as long as it is a mixture of amino acids and low-molecular-weight peptides obtained by hydrolyzing proteins with digestive enzymes or acids. Examples of protein sources include casein, plant peptones from soybeans, potatoes, wheat, peas, etc., and meat from pigs, cattle, poultry, etc. Mixed peptones obtained by mixing these may also be used. Examples of the digestive enzymes include pancreatin, trypsin, pepsin, papain, and bacterial enzymes (proteinases).

[0028] The peptone is preferably a peptone containing a lac inducer. To assess whether a peptone contains a lac inducer, a pET vector containing a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 at the 3' end of the T7lac promoter is introduced into Escherichia coli BL21(DE3) and the resulting transformant is cultured in a designated medium at 37°C for 12 hours with shaking at 150 rpm. Peptone is then added and the culture is further cultured at 37°C for 6 hours with shaking at 150 rpm. The relative expression level of luciferase to that obtained under the same conditions but with 1 mM IPTG added instead of the peptone is then determined. If the relative expression level is 0.2 or higher, preferably 0.5 or higher, more preferably 0.8 or higher, even more preferably 1.0 or higher, and most preferably 1.2 or higher, the peptone can be assessed as containing a lac inducer. Alternatively, enzyme activity, mRNA amount, or protein amount may be used for evaluation. Examples of the designated medium include LB medium, peptone, yeast extract, and a mixture thereof.

[0029] Furthermore, as another method for evaluating whether a peptone contains a lac-inducing substance, a pET vector containing a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 located at the 3' end of the T7lac promoter is introduced into E. coli BL21(DE3) strain, and the transformant is cultured in a predetermined medium at 37°C and 150 rpm for 12 hours with shaking, and then peptone is added and the culture is further cultured at 37°C and 150 rpm for 6 hours with shaking. The amount of luciferase expressed when 1 mM IPTG is administered instead of the peptone under the same conditions is examined to determine the amount of luciferase expressed (RLU / OD / mL). If the amount of luciferase expressed is 0.2 or more, preferably 0.5 or more, more preferably 0.8 or more, even more preferably 1.0 or more, and most preferably 1.2 or more, the peptone can be evaluated as containing a lac-inducing substance.

[0030] [Yeast extract] The yeast extract in this specification can be obtained by extracting the contents of yeast cells by autolysis using protease present in the yeast cells, enzymatic hydrolysis, acid or alkali hydrolysis, hot water extraction, or the like.

[0031] The yeast extract is preferably a peptone containing a lac inducer. To assess whether a peptone contains a lac inducer, a pET vector containing a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 at the 3' end of the T7lac promoter is introduced into Escherichia coli BL21(DE3) and the resulting transformant is cultured in a designated medium at 37°C for 12 hours with shaking at 150 rpm. The peptone is then added and the culture is further cultured at 37°C for 6 hours with shaking at 150 rpm. If the luciferase expression level is 0.2 or higher, preferably 0.5 or higher, more preferably 0.8 or higher, even more preferably 1.0 or higher, and most preferably 1.2 or higher, the yeast extract can be assessed as containing a lac inducer. Alternatively, enzyme activity, mRNA level, or protein level may be used for evaluation. Examples of the designated medium include LB medium, peptone, yeast extract, and a mixture thereof.

[0032] Furthermore, as another method for evaluating whether a yeast extract contains a lac-inducing substance, a pET vector containing a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 located at the 3' end of the T7lac promoter is introduced into the Escherichia coli BL21(DE3) strain, and the transformant is cultured in a designated medium at 37°C and 150 rpm for 12 hours with shaking, and then yeast extract is added and the culture is further cultured at 37°C and 150 rpm for 6 hours with shaking. The luciferase expression level (RLU / OD / mL) is then examined, and if the luciferase expression level is 0.2 or higher, preferably 0.5 or higher, more preferably 0.8 or higher, even more preferably 1.0 or higher, and most preferably 1.2 or higher, the yeast extract can be evaluated as containing a lac-inducing substance.

[0033] When the peptone and yeast extract are mixed, the composition ratio of peptone to yeast extract can be 9:1 to 1:9, 8:2 to 2:8, 7:3 to 3:7, 4:6 to 6:4, or 5:5. Preferably, the ratio of peptone to yeast extract can be 8:1, 6:1, 3:1, 2:1, or 1:1.

[0034] From the viewpoint of proliferation or expression of the target protein, the additive composition for inducing expression of the present protein may contain vitamins such as biotin, pyridoxine, pantothenic acid, nicotinic acid, thiamine, etc., inorganic salts such as zinc, magnesium, phosphorus, iron, calcium, sodium, potassium, etc., monosaccharides such as glucose, disaccharides such as sucrose and lactose, sugar alcohols such as sorbitol, oligosaccharides, etc.

[0035] 2. Methods for producing target proteins The expression cassettes A and B and bacteria in the method for producing the target protein of the present invention are the same as those described for the additive composition for inducing protein expression of the present invention.

[0036] The method for producing the target protein of the present invention preferably does not include a step of adding isopropyl-β-thiogalactopyranoside.

[0037] The step of adding an additive composition for inducing protein expression to a medium at a timing when the expression of the target protein is desired is performed by culturing the bacteria in the medium and adjusting the OD of the medium. 600 Preferably, the additive composition for inducing expression of the present protein is added to the medium when the pH is 0.3 to 1.2, more preferably 0.4 to 0.8.

[0038] The medium is preferably a low-induction medium primarily containing yeast extract, peptone, or a mixture thereof, and the yeast extract, peptone, or mixture thereof is selected from the group consisting of Escherichia coli BL21(DE3) transformed with a pET vector containing a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 located at the 3' end of the T7lac promoter, cultured in a designated medium at 37°C for 12 hours with shaking at 150 rpm, and then the yeast extract, peptone, or mixture thereof is added and cultured further at 37°C for 6 hours with shaking at 150 rpm. The luciferase expression level, relative to the luciferase expression level when 1 mM IPTG is administered instead of the yeast extract, peptone, or mixture thereof under the same conditions, is preferably 0.1 or less, preferably 0.05 or less, more preferably 0.02 or less, and even more preferably 0.01 or less. Note that the expression level may also be evaluated in terms of enzyme activity, mRNA amount, or protein amount.

[0039] When the additive composition for inducing protein expression of the present invention is added to a culture medium, if the additive composition for inducing protein expression of the present invention contains peptone as an active ingredient, it can be added so that the content of peptone in the culture medium is 0.5 to 6%, preferably 1.0 to 5%, more preferably 1.5 to 4.5%, and particularly preferably 2 to 4%.

[0040] When the present additive composition for inducing protein expression is added to a medium, and the additive composition for inducing protein expression contains peptone and yeast extract as active ingredients, the peptone content in the medium is 0.5 to 6%, preferably 1.0 to 5%, more preferably 1.5 to 4.5%, and particularly preferably 2 to 4%, and the yeast extract content is 0.05 to 4%, preferably 0.1 to 3%, and more preferably 0.2 to 2%. Specifically, the peptone and yeast extract can be added at 1.5 to 2.5% and 0.1 to 0.3%, respectively.

[0041] When the present additive composition for inducing protein expression is added to a medium, and the additive composition for inducing protein expression contains peptone and yeast extract as active ingredients, the composition ratio of peptone:yeast extract can be 9:1 to 1:9, 8:2 to 2:8, 7:3 to 3:7, 4:6 to 6:4, or 5:5. Preferably, the ratio of peptone:yeast extract can be 8:1, 6:1, 3:1, 2:1, or 1:1. [Example]

[0042] The present invention will be described in more detail below with reference to examples. The examples are not limiting.

[0043] 1. Experimental Method In the following examples, "strain and medium," "plasmid construction," "E. coli culture," "SDS-PAGE," and "luciferase assay" were carried out by the following methods.

[0044] (Strain and medium) E. coli BL21 strain (F - ompT dcm hsdS (r B - m B - ) gal) and BL21(DE3) strain (F - ompT dcm hsdS (r B - m B -) gal λ (DE3)) were obtained from Takara Bio Inc. and Merck, respectively. Escherichia coli strain DH5α (F - Φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(r K - , m K + ) phoA supE44 λ - The thi-1 gyrA96 relA1 vector was obtained from Takara Bio Inc. and used for plasmid cloning. Commercially available premixed LB media included Difco LB broth (244620: Becton Dickinson (BD)) (hereinafter referred to as "BD Difco"), BBL Select APS LB broth (292438: BD) (hereinafter referred to as "BD BBL"), LB Broth Lennox (20066-95: Nacalai Tesque) (hereinafter referred to as "Nacalai"), LB Broth Miller (71753-5: Merck) (hereinafter referred to as "Novagen"), and LB Broth Lennox (L7275: Merck) (hereinafter referred to as "Sigma"). The premixed powder was weighed, dissolved in water, and then autoclaved according to the manufacturer's instructions. The yeast extracts used were 212750 (BD), A1202 (Biokar), 551-01310 (Kyokuto), 70161 (Millipore), 36802-1 (Nacalai Tesque), and Y1625 (Sigma).The peptones used were Bacto Peptone (211677: BD), Bacto Tryptone (211705: BD), casein peptone (26440-95: Nacalai Tesque), meat peptone (26442-75: Nacalai Tesque), soybean peptone (2644365: Nacalai Tesque), and Hipolypeptone (396-02116: Fujifilm Wako Pure Chemical Industries). The LB medium contained 5 g of yeast extract (Kyokuto), 10 g of casein peptone (Nacalai Tesque), and 5 g of NaCl per liter (LBKyNc Ampicillin may be added to the LB medium as described above to a final concentration of 100 μg / mL. KyNc The LBamp medium contained 15 g of agar per liter.

[0045] (Plasmid construction) The oligonucleotide primers used in the following examples were obtained from Eurofins Genomics and are shown in Table 1.

[0046] [Table 1]

[0047] Plasmid construction was performed according to the T5 exonuclease-mediated assembly method (Xia, Y., et al., Nucleic. Acids Res., 47, e15 (2019)). Briefly, vector and insert DNA fragments were designed to contain 12- or 15-bp overlapping sequences for recombination and amplified by PCR using GXL polymerase (Takara Bio). PCR was performed according to the manufacturer's instructions using a three-step program: initiation at 98°C for 4 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 68°C for 30–150 seconds, for 30 cycles. The PCR-amplified vector and insert fragments (1 μL each) were mixed and treated with T5 exonuclease (New England Biolabs) at 30°C for 5 minutes. The treated mixture was mixed with 20 μL of E. coli DH5α competent cells, incubated at 45°C for 30 seconds, and plated on an LB amp plate. Plasmids were isolated from the transformants using a plasmid preparation kit (Qiagen) according to the manufacturer's instructions. DNA concentrations were measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific).

[0048] The pET21a vector fragment was prepared by PCR using pET21a+5240c (SEQ ID NO: 11) primer and pET21a+5308 (SEQ ID NO: 12) primer and pET21a plasmid (Merck) as a template.

[0049] The 711-bp eEmRFP fragment was inserted downstream of the T7lac promoter in the pET21a vector to construct pET21a-T7lacp-eEmRFP. The eEmRFP fragment was amplified by PCR from the pAmp-srlAp-eEmRFP plasmid (GenBank accession number LC731004) using the pET21a+5240(15)-eEmRFP+1 (SEQ ID NO: 13) and pET21a+5308c(15)-eEmRFP+711c (SEQ ID NO: 14) primers. The sequence of T7lacp-eEmRFP was confirmed by sequencing.

[0050] A polynucleotide encoding the P. luminescens luciferase operon sequence (SEQ ID NO: 1) was inserted downstream of the T7lac promoter in the pET21a vector to construct pET21a-T7lacp-Lux. The luciferase operon fragment was amplified by PCR from the pLux plasmid (a gift from Dr. Kenyoshi Yamamoto, Hosei University) using the pET21a+5240(15)-luxC+1 (SEQ ID NO: 15) and pET21a+5308c(15)-luxE+1113c (SEQ ID NO: 16) primers. The T7lac promoter sequence was confirmed by sequencing, and luciferase activity was measured using the luciferase assay described below. Note that pET21a-T7lacp-Lux encodes luciferase and a set of enzymes that synthesize luminescence from fatty acids, allowing activity to be measured without substrate.

[0051] The 4107-bp Cas9 sequence (SEQ ID NO: 17) was inserted downstream of the T7lac promoter in the pET21a vector to construct pET21a-T7lacp-Cas9. The Cas9 fragment was amplified by PCR from the pCas plasmid (Jiang, Y., at al., Appl. Environ. Microbiol., 81, 2506-2514 (2015)) using the pET21a+5240(15)-Cas9+1 (SEQ ID NO: 18) primer and the pET21a+5308c(15)Cas9+4107c primer (SEQ ID NO: 19). pCas was provided by RIKEN RBC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project.

[0052] (SDS-PAGE) A suspension of pET-T7lacp-eEmRFP transformed cells was inoculated into 1 mL of LBG amp in a test tube and cultured overnight with shaking at 37°C. 50 μL of the overnight culture was inoculated into 10 mL of LBG amp in a 125 mL flask and cultured with shaking at 37°C. OD 600 The value was 0.5-0.8 and the cells were induced with 0.5 mM IPTG.

[0053] For time-course analysis of eEmRFP and Cas9 production, one transformed colony was selected and inoculated into 6 mL of 0.5% yeast extract (Biokar) and 1% Bactotryptone medium (BD). After 12 hours of shaking incubation at 37°C, 1 mL of the culture was dispensed into test tubes. For induction, each culture was mixed with either 10 μL of 100 mM IPTG or 25 μL of 10% yeast extract and 200 μL of 10% peptone. At the indicated time points, 20 μL of cell culture was mixed with 20 μL of 2x sample buffer (Bio-RAD) and lysed by heating at 95°C for 5 minutes. 5 μL of the whole cell lysate was separated on a 5-20% SuperSepAce gel (Fujifilm Wako Pure Chemical Industries), and proteins were detected using Coomassie Brilliant Blue staining.

[0054] (Luciferase assay) To measure luciferase activity, E. coli was cultured in 1 mL of medium at 37°C for 24 hours with shaking. The culture was diluted 1000-fold with water, and 50 μL of the diluted culture was dispensed into a 96-well black microplate for luminescence measurement. Luminescence was measured using a SpectraMax iD5 (Molecular Devices) with an integration time of 1 second. Luciferase activity was measured as the OD per mL of culture. 600 The concentration was calculated as relative luminescence units (RLU) per unit area.

[0055] For time-course analysis of luciferase production, five independent transformed colonies were selected and inoculated into 6 mL of 0.5% yeast extract (Biokar) and 1% Bactotryptone medium (BD). After 12 hours of shaking incubation at 37°C, the OD of the five transformed cultures was 600 The values ​​ranged from 0.3 to 1.2. The cultures were dispensed into test tubes (1 mL each). For induction, each 1 mL culture was mixed with 10 μL of 100 mM IPTG, 100 μL of 10% lactose, or 25 μL of 10% yeast extract and 200 μL of 10% peptone. Cell cultures were harvested at the designated time points and diluted 1000-fold with water, and 50 μL of the diluted sample was used to measure luciferase activity. Student's t-test was used to compare premixed LB broth and yeast extract and peptone mixtures.

[0056] [Example 1] Expression in various LB media with or without IPTG First, the strain was cultured alone in commercially available LB medium or medium ingredients such as yeast extract and peptone, and the effect of the medium on pET expression was examined.

[0057] The E. coli BL21 strain or the E. coli BL21(DE3) strain containing galλ(DE3) in its genome was transformed with the pET21 vector containing the above-mentioned pET21a-T7lacp-eEmRFP downstream of the T7lac promoter. KyNc The cells were cultured in LBamp medium supplemented with ampicillin. The results are shown in Figure 1A. eEmRFP is a red fluorescent protein optimized for E. coli codons.

[0058] In Figure 1A, the upper panel shows the introduced expression vector, and the lower panel shows the results of culturing the transformant in LBamp medium. Most of the colonies (right side of the photograph) of E. coli BL21(DE3) transformants harboring pET21a-T7lacp-eEmRFP turned red on LBamp plates without IPTG induction, indicating strong eEmRFP expression on LBamp plates. In contrast, colonies (left side of the photograph) of the parent strain E. coli BL21, in which DE3 was not lysogenized, remained white on LBamp plates. This confirms that expression in E. coli BL21(DE3) is driven by T7 RNA polymerase and that eEmRFP expression can be induced even without IPTG.

[0059] Next, IPTG was added to liquid LBamp medium to final concentrations of 0, 0.01, and 1 mM, and the E. coli BL21(DE3) / pET21a-T7lacp-eEmRFP transformant was cultured for 24 hours. The results are shown in Figure 1B.

[0060] RFP expression was also observed in liquid LBamp medium without IPTG (0 mM). Surprisingly, the addition of IPTG to liquid LBamp medium attenuated the color intensity in a concentration-dependent manner (0.01 and 1 mM).

[0061] Next, to evaluate protein production, the above E. coli BL21(DE3) / pET21a-T7lacp-eEmRFP transformant was pre-cultured overnight in LBG amp, and pET expression was suppressed according to the manufacturer's instructions. 50 μL of the overnight culture was inoculated into 10 mL of LBG amp and cultured with shaking. OD 600 At pH 0.5-0.8, IPTG was added to a final concentration of 0.5 mM. The same culture without IPTG was used as a control (Figure 1C, LBamp). The time course of eEmRFP production was assessed using SDS-PAGE. The results are shown in Figure 1C. In Figure 1C, LBamp + 0.5 mM IPTG on the right represents the case where IPTG was added to a final concentration of 0.5 mM, while LBamp on the left represents the case where IPTG was not added.

[0062] A strong eEmRFP band was detected without IPTG addition, but only a weak band was observed with IPTG addition, indicating that IPTG represses eEmRFP expression even when the manufacturer's recommended protocol was followed.

[0063] Furthermore, various LB media were used to culture the strain without IPTG addition, and luciferase activity was evaluated. KyNc In addition, different commercially available premixed LB media were used, including those from BD Difco, BD BBL, Nacalai, Novagen, and Sigma. To quantify expression, a polynucleotide (SEQ ID NO: 1) encoding the P. luminescens luciferase operon sequence was inserted into the pET21a vector downstream of the T7lac promoter to create pET21a-T7lacp-Lux (Figure 1D). E. coli BL21(DE3) was transformed with this pET21a-T7lacp-Lux to create a T7lacp-Lux transformant (E. coli BL21(DE3) / pET21a-T7lacp-Lux transformant). The resulting transformant was cultured in the premixed LB medium without the addition of IPTG, and the luciferase activity of T7lacp-Lux (RUL / OD / mL (×10)) was measured. 6 The results are shown in Figure 1E.

[0064] Cells grown in LB medium from BD Difco and BD BBL were KyNc The luciferase activity was similar to that of the lac inducer. In contrast, cells cultured in Nacalai (P = 0.0004), Novagen (P = 0.0295), and Sigma (P = 0.0013) LB media showed low expression. These results suggest that different types of LB media contain different concentrations of lac inducers. LB media consists of yeast extract, peptone, and NaCl. Since NaCl is clearly not a lac inducer, it is clear that the yeast extract or peptone contains a lac inducer.

[0065] [Example 2] Measurement of pET-Lux expression in a medium containing only yeast extract or peptone To investigate leaky expression in yeast extract or peptone individually, it was necessary to examine whether E. coli cells would grow in media containing yeast extract or peptone alone. Therefore, we first examined the growth of E. coli BL21(DE3) in media containing yeast extract or peptone alone. The yeast extracts used were from BD (Dy), Biokar (By), Millipore (My), Kyokuto (Ky), Nacalai Tesque (Ny), and Merck (Sy). Capital letters in parentheses indicate the supplier's abbreviation, and a lowercase "y" indicates yeast extract. Because peptones are produced from different sources, such as casein, meat, and soybeans, the abbreviations are "c," "m," and "s," respectively. The peptones used were Nakarai Casein (Nc), Nakarai Meat (Nm), and Nakarai Soy (Ns) from Nacalai Tesque, Casein (Wc) from Fujifilm Wako Pure Chemical Industries, Animal Peptone (Da; the source of the animal peptone is unknown, so "a" is used), and Casein (Dc) from BD. The medium was prepared using only single ingredients, without any components such as NaCl or NaOH. The concentration was varied from 0.5% to 8%. The results are shown in Figure 2A. In Figure 2A, the upper panel shows the case of culturing with yeast extract, and the lower panel shows the case of culturing with peptone. The horizontal axis represents the concentration of the medium added, and the vertical axis represents the OD. 600 is.

[0066] As a control, LB KyNc is the OD after 24 hours of culture. 600 Growth was observed at an OD of 2.6 ± 0.7 (data not shown). Interestingly, almost all yeast extracts and peptones, except for Ny, showed growth at an OD of 2.6 ± 0.7 at 4%. 600 showed values ​​higher than 1.0 (Fig. 2A).

[0067] In Figure 2A, 600The E. coli BL21(DE3) / pET21a-T7lacp-Lux transformant was cultured under the same conditions as above for the yeast extracts By, Dy, My, and Sy, and the peptones Nc, Nm, Ns, Wc, and Dc, which all showed high luciferase activity (4%), and the luciferase activity in each medium was measured. The results for the peptones are shown in Figure 2B.

[0068] Luciferase activity was low when cultured in yeast extract media By, Dy, and Sy. On the other hand, high activity was observed when cultured in yeast extract media My (not shown). Furthermore, luciferase activity was higher when cultured in peptone than when cultured in yeast extract. Peptone media Nc, Nm, Ns, and Wc had high activity, while Dc medium showed the lowest activity among the peptones. These results indicated that yeast extract and peptone contain different concentrations of lac inducers.

[0069] [Example 3] High-expression medium and low-expression medium containing a mixture of yeast extract and peptone Based on Figure 2B, it was suggested that peptone contains lac inducers. Therefore, we wondered whether it might be possible to prepare a mixed LB-like medium with a low or high lac inducer content. We hypothesized that mixing yeast extract with a low lac inducer content and peptone could produce a low-expression medium that could be used for leak-free culture before lac induction. Alternatively, mixing yeast extract with a high lac inducer content and peptone could produce a lac inducer.

[0070] To prepare a low-expression medium, we mixed By with Dc (ByDc). Similarly, we prepared a high-expression medium by mixing My and Nc (MyNc). Furthermore, we attempted to prepare a medium with intermediate lac expression levels by mixing low-expressing By with high-expressing Nc, or high-expressing My with low-expressing Dc (ByNc, MyDc). The pET21a-T7lacp-Lux transformant was cultured in each medium and the luciferase activity was examined. The results are shown in Figure 3A.

[0071] Luciferase expression was low in the medium containing a mixture of By and Dc and in the medium containing a mixture of My and Dc, moderate in the medium containing a mixture of By and Nc, and high in the medium containing a mixture of My and Nc.

[0072] The expression of Lux was further confirmed by examining the expression of eEmRFP in E. coli BL21(DE3), and the results are shown in Figure 3B.

[0073] Transformed colonies were cultured in LB ByDc It was white on the plate, but MyNc The plate showed a strong red color. These results demonstrate that gene expression using pET vectors can be suppressed or induced by mixing a specific yeast extract and peptone. Proteins that are toxic to E. coli can be expressed without induction (leaky expression), resulting in extremely poor growth and, in some cases, even the isolation of transformant colonies. However, this problem can be avoided by using a low-expression medium that suppresses expression.

[0074] [Example 4] Consistent high expression medium In the above example, a medium with consistently high expression was prepared by mixing My and Nc, which have a high expression-inducing effect. Therefore, to optimize the mixture ratio, various concentrations of My were mixed with 1% Nc. The E. coli BL21(DE3) / pET21a-T7lacp-Lux transformant was cultured in these media, and luciferase activity was examined. The results are shown in Figure 4.

[0075] It was confirmed that the addition of My and Nc increased cell proliferation and protein expression.

[0076] [Example 5] Induction profile of IPTG, lactose, and induction medium According to the manufacturer's instructions for the pET system version 8, cells should be harvested 2-3 hours after induction. We speculated that IPTG is suitable for rapid expression induction. Therefore, we investigated the kinetics of protein induction using E. coli BL21(DE3) / pET21a-T7lacp-Lux transformants. The transformants were pre-cultured in a low-induction medium containing 0.5% By and 1% DC at 37°C with shaking for 12 hours, and the OD 600 The pH was adjusted to 0.3-1.2. Subsequently, 1 mM IPTG, 0.9% lactose, 0.2% My / 1.6% Nc, or 0.2% By / 1.6% Dc was added as an inducer, and luciferase activity was measured. The results are shown in Figure 5A.

[0077] When pre-cultured in the low-induction medium and then 0.2% By / 1.6% DC was added at the same time as adding the inducer, luciferase activity was not induced. When IPTG was added, activity was detected 15 minutes after addition and increased within one hour. On the other hand, My / Nc showed no activity within one hour, but showed higher activity than IPTG 4 to 8 hours after addition. Therefore, My / Nc was confirmed to be an inducer with a stronger expression-inducing ability than IPTG.

[0078] To examine the expression profiles of other proteins, we analyzed the expression of eEmRFP and SpCas9 (a genome editing enzyme from Streptococcus pyogenes) by SDS-PAGE. For eEmRFP expression, we used the E. coli BL21(DE3) / pET21a-T7lacp-eEmRFP transformant described above. For SpCas9 expression, we used the E. coli BL21(DE3) / pET21a-T7lacp-Cas9 transformant, which was created by transforming the E. coli BL21(DE3) strain with pET21a-T7lacp-Cas9. The results are shown in Figure 5B.

[0079] The production of eEmRFP was detected 15 min after IPTG addition and reached a plateau after 2 h. My / Nc induction slowed the production but resulted in higher expression levels than IPTG. My / Nc also induced Cas9 protein production.

[0080] From the above, it was demonstrated that by culturing in a low-induction medium and then adding a mixture of a specific yeast extract and peptone that has expression-inducing properties, it is possible to produce proteins at levels comparable to or greater than those produced by IPTG without using IPTG. Furthermore, the above results demonstrated that it is possible to produce proteins that are toxic to E. coli by controlling the expression induction.

[0081] [Example 7] Induction with yeast extract alone or peptone alone In Example 6, a mixture of yeast extract and peptone was used as an expression inducer, but each was used alone to examine its activity.

[0082] The E. coli BL21(DE3) / pET21a-T7-Lux transformant was inoculated into 30 mL of medium (0.5% Biokar yeast extract, 1% BD tryptone) in a 125 mL baffled flask and cultured at 37°C and 150 rpm for 12 hours with shaking. One mL of the culture was dispensed into test tubes, and IPTG, yeast extract, peptone, and distilled water (DW) were added. After induction, culture was cultured at 37°C and 150 rpm for 6 hours with shaking. A luciferase assay was then performed. The results are shown in Figure 6. The numbers on the horizontal axis in Figure 6 indicate the concentration of the added substances.

[0083] In Figure 2B, Nc, Nm, Ns, and Wc, which showed high activity when used as medium, were confirmed to induce expression even when added alone. On the other hand, yeast extract induced expression, but at a lower level than peptone.

[0084] The above examples demonstrate that expression can be induced without using IPTG by culturing E. coli while minimizing pET expression by using commercially available LB medium with low content of inducers, or yeast extract or peptone with low inducibility as a basal medium, and then adding peptone as an inducer at the desired timing.

Claims

1. An additive composition for inducing protein expression is used to induce expression of a target protein in a bacterium containing an expression cassette A in which a polynucleotide encoding T7 RNA polymerase is located on the 3'-end of a lac promoter, and an expression cassette B in which a polynucleotide encoding the target protein is located on the 3'-end of a T7 promoter, the additive composition containing peptone as an active ingredient but not containing isopropyl-β-thiogalactopyranoside, and which is used by being added to a culture medium at a timing when it is desired to induce expression of the target protein during the cultivation of the bacterium.

2. The additive composition for inducing protein expression according to claim 1 , wherein the bacterium is Escherichia coli or Bacillus subtilis.

3. 2. The additive composition for inducing protein expression according to claim 1, wherein the bacterium is Escherichia coli BL21(DE3) strain.

4. The protein expression induction composition according to any one of claims 1 to 3, wherein the expression cassette B is incorporated into a pET vector, a pCold vector, a pQE vector, or a pGEX vector.

5. 5. The additive composition for inducing protein expression according to claim 1, wherein a pET vector in which a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 is located at the 3'-end of a T7lac promoter is introduced into an Escherichia coli BL21(DE3) strain, the transformant is cultured in a predetermined medium at 37°C and 150 rpm for 12 hours with shaking, and then peptone is added and the culture is further cultured at 37°C and 150 rpm for 6 hours with shaking, and the amount of luciferase expressed is 0.2 or more relative to the amount of luciferase expressed when 1 mM IPTG is added instead of the peptone under the same conditions.

6. The additive composition for inducing protein expression according to any one of claims 1 to 5, further comprising a yeast extract.

7. 7. The additive composition for inducing protein expression according to claim 6, wherein a pET vector containing a polynucleotide encoding the luciferase operon sequence set forth in SEQ ID NO: 1 located at the 3'-end of a T7lac promoter is introduced into an Escherichia coli BL21(DE3) strain, the transformant is cultured in a predetermined medium at 37°C and 150 rpm for 12 hours with shaking, and then yeast extract is added and the culture is further cultured at 37°C and 150 rpm for 6 hours with shaking, and the amount of luciferase expressed is 0.2 or more relative to the amount of luciferase expressed when 1 mM IPTG is added instead of the yeast extract under the same conditions.

8. 10. A method for producing a target protein by inducing expression of the target protein in a bacterium containing an expression cassette A in which a polynucleotide encoding T7 RNA polymerase is located on the 3'-end of a lac promoter, and an expression cassette B in which a polynucleotide encoding the target protein is located on the 3'-end of the T7lac promoter, the method comprising the steps of culturing the bacterium in a medium and adding the additive composition for inducing protein expression according to any one of claims 1 to 7 to the medium at a time when it is desired to induce the expression of the target protein.

9. 9. The method according to claim 8, which does not include a step of adding isopropyl-β-thiogalactopyranoside.

10. The step of adding the additive composition for inducing protein expression according to any one of claims 1 to 7 to a culture medium at a timing when it is desired to induce the expression of the target protein comprises culturing the bacteria in the culture medium and measuring the OD of the culture medium. ６００ The method according to claim 8 or 9, wherein the additive composition for inducing protein expression is added to the medium at a stage of 0.3 to 1.

2.

11. The medium is a low-induction medium mainly containing yeast extract, peptone, or a mixture thereof, The yeast extract, peptone, or mixture thereof The method according to any one of claims 8 to 10, wherein a transformant obtained by introducing a pET vector, in which a polynucleotide encoding luciferase set forth in SEQ ID NO: 1 is located at the 3'-end of a T7lac promoter, into an Escherichia coli BL21(DE3) strain is cultured in a predetermined medium with shaking at 37°C and 150 rpm for 12 hours, and then the yeast extract, peptone, or a mixture thereof is added and the culture is further cultured with shaking at 37°C and 150 rpm for 6 hours, wherein the expression level of luciferase is 0.1 or less as a relative value to the expression level of luciferase when 1 mM IPTG is administered instead of the yeast extract, peptone, or mixture thereof under the same conditions.

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