JP5013375B2 - Single protein production in living cells promoted by messenger RNA interference enzyme - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is directed to US Provisional Application No. 60/624976, filed Nov. 4, 2004, entitled “Single Protein in Live Cells Promoted by mRNA Interfering Enzymes” by Inouye et al. Claim priority. The entire disclosure of this application is incorporated herein by reference.

  The present invention relates to a single protein production system in living cells that is facilitated by single-stranded RNA and mRNA-interfering enzyme, a sequence specific endoribonuclease.

  Most bacteria contain a suicide gene that is expressed upon exposure to cellular stress, resulting in growth arrest and ultimately death (Elenberg-Kulka and Gerdes, Ann. Rev. Microbiol. 53: 43-70). 1999), Engelberg-Kulka et al., Trends Microbiol. 12: 66-71 (2004)). These toxin genes are usually co-expressed with a cognate antitoxin gene in the same operon (referred to as addiction module or antitoxin-toxin system). E. coli has five addiction modules (Christensen et al., J. Mol. Biol. 332: 809-19 (2003)), of which the MazE / MazF module is the most thorough. It has been investigated. The x-ray structure of the MazE / MazF complex (Kamada et al., Mol. Cell 11: 875-84 (2003)) is known, and the enzymatic activity of MazF has recently been characterized (Zhang et al., J Biol.Chem.278: 32300-306 (2003)).

MazF is a sequence-specific endoribonuclease that specifically cleaves single-stranded RNA (ssRNA) at the sequence ACA. Endonucleases are one of a large group of enzymes that cleave nucleic acids at various positions within a nucleic acid strand. An endoribonuclease or ribonuclease is specific for RNA. MazF is called an mRNA interference enzyme because its main target is messenger RNA (mRNA) in vivo. Transfer RNA (tRNA) and ribosomal RNA (rRNA) appear to be protected from cleavage, respectively, due to their secondary structure or binding to ribosomal proteins. Thus, expression of MazF causes almost complete degradation of mRNA, leading to a drastic decrease in protein synthesis and ultimately cell death (Zhang et al., Mol. Cell 12: 913-23 (2003)). . MazF has been found in selected bacteria, and recently the E. coli protein PemK (encoded by plasmid R100) has also been shown to be a sequence-specific endoribonuclease (Zhang et al., J. Biol. Chem). 279: 20678-20684 (2004)). PemK cleaves RNA with a specific nucleic acid sequence, ie UAX, with high specificity. Here, X is C, A or U. See WO 2004/018571, which is incorporated herein by reference. These sequence-specific endoribonucleases are conserved in various species, strongly indicating that they play an essential role in physiology and evolution. We refer to this family of sequence-specific endoribonuclease toxins as “mRNA interference enzymes” (Zhang et al., J. Biol. Chem. 279: 20678-20684 (2004)).
US Provisional Application No. 60/624976 Elenberg-Kulka and Gerdes, Ann. Rev. Microbiol. 53: 43-70 (1999) Engelberg-Kulka et al., Trends Microbiol. 12: 66-71 (2004) Christensen et al. Mol. Biol. 332: 809-19 (2003) Kamada et al., Mol. Cell 11: 875-84 (2003) Zhang et al. Biol. Chem. 278: 32300-306 (2003) Zhang et al., Mol. Cell 12: 913-23 (2003) Zhang et al. Biol. Chem. 279: 20678-20684 (2004) International Publication No. 2004/018571 J. et al. Sambrook and D.C. W. Russell, Molecular Cloning: A Laboratory Manual 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.A. Y. (2001) Konigsberg et al., Proc. Nat'l. Acad. Sci. U. S. A. 80: 687-91 (1983) US Pat. No. 5,656,493 US Pat. No. 5,336,675 US Pat. No. 5,234,824 US Pat. No. 5,187,083 Thieringer et al., Bioassays 20 (1): 49-57 (1998). Qing et al., Nat. Biotechnol. 22: 877-882 (2004) Tokuda and Matsuyama, Biochem. Biophys. Acta 1693: 5-13 (2004) Yamaguchi and Inouye, Cell 53: 423-432 (1988) Wolfe et al. Biol. Chem. 257: 7898-7902 (1982) Pedersen et al., Mol. Microbiol. 45: 501-10 (2002) Amitai et al. Bacteriol. 186: 8295-8300 (2004)

  In this study, we designed a single protein production (SPP) system in live E. coli cells utilizing the unique cleavage properties of MazF. Expression of a gene engineered to express ACA mRNA without changing the amino acid sequence maintained target protein synthesis at a high level for at least 96 hours in the absence of background cell protein synthesis. Thus, the toxic effects of MazF target mRNA with minimal side effects on cell physiology. Indeed, despite the state of growth arrest, these cells retain the metabolic and biosynthetic activities essential for energy metabolism (ATP production), amino acid and nucleotide biosynthesis and transcription and translation. While showing that the SPP system is effective for human and yeast proteins, this technique is also effective for overexpression of endogenous inner membrane proteins with relatively low natural expression levels. This SPP system eliminates the need for a protein purification step in experiments that require the recovery of isolated proteins, and more importantly, structural and functional studies of proteins in intact living cells. An unprecedented signal / noise ratio is obtained, which makes it possible.

  The present invention provides for the efficient and selective degradation of all cellular mRNAs in vivo by mRNA interfering enzymes, such as the single-stranded RNA- and ACA-specific endoribonuclease, the bacterial toxin MazF, in vivo to efficiently and selectively degrade all cellular mRNAs. Describes a single protein production (SPP) system in living E. coli cells that takes advantage of the unique property of causing a plunging. In one embodiment of the present invention, a system for reducing non-target cell protein synthesis and expressing a single target protein in live transformable cells comprises the following (a)-(c): (A) an isolated transformable living cell comprising cellular mRNA having at least one first mRNA interfering enzyme recognition sequence; (b) comprising an isolated nucleic acid sequence encoding an mRNA interfering enzyme polypeptide. At least one second mRNA interference enzyme such that the isolated nucleic acid sequence encoding the first expression vector, wherein the mRNA interference enzyme polypeptide yields a mutant nucleic acid sequence encoding the mutant mRNA interference enzyme polypeptide. (C) a second expression vector, optionally comprising an isolated nucleic acid sequence encoding the target protein, wherein the target protein is mutated by replacing the recognition sequence with another triplet codon sequence; The isolated nucleic acid sequence encoding Both are those mutated by replacing one third mRNA interfering enzyme recognition sequence with a different triplet codon sequence. Here, the isolated cells are transformed with the first expression vector and the second expression vector, and the isolated cells are maintained under conditions that allow expression of the mutant target protein in the cells. The

  In another embodiment, the present invention provides a method for increasing the expression of a target protein in isolated living cells, comprising the following steps (a)-(g). (A) Mutating an isolated nucleic acid sequence encoding an mRNA interfering enzyme polypeptide by substituting at least one first mRNA interfering enzyme recognition sequence with another triplet codon sequence, (B) mutating the isolated nucleic acid sequence encoding the target protein by replacing at least one second mRNA interference enzyme recognition sequence with an alternative triplet codon sequence. Generating a mutant nucleic acid sequence encoding a mutant target protein, (c) a first expression vector comprising the mutant nucleic acid sequence of step (a) and a second expression vector comprising the mutant nucleic acid sequence of step (b) Providing (d) an isolated having a cellular messenger RNA sequence comprising at least one third mRNA interfering enzyme recognition sequence. Preparing a viable transformable cell, (e) introducing the first expression vector and the second expression vector into the isolated viable cell, (f) a mutant mRNA interference enzyme Expressing the polypeptide, and (g) maintaining the isolated cell under conditions that permit expression of the mutant target protein in the cell.

The following definitions describe the parameters of the present invention.
The abbreviation “ACA” refers to the adenine-cytosine-adenine sequence.
As used herein, with respect to a particular nucleic acid, the term “encode”, “encoding” or “encoded” is conserved in the nucleic acid for translation into a particular protein. Information. A nucleic acid encoding a protein may include untranslated sequences (eg, introns) within the translated region of the nucleic acid, or may lack such intervening untranslated sequences (eg, as in cDNA). Information encoding proteins is specified by the use of codons. Typically, an amino acid sequence is encoded by a nucleic acid using a “universal” genetic code.

  The term “codon” as used herein refers to a triplet of nucleotides that identifies a single amino acid residue in a polypeptide chain. Most organisms use 20 or 21 amino acids to produce their polypeptides that are proteins or protein precursors. Since there are 4 possible nucleotides in DNA, adenine (A), guanine (G), cytosine (C) and thymine (T), 64 types are recognized to recognize only 20 amino acids and termination signals. There are possible triplets. Because of this redundancy, most amino acids are encoded by more than one triplet. Codons that specify a single amino acid are not used with equal frequency. Different organisms often show a “preferred tendency” in particular for one of several codons encoding the same given amino acid. If the coding region contains rare codons at a high level or as a cluster, expression may increase if the rare codon is removed by gene resynthesis or mutagenesis. Which is incorporated herein by reference. Sambrook and D.C. W. Russell, Molecular Cloning: A Laboratory Manual 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.A. Y. (2001), see 15.12. Thus, “codon selection” may be performed to optimize expression in the selected host. The most preferred codons are those frequently found in highly expressed genes. For “codon preference” in E. coli, see Konigsberg et al., Proc. Nat'l. Acad. Sci. U. S. A. 80: 687-91 (1983).

  Those skilled in the art will be able to make individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence, such as changing, adding or deleting one amino acid or a small percentage of amino acids in the encoded sequence. It will be understood that a change is a “conservatively modified variant” in which one amino acid is replaced with a chemically similar amino acid. The term “conservatively modified variant” applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same amino acid sequence or a conservatively modified variant. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode a given protein. For example, the UUA, UUG, CUU, CUC, CUA and CUG codons all encode the amino acid leucine. Thus, at any position where a leucine is specified by a codon, it can be changed to any of the corresponding codons described above without changing the polypeptide encoding the codon. Such nucleic acid mutations are “silent mutations” and represent a type of mutation conservatively altered. In this specification, every nucleic acid sequence encoding a polypeptide describes all possible silent variations of the nucleic acid by reference to the genetic code. One skilled in the art will understand that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and UGG, which is usually the only codon for tryptophan) can be altered to yield a functionally identical molecule. Like. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide of the present invention is within the scope of the present invention.

  The term “eotaxin” as used herein refers to 74 amino acid residues that are associated with animal and human eosinophilic inflammatory conditions belonging to the CC (or β) chemokine family. A chemotaxis factor consisting of

  The present invention includes active portions, fragments, derivatives, variants and functional variants of mRNA interference enzyme polypeptides, wherein such active portions, fragments, derivatives and functional variants retain some biological properties of mRNA interference enzymes. Anything to do. By “active portion” of an mRNA interfering enzyme polypeptide is meant a peptide that is shorter than a full-length polypeptide but retains measurable biological activity. A “fragment” of an mRNA interfering enzyme is at least 5 to 7 consecutive amino acids, often at least about 7 to 9 consecutive amino acids, typically at least about 9 to 13 consecutive amino acids, most preferably Means a range of amino acid residues of at least about 20 to 30 consecutive amino acids. A “derivative” or fragment thereof of an mRNA interfering enzyme refers to a polypeptide that has been altered by altering the amino acid sequence of the protein, eg, by manipulating the nucleic acid encoding the protein, or by altering the protein itself. To do. Such derivatives of the native amino acid sequence may include insertions, additions, deletions or substitutions of one or more amino acids and may or may not alter the intrinsic activity of the original mRNA interference enzyme. .

  The term “gene” refers to an ordered sequence of nucleotides that encodes a specific functional product (ie, a protein or mRNA molecule) located at a specific position in a segment of DNA. This may include regions before and after the coding DNA, as well as introns between exons.

  The term “induces” or “inducible” refers to a gene or gene product whose transcription or synthesis is increased by exposing the cell to an inducer or to a condition, such as heat.

  The term “inducer” or “inducer” refers to a low molecular weight compound or physical factor that binds to a repressor factor to form a complex that cannot bind to the operator.

  The term “induction” refers to an action or process that causes some specific effect by an organism after exposure to a specific stimulus, such as transcription of a specific gene or operon or production of a protein.

  In the context of inserting a nucleic acid into a cell, the terms “introduced”, “transfection”, “transformation”, “transduction” refer to the nucleic acid in the cell's genome (eg, chromosome, plasmid, plastid or mitochondrial DNA). A reference to the incorporation of nucleic acids into prokaryotic or eukaryotic cells, which can be integrated into an autonomous replicon, or can be transiently expressed (eg, transfected mRNA).

  The term “isolated” refers to a substance, such as a nucleic acid or protein, that is substantially free of components that normally accompany or interact with it when found in its naturally occurring environment. Isolated material optionally contains material not found in its natural environment. Or, if the material is in its natural environment, the material has been altered synthetically (non-naturally) by deliberate human intervention. For example, an “isolated nucleic acid” can include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or incorporated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. As applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that is well separated from other nucleic acids to which it is generally associated in its natural state (ie, in a cell or tissue). Isolated nucleic acid (either DNA or RNA) may further represent a molecule made directly by biological or synthetic means and separated from other components present at the time of production.

  The abbreviation “IPTG” refers to a synthetic inducer, isopropyl-β-D-thiogalactopyranoside, which induces β-galactosidase, an enzyme that promotes lactose utilization, by binding to and inhibiting the lac repressor. For example, IPTG is used in combination with a synthetic chromogenic substrate Xgal in a cloning strategy using a plasmid vector containing the lacZ gene to distinguish recombinants from non-recombinant bacterial colonies.

  The term “MazF” as used herein refers to the general class of endoribonucleases, specific enzymes with specific names, and structural and sequence homology consistent with the role of MazF polypeptides in the present invention. It refers to active fragments and derivatives thereof.

  The abbreviation “lspA” refers to the gene responsible for signal peptidase II activity in E. coli.

  The abbreviation “LspA” refers to the gene responsible for lipoprotein signal peptidase activity in E. coli.

  The family of enzymes encompassed by the present invention is referred to as “mRNA interfering enzymes”. The present invention is intended to extend to molecules with structural and functional similarities consistent with the role of this enzyme family in the present invention.

  As used herein, the term “nucleic acid” or “nucleic acid molecule” includes any DNA or RNA molecule, either single-stranded or double-stranded, and when single-stranded, linear or It also includes molecules of its complementary sequence in either circular form. In the discussion of nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule can be described herein in the 5 'to 3' direction according to the usual conventions for providing the sequence. Unless otherwise limited, the term includes known analogs.

  The term “oligonucleotide” refers to a nucleic acid molecule composed of two or more, preferably more than three ribo or deoxyribonucleotides linked by phosphodiester bonds.

  The term “operator” refers to the DNA (s) to which one or more regulatory protein (s) (repressor or activator) upstream (5 ′) of the gene (s) binds and regulates gene expression. An area.

  As used herein, the term “operon” refers to a genetic unit that is functionally united for the control of gene expression. It consists of one or more genes encoding one or more polypeptides and adjacent sites (promoter and operator) that control their expression by controlling transcription of the structural gene. The term “expression operon” refers to a nucleic acid segment that facilitates the expression of a polypeptide coding sequence in a host cell or organism that may have transcriptional and translational control sequences such as promoters, enhancers, translation initiation signals, polyadenylation signals, terminators, and the like. .

  The phrase “operably linked” refers to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates transcription of a DNA sequence corresponding to the second sequence and Mediate. In general, “operably linked” means that the nucleic acid sequences to be linked are contiguous, and if necessary to join two protein coding regions, they are contiguous and in the same reading frame. Means that

  The abbreviation “ORF” represents an “open reading frame” that is a portion of a sequence of a gene containing a sequence of bases capable of encoding a peptide or protein that is not interrupted by an internal stop sequence. An open reading frame begins with a start codon and ends with a stop codon. A stop or stop codon determines the end of the polypeptide.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. This term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues have been changed from a natural amino acid to its artificial chemical analog.

  The abbreviation “PCR” refers to the polymerase chain reaction, which is a technique that amplifies the amount of DNA to make it easier to isolate, clone and sequence DNA. See, for example, US Pat. Nos. 5,656,493, 5,333,675, 5,234,824, and 5,187,083. These are incorporated herein.

  As used herein, the term “promoter” is the region of DNA upstream (5 ′) of transcription initiation that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Point. The term “inducible promoter” refers to the activation of a promoter in response to either the presence of a specific compound, ie, an inducer or inducer, or a specific external condition (eg, elevated temperature).

  The phrase “site-directed mutagenesis” refers to an in vitro technique using recombinant DNA methods whereby a base change, ie mutation, is introduced into a piece of DNA at a specific site.

  The term “untranslated region” or UTR, as used herein, refers to the portion of DNA whose base is not involved in protein synthesis.

  The terms "variant", "variant" and "derivative" of a particular sequence of nucleic acid refer to a nucleic acid sequence that is closely related to the particular sequence, but that has changes in sequence or structure, either naturally or by design. . “Closely related” means that at least 60%, often more than 85% of the nucleotides of a sequence match over a defined length of a nucleic acid sequence. Nucleotide sequence changes or differences between closely related nucleic acid sequences may represent nucleotide changes in the sequence that a particular natural nucleic acid sequence occurs during normal replication or duplication processes. Other changes can also be specifically designed and introduced into the sequence for specific purposes. Such specific changes can be made in vitro using various mutagenesis techniques. Such specifically generated sequence variants may be referred to as “variants” or “derivatives” of the original sequence.

  Those skilled in the art can similarly produce protein variants with single or multiple amino acid substitutions, deletions, additions or substitutions. These variants can include, among others: (A) a variant in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) a variant in which one or more amino acids are added, (c) at least one amino acid is a substituent. (D) a variant in which an amino acid residue from one species is replaced with a corresponding residue from another species in either a conservative or non-conservative position, and (d) a target protein, A variant fused to another peptide or polypeptide, such as a fusion partner, a protein tag or other chemical moiety, etc., which may confer useful properties on the target protein, eg, an epitope for an antibody. Techniques for obtaining such variants include genetic (repression, deletion, mutation, etc.), chemical and enzymatic techniques and are known to those skilled in the art.

  As used herein, the terms “vector” and “expression vector” refer to a replicon, ie, any factor that acts as a carrier or transporter, such as a phage, plasmid, cosmid, bacmid, phage or virus. When combined with another gene sequence or element (either DNA or RNA), the sequence or element can be replicated and transferred to the host cell. The E. coli SPP system described herein utilizes a pColdI vector that induces protein production at low temperatures.

  It should be noted that as used herein and in the appended claims, a noun includes a plurality of instructions unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned in this specification are herein incorporated by reference and disclose and describe the methods and / or materials from which the publications are cited.

〔Example〕
The following examples are submitted to provide those skilled in the art with a complete disclosure and description of how to make and use the invention and are intended to limit the scope of what the inventors regard as their invention. It is not intended, nor is it intended to represent that the following experiment is all or the only experiment performed. While accurate with respect to numbers used (eg, amounts, temperatures, etc.), some experimental error and bias should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Strains and Plasmids In the following experiments, E. coli BL21 (DE3) cells were used. The mazF gene was cloned into the NdeI-XhoI site of pACYCDuet (Novagen) to create plasmid pACYCmazF. pACYCmazF (-9ACA) was constructed by site-directed mutagenesis using pACYCmazF as a template. The eotaxin gene was synthesized based on the optimal E. coli codon usage frequency (see FIG. 2A , SEQ ID NO: 1 ), cloned into the NdeI-HindIII site of pColdI (SP-1), and plasmid pColdI (SP-1) eotaxin. Was made. pColdI (SP-1) eotaxin was constructed as described in the text by site-directed mutagenesis using pColdI (eotaxin) as a template. Mutagenesis was performed using Pfu DNA polymerase (Stratagene) according to the instructions for the QuickChange site-directed mutagenesis kit (Stratagene). pColdI (SP-2) eotaxin was also constructed by site-directed mutagenesis using pColdI (SP-1) eotaxin as a template. pColdI (SP-1) eotaxin (+ ACA) was constructed by site-directed mutagenesis using pColdI (SP-1) eotaxin as a template. The wild type Hsp10 gene was amplified by PCR using the yeast chromosome as a template and cloned into the NdeI-BamHI site of pColdI (SP-2) to produce plasmid pColdI (SP-2) Hsp10. The ACAHsp10 gene was amplified by two-step PCR using a yeast chromosome as a template, and cloned into the NdeI-BamHI site of pColdI (SP-2) to produce plasmid pColdI (SP-2) Hsp10 (-ACA). The wild type and no ACARpb12 gene was amplified by PCR using wild type Rpb12 plasmid as template and 5 'and 3' oligonucleotides containing the altered sequence and cloned into the NdeI-BamHI site of pColdI (SP-2) Plasmids pColdI (SP-2) Rpb12 and pColdI (SP-2) Rpb12 (-ACA) were prepared respectively. The ACALspA gene was amplified by two-step PCR and cloned into the NdeI-BamHI site of pColdI (SP-2) to produce plasmid pColdIV (SP-2) lspA (-ACA).

E. coli BL21 (DE3) carrying these plasmids was grown in M9-glucose medium. When the OD ₆₀₀ of the culture reached 0.5, the culture was transferred to 15 ° C. for 45 minutes and 1 mM IPTG was added to the culture. At the indicated time interval, 1 ml of culture was added to a test tube containing 10 mCi [ ³⁵ S] -methionine. After 15 minutes of incubation (pulse), 0.2 ml of 40 mg / ml methionine was added and incubated for an additional 5 minutes (chase). Labeled cells were washed with M9-glucose medium and resuspended in 100 μl SDS-PAGE loading buffer. 10 μl of each sample was analyzed by SDS-PAGE followed by autoradiography.

Preparation of membrane fraction Cells collected from 1 ml culture by centrifugation (10,000 × g for 5 minutes) were suspended in 10 mM Tris-HCl (pH 7.5) and disrupted by ultrasound. After removing unbroken cells, the total membrane fraction was obtained by centrifugation (60,000 × g for 60 minutes).

Effect of MazF induction of cellular protein synthesis E. coli BL21 (DE3) cells carrying pACYCmazF were transformed into pColdI (SP-1) eotaxin (1A and 1B left panel) or pColdI (SP-2) eotaxin (1B right panel). And 1C). Cells were grown at 37 ° C. in M9 medium. The OD ₆₀₀ is 0.5 and the culture is transferred to 15 ° C. and incubated at 15 ° C. for 45 minutes to acclimate the cells to low temperature, then IPTG (1 mM) is added to reduce both eotaxin and MazF expression. Induced (time 0). At the time indicated at the top of each gel, cells were pulse labeled with ³⁵ S-methionine for 15 minutes and total cellular proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.

  pACYCmazF was created by cloning the mazF gene into pACYC, a low copy number plasmid containing the IPTG inducible phage T7 promoter. Cloning techniques are generally described in J. Org. Sambrook and D.C. W. Russell, Molecular Cloning: A Laboratory Manual 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.A. Y. (2001). Since no colonies were formed on agar plates containing IPTG, E. coli BL21 (DE3) transformed with pACYCmazF was sensitive to IPTG, a lac inducer (not shown).

FIG. 1 shows the expression of human eotaxin using pColdI (SP-1) and pColdI (SP-2) with or without MazF co-expression by SDS-PAGE. FIG. 1B shows the results of cells transformed with pColdI (SP-1) eotaxin (left panel) and cells transformed with pColdI (SP-2) eotaxin (right panel). FIG. 1C shows the results of cells transformed with pACYCmazF and pColdI (SP-2) eotaxin and incubated in LB (left panel) or M9 medium (right panel). Cells were treated as in FIGS. 1A and 1B and at the indicated time points, total cellular proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. Note that the same volume of culture was taken for analysis. The position of the molecular weight marker is indicated on the left side of the gel, and the position of eotaxin is indicated by an arrow. Since MazF effectively cleaves mRNA at the ACA sequence, cellular protein synthesis is shown upon induction of MazF at 37 ° C. (Zhang et al., Mol. Cell 12: 913-23 (2003)) or in FIG. 1A. As shown, it was dramatically inhibited at 15 ° C. In this cold shock experiment, cells carrying pACYCmazF were first incubated at 15 ° C. for 45 minutes to induce the cold shock protein required for cold shock acclimation (Thieringer et al., Bioassays 20 (1): 49-57 (1998)). IPTG was then added to the culture to induce MazF (FIG. 1A, time 0 in the left panel). At the time indicated at the top of the gel, the cells were pulse labeled with [ ³⁵ S] methionine for 15 minutes. Panel A left panel shows the results for cells transformed with pACYC eotaxin alone. Panel A center panel shows results for cells transformed with pColdI (SP-1) eotaxin alone. Panel A right panel shows the results for cells transformed with both plasmids.

  At time 0, a pattern very similar to that of cells without IPTG (shown as control, C) was observed, but cellular protein synthesis was dramatically inhibited 1 hour after the addition of IPTG. After 6 hours, almost all cellular protein synthesis was almost completely blocked.

Expression of ACA-free mRNA in MazF-induced cells We have found that when mRNA that has been engineered to contain no ACA sequences is expressed in MazF-induced cells, the mRNA is stably maintained in the cell and any other It was speculated that the protein encoded by the mRNA could be produced without producing cellular proteins. To test this possibility, we synthesized a gene for human eotaxin, excluding all ACA sequences in the gene without changing the amino acid sequence. FIG. 2A shows the amino acid sequence of human eotaxin and the nucleotide sequence of the gene. The nucleotide sequence was designed with the preferred E. coli codon and the underlined triplet was changed to ACA in the following experiment. The ACA sequence can be changed to other MazF non-cleavable sequences without changing the amino acid sequence of the protein, regardless of the position of the ACA sequence in the reading frame. This is unique among the 64 possible triplet sequences.

The eotaxin gene shown in FIG. 2A (SEQ ID NO: 1) is a sequence from the translation-promoting element of the major cold shock protein CspA cspA gene (Qing et al., Nat. Biotechnol. 22: 877-882 (2004)). ), A His-Met sequence consisting of 6 His residues, a factor Xa cleavage site, and an NdeI site for gene insertion. The entire coding region of the fusion protein is transformed into the cold shock vectors pColdI (SP-1) and pColdI (SP-2) vectors (Qing et al., Nat. Biotechnol. 22: 877, which allow high protein expression during cold shock. 882 pages (2004)). In pColdI (SP-1), two ACA sequences, one between the Shine-Dalgarno sequence and the start codon, and one in the translation enhancing element were converted to AUA. In pColdI (SP-2), in addition to the two ACA sequences in pColdI (SP-1), three other ACA sequences in the 5′-untranslated region (5′-UTR) also have base substitutions. Was changed to a MazF non-cleavable sequence (in order from 5′ACA to 3′ACA, GCA, AUA and GCA). E. coli BL21 (DE3) cells were transformed with the resulting constructs, pColdI (SP-1) eotaxin and pColdI (SP-2) eotaxin, respectively.

Cells transformed with pColdI (SP-1) eotaxin were subjected to a cold shock at 15 ° C. and acclimated to a low temperature for 1 hour, and then IPTG was added to induce eotaxin production. The cells were then pulse labeled with [ ³⁵ S] methionine for 15 minutes (time 0, FIG. 1A, middle panel). During the incubation from time 0 to 72 hours, eotaxin was produced at an almost constant level along with other cellular proteins. Production of eotaxin at 12 hours was approximately 11% of total cellular protein synthesis, as judged from [ ³⁵ S] methionine uptake.

  When both eotaxin and mazF genes were co-expressed with E. coli BL21 (DE3) containing both pACYCmazF and pColdI (SP-1) eotaxin, background cellular protein synthesis was dramatically reduced after 3 hours induction However, eotaxin production persisted at an almost constant level for 72 hours (FIG. 1A, right panel). Interestingly, the level of eotaxin production in this experiment was higher than that without MazF induction (FIG. 1A, middle panel, 47% at 12 hours) (FIG. 1A, right panel, 11% of total protein production at 12 hours). %). This approximately 5-fold enrichment is likely due to the availability of more ribosomes for eotaxin mRNA translation as cellular mRNA is degraded by MazF. Of note, no intrinsic protein band was observed after 12 hours.

When the same experiment was performed using cells containing both pACYCmazF and pColdI (SP-2) eotaxin, almost only eotaxin was produced (FIG. 1B, right panel). Of note, eotaxin production was substantially higher than that using pColdI (SP-1) eotaxin (FIG. 1B, left panel). This higher production of eotaxin appears to be due to stabilization of eotaxin mRNA by removing the 5'-UTR ACA sequence in pColdI (SP-1). Approximately 90% of [ ³⁵ S] methionine is incorporated into eotaxin 12 hours after MazF induction, notably no clear cellular protein band is observed (FIG. 1B, right panel) and the SPP system Show a dramatic improvement in the signal-to-noise ratio of eotaxin. It is noteworthy that high levels of eotaxin production did not decrease after 96 hours of induction. Furthermore, background cellular protein synthesis decreased faster (in 3 hours) than with pColdI (SP-1) eotaxin (in 6 hours) (compare the left panel in FIG. 1B with the right panel).

With both vectors (FIGS. 1A and 1B), cell proliferation was completely blocked upon MazF induction as judged by OD ₆₀₀ and also [ ³⁵ S] methionine incorporation into cellular proteins. These results show that the cells arrested for growth by MazF induction are not physiologically killed, but rather have the full capacity to synthesize proteins if their mRNA does not have an ACA sequence. In other words, the cell integrity of E. coli BL21 (DE3) cells has been maintained intact for a long time, and not only energy metabolism but also amino acid and nucleotide biosynthesis functions are completely completed in cells whose growth has been stopped. Is active. In addition, transcription and translation mechanisms, including RNA polymerase, ribosomes, tRNA, and all other factors required for protein synthesis are also well maintained.

Production of eotaxin using pColdI (SP-2) eotaxin appears as a major band by Coomassie blue staining after SDS polyacrylamide gel electrophoresis (FIG. 1C). At 0 hours, almost no eotaxin band is observed, but at 12 hours it becomes a major band and its concentration increases after 24 hours. However, even longer incubations did not significantly promote their production levels, suggesting that there is a threshold level of eotaxin production in MazF induced cells. Since [ ³⁵ S] methionine uptake remained constant over 96 hours (FIG. 1B), eotaxin production and degradation in the SPP system appears to reach equilibrium after 24 hours. It is important to note that the concentration of cellular protein bands remained constant as expected from complete growth inhibition upon MazF induction. The present inventors examined whether eotaxin production is affected by a rich medium such as LB medium, but when pColdI (SP-2) was used, eotaxin production was limited by the use of LB medium. There was no enhancement from the level obtained with M9 medium.

Negative Effect of ACA Sequence on Protein Production To confirm that the exclusive eotaxin production in MazF-induced cells observed in FIG. 1 is due to eotaxin mRNA without ACA, as shown in FIG. Five original ACA sequences were added without changing the amino acid sequence. The eotaxin gene was expressed by use of pColdI (SP-2), cells were treated as described in FIG. 1 and labeled with [ ³⁵ S] -methionine. The left panel shows the results for the ACA-free eotaxin gene (same as the left panel in FIG. 1B) and the right panel shows the results for the eotaxin gene with 5 ACA sequences.

  When this gene was expressed by using both pColdI (SP-1) and pACYCmazF under the same conditions as described in FIG. 1, only low levels of eotaxin production were observed for the first 2 hours. At a later time point, production was further reduced to background levels (FIG. 2B, right panel) compared to expression with ACA-free mRNA (FIG. 2B, left panel).

  Curiously, the mazF gene encodes an mRNA with an unusually high ACA content (9 ACA sequences in a 111 residue protein) (only 2 ACA sequences in 82 amino acid residues in MazE) and mazF expression in cells Is negatively regulated. Therefore, we constructed a non-ACAmazF gene [pACYCmazF (-9ACA)] and tested whether removing these ACA sequences from the mazF coding region could more effectively reduce background cell protein production. did.

  FIG. 3 shows the effect on eotaxin expression when all ACA sequences in the mazF ORF are removed. Panel A shows the amino acid sequence of MazF and the nucleotide sequence of its ORF. The underlined triplet sequence (9 total) was originally ACA in the wild type mazF gene and changed to a MazF non-cleavable sequence. Panel B shows the expression of eotaxin by pColdI (SP-2) eotaxin using the wild-type mazF gene (left panel) and the non-ACAmazF gene (right panel). The experiment was performed as described in FIG.

As shown in FIG. 3A (SEQ ID NO: 2) , none of the base substitutions changes the amino acid sequence of MazF. Cells containing pYCACmazF (-9ACA) grew slightly slower in M9 media than cells containing pYCACmazF, but background protein synthesis was further reduced without significantly affecting eotaxin production (FIG. 3B). . These results clearly show that the ACA sequence in the mRNA plays an important role in protein production in MazF-induced cells.

Application of the SPP system to yeast proteins We applied the SPP system to two yeast proteins, heat shock factor Hsp10 and RNA polymerase subunit Rpb12. The Hsp10 and Rpb12 ORFs contained three and one ACA, respectively, which were converted to a MazF non-cleavable sequence without changing their amino acid sequence (FIG. 4A). These and wild type sequences were each inserted into pColdI (SP-2). The obtained plasmids were respectively pColdI (SP-2) Hsp10 for wild type Hsp10, pColdI (SP-2) Hsp10 (-1ACA) for mutant Hsp10, and pColdI (SP-2) for wild type Rpb12. ) Rpb12, and pColdI (SP-2) Rpb12 (-3ACA). These plasmids were individually transformed into E. coli BL21 (DE3) containing pACYCmazF. The protein expression pattern was then examined at 15 ° C. for 48 hours.

  The expression of yeast proteins in the SPP system is shown in FIG. Using pColdI (SP-2), yeast Hsp10 and Rpb12 were expressed in the SPP system with and without ACA sequences in their genes. The experiment was performed as described above with respect to FIG. FIG. 4A shows the expression of Hsp10 using wild-type and no ACAHsp10 genes. The hsp10 ORF consisting of 106 codons contains three ACA sequences (A25-Q26 GCA-CAA, T29 ACA and P76-Q77 CCA-CAG), which are in GCC-CAA, ACC and CCC-CAG, respectively. Converted (changed bases are shown in bold). These base substitutions do not change the amino acid sequence of Hsp10. FIG. 4B shows the expression of Rpb12 using wild type and no ACA genes. The rpb12 ORF consisting of 70 codons contained one ACA at T10, which was converted to an ACC for threonine.

FIG. 4A shows that even with the three natural ACA sequences (WT), Hsp10 is expressed at reasonable levels. However, removal of all ACA sequences significantly increased Hsp10 synthesis several fold. It is noteworthy that the background was also significantly reduced in the case of ACAHsp10, probably because more ribosomes were exclusively used for the production of Hsp10. In FIG. 4B, almost no ³⁵ S-methionine uptake was observed in the wild type panel, and some uptake was seen in the non-ACARpb12. Thus, although Rpb12 contains only one ACA, it shows that this causes a devastating effect on production in the SPP system. These results suggest that mRNA sensitivity to MazF can be governed not only by the number of ACA sequences in the mRNA, but also by the effective sensitivity of the ACA sequences to MazF. Since ribosomes are thought to protect mRNA from its cleavage by MazF, ACA sequence sensitivity appears to be determined by its location in the single stranded region of the mRNA as well as by efficient translation of the mRNA by the ribosome.

Application of SPP system to integral membrane proteins We have attempted to apply the SPP system to trace integral membrane proteins. We selected the signal peptidase II gene lspA in E. coli specifically required for cleavage of the lipoprotein signal peptide (Tokuda and Matsuyama, Biochem. Biophys. Acta 1693: 5-13). (2004)). E. coli contains a total of 96 lipoproteins, either in the inner or outer membrane, depending on the nature of the second amino acid residue of the mature lipoprotein (whether it is acidic or neutral). (Yamaguchi and Inouye, Cell 53: 423-432 (1988), Tokuda and Matsuyama, Biochem. Biophys. Acta 1693: 5-13 (2004)). The signal peptide of all other secreted proteins is cleaved by signal peptidase I (leader peptidase), which is presumed to be present at a level of 500 molecules per cell in E. coli (Wolfe et al., J. Biol. Biol.Chem.257: 7898-7902 (1982)).

  Lipoprotein signal peptidase (LspA) is also thought to be a very small amount of protein in the inner membrane. It consists of 164 amino acid residues and contains 4 putative transmembrane domains, indicating that LspA is an integral inner membrane protein. The three ACA sequences in the IspA ORF were changed to non-MazF cleavable sequences without changing their amino acid sequence, and in the SPP system using mazF (-9ACA), no ACALspA was used using pColdI (SP-2) Was expressed.

  The expression of the inner membrane protein LspA in the SPP system using pColdL (SP-2) is shown in FIG. LspA (signal peptidase II, lipoprotein signal peptidase) was expressed in the SPP system as described in FIG. Panel A shows total cellular protein. Panel B shows the membrane fraction. The position of LspA is indicated by an arrow.

As shown in FIG. 5A, the expression of LspA in the SPP system is apparently toxic to cells since ³⁵ S-methionine uptake lasted only 1 hour after IPTG induction. However, as shown in FIG. 5B, the LspA band concentration at 0 and 1 hour was the highest compared to other cellular protein bands (compare C lane in FIG. 5A), so Of ³⁵ S-methionine appears to be incorporated into LspA. Background cellular protein synthesis observed at 0 and 1 hour was easily removed by ultracentrifugation and ³⁵ S-methionine incorporation was highly enriched in the membrane fraction.

DISCUSSION This study shows that complete inhibition of cellular protein synthesis by mRNA interfering enzymes has no effect on cell physiology degradation. As a result of almost all cellular mRNA being cleaved at the ACA sequence by MazF, cellular protein synthesis is completely blocked, which in turn leads to complete cell growth arrest. Surprisingly, however, cells that have been arrested by MazF induction undergo high levels of protein synthesis for extended periods of time (at least 96 hours at 15 ° C.) if the mRNA has been modified to have no ACA sequences. I found that I have the ability. In this way, the inventors have succeeded for the first time in establishing single protein production (SPP) in vivo.

  Our results demonstrate that MazF-induced cells are not dead. Inducing MazF maintains energy metabolism that produces sufficient ATP required for various cellular functions, including RNA and protein synthesis, and does not hinder cell integrity. In addition, biosynthesis of amino acids and nucleotides is also maintained intact. In the absence of new cellular protein synthesis, all protein factors required for these cellular functions (eg, protein factors required for protein synthesis) and cell metabolism remain stable at 15 ° C for at least 96 hours. It's amazing how it was found. It has not yet been determined how long these cell functions can be retained without affecting SPP capacity. Although they appear to be dormant at first glance, they are completely capable of RNA and protein synthesis and are clearly different from dormancy caused by quiescence due to nutritional deficiencies. We propose to call the physiological state caused by MazF induction a “pseudo-pause” state. It has not yet been determined whether pseudo-resting cells are dead or not. Bacterial viability is often determined by the ability of the cells to colonize after various treatments. The viability of E. coli cells after MazF induction was examined in this way, and it was shown that when MazE is induced, growth resumes during the limited time incubation after MazF induction ( Pedersen et al., Mol. Microbiol. 45: 501-10 (2002), Amitai et al., J. Bacteriol. 186: 8295-8300 (2004)). Thus, while the effect of MazF is reversible to some extent, it is claimed that there is a “return limit” that causes all cells to die (Amitai et al., J. Bacteriol. 186: 8295-8300 (2004). )). Importantly, the MazE gene used by both groups contains two ACA sequences in its ORF. This result clearly shows that for any gene to be expressed in MazF-induced cells, the ACA sequence in these genes must be converted to a MazF non-cleavable sequence. Therefore, pseudo-resting cells expressing MazF are likely not to express MazE unless all ACA sequences are excluded from the ORF of MazE.

The ability to produce only a single protein of interest in living or non-dead cells provides a novel approach to study various aspects of proteins in living cells that could not be achieved previously. Since the protein of interest can be exclusively labeled with isotopes ( ¹⁵ N and ¹³ C) in living cells using the SPP system, it may be possible to examine the NMR structure of the protein in living cells. Recently, the inventors have shown that expression of a protein of interest with a high expression cold shock vector pCold can determine the NMR structure of the protein using cell lysates without protein purification (Qing et al., Nat. Biotechnol). 22: 877-882 (2004)). We now show that when MazF and pCold vector are used together, the signal-to-noise ratio is dramatically reduced since background cell protein synthesis is almost completely blocked by MazF induction. In these experiments, we have shown that removal of the ACA sequence from the pColdI vector itself is also very important, thereby improving eotaxin production by a factor of 5. When combined with MazF, the rate of eotaxin synthesis was at a level of 90% of total cellular protein synthesis, as judged by ³⁵ S-methionine incorporation. The remaining 10% constituted a general background without being incorporated into any particular protein band. This in turn makes it possible to conduct structural studies of very small amounts of proteins that are toxic when expressed in large quantities and thus have limited production. The inventors have now demonstrated that LspA, indeed a very small amount of inner membrane protein, can be expressed exclusively in the membrane fraction. Since some proteins can only fold in living cells, their structural studies can only be accomplished through the use of the SPP system.

  Another unique advantage of the SPP system is that cell growth is completely blocked upon MazF induction, so that the protein of interest can be produced or labeled with isotopes in highly concentrated cultures. . The SPP system can be used to produce not only proteins but also other non-protein compounds. Furthermore, the SPP system is not limited to bacteria, and MazF and other mRNA interference enzymes can be applied to eukaryotic cells to create SPP systems in yeast and mammalian cells.

  Where values in a range are provided herein, between the upper and lower limits of the range, up to one-tenth of the lower limit unit, unless otherwise specified by context. It is understood that each of the values in and any other stated or in between values in the stated range are encompassed by the invention. Subject to any specifically excluded limits of the stated ranges, upper and lower limits of smaller ranges that can be independently included in these smaller ranges are also encompassed by the present invention. Where the stated range includes one or both of the limits, ranges excluding both those included limits are also included in the invention.

  The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the date of issue provided may be different from the date of actual issue and may need to be individually verified.

  Although the invention has been described with reference to particular embodiments, it will be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. It should be. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method or steps, to the intended spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto.

FIG. 2 shows the expression of human eotaxin using pColdI (SP-1) and pColdI (SP-2) with or without MazF co-expression. FIG. 2 shows the expression of human eotaxin using pColdI (SP-1) and pColdI (SP-2) with or without MazF co-expression. FIG. 2 shows the expression of human eotaxin using pColdI (SP-1) and pColdI (SP-2) with or without MazF co-expression. FIG. 5 shows the effect of ACA sequence on eotaxin expression. FIG. 5 shows the effect of ACA sequence on eotaxin expression. FIG. 5 shows the effect of removal of all ACA sequences in the MazF ORF on eotaxin expression. FIG. 5 shows the effect of removal of all ACA sequences in the MazF ORF on eotaxin expression. It is a figure which shows the expression of the yeast protein in a SPP system. It is a figure which shows the expression of LspA which is an inner membrane protein in the SPP system using pColdIV (SP-2).

Claims (32)

A system for expressing a single target protein while reducing non-target cellular protein synthesis in a transformable prokaryotic living cell, comprising:
(A) an isolated transformable prokaryotic living cell;
(B) an isolated nucleic acid sequence encoding an mRNA interference enzyme polypeptide that recognizes an mRNA interference enzyme recognition sequence and specifically cleaves a single-stranded mRNA at the mRNA interference enzyme recognition sequence, and the isolated nucleic acid A first expression vector comprising an inducible promoter operably linked to a sequence, wherein said mRNA when said isolated nucleic acid sequence encoding an mRNA interfering enzyme polypeptide comprises said mRNA interfering enzyme recognition sequence nucleic acid sequence is released the single encoding interference enzyme polypeptide by replacing sequences of alternative at least one mRNA interfering enzyme recognition sequences were mutated without changing the amino acid sequence encoded, the mRNA It is mutated nucleic acid sequence encoding the interference enzyme polypeptide;
(C) a second expression vector comprising an isolated nucleic acid sequence encoding the target protein , wherein when the isolated nucleic acid sequence encoding the target protein comprises the mRNA interference enzyme recognition sequence, the target protein The isolated nucleic acid sequence encoding is a mutated nucleic acid sequence encoding the target protein that has been mutated without altering the encoded amino acid sequence by replacing the mRNA interference enzyme recognition sequence with an alternative sequence. Is ;
Comprise becomes viable cells isolated transformable prokaryotic (a) is transformed with the first expression vector and the second expression vector, transformation resulting cells, a system characterized in that it is maintained under conditions that allow expression of the target protein in said transformed cells. The system of claim 1 , wherein the second expression vector comprises at least one inducible promoter operably linked to a mutated nucleic acid sequence encoding the target protein. The system according to claim 1 , wherein the mRNA interference enzyme recognition sequence is adenine-cytosine-adenine. Variant nucleic acid sequences encoding the target protein, rare codon a double mutant nucleic acid sequences have been further mutated to be substituted is preferred codons, said double mutant nucleic acid sequence, amino acid sequence identical to the amino acid sequence of the target protein The system of claim 1, which encodes a protein having 5. The system of claim 4 , wherein the inducible promoter is operably linked to a double mutant nucleic acid sequence. variant nucleic acid sequences encoding mRNA interference enzyme polypeptide is a double mutant nucleic acid sequences have been further mutated to be substituted on rare codons are preferred codons, said double mutant nucleic acid sequence, the amino acid of the mRNA interfering enzyme polypeptide 2. The system of claim 1 which encodes an mRNA interference enzyme polypeptide having an amino acid sequence identical to the sequence. The system of claim 1 , wherein the transformable prokaryotic live cell is an E. coli cell. The system of claim 1, wherein the mRNA interference enzyme polypeptide is MazF. The system of claim 1, wherein the target protein is a mammalian protein. The system of claim 9 , wherein the mammalian protein is a human protein. The system of claim 1, wherein the target protein is a yeast protein. The system of claim 1, wherein the target protein is a trace bacterial protein. 13. The system according to claim 12 , wherein the target protein is a toxic minor protein. The system of claim 1, wherein the transformed cells are maintained in a medium comprising at least one radiolabeled isotope. 15. The system of claim 14 , wherein the target protein is radioisotopically labeled when expressed. An isolated nucleic acid sequence encoding the target protein is one that is amplifiable by polymerase chain reaction system of claim 1. (A) An isolated nucleic acid sequence encoding an mRNA interference enzyme polypeptide that recognizes an mRNA interference enzyme recognition sequence and specifically cleaves single-stranded mRNA with the mRNA interference enzyme recognition sequence is the mRNA interference enzyme recognition sequence. The nucleic acid sequence is mutated to replace at least one mRNA interfering enzyme recognition sequence with an alternative sequence without altering the encoded amino acid sequence to encode the mRNA interfering enzyme polypeptide. Producing a nucleic acid sequence;
(B) When the isolated nucleic acid sequence encoding the target protein contains the mRNA interference enzyme recognition sequence, the isolated nucleic acid sequence encoding the target protein is mutated to change the encoded amino acid sequence. Without replacing the mRNA interfering enzyme recognition sequence with an alternative sequence, resulting in a mutated nucleic acid sequence encoding the target protein;
Step (c) a first expression vector comprising at least one inducible promoter is operably linked to a nucleic acid sequence or variant nucleic acid sequence and nucleic acid sequence or variant nucleic acid sequences of (a), and step (b) Providing a second expression vector comprising a nucleic acid sequence of
(D) providing an isolated transformable prokaryotic living cell,
(E) introducing the first expression vector and the second expression vector into the isolated live transformable prokaryotic cell and co-transfecting;
(F) Inducing an inducible promoter contained in the first vector with an inducing factor that expresses the mRNA interference polypeptide, and transforming the mRNA interference enzyme in the transformed cell obtained in step (e) Expressing the prokaryotic organism, comprising: expressing the peptide; and (g) maintaining the transformed cell of step (f) under conditions that allow expression of the target protein in the cell. A method of expressing a target protein in a cell or increasing the expression of a target protein. The second expression vector comprises further at least one inducible promoter is operably linked to a nucleic acid sequence or variant nucleic acid sequence encoding a target protein, method of claim 17. 19. The method of claim 18 , further comprising inducing an inducible promoter operably linked to a nucleic acid sequence encoding a target protein or a mutated nucleic acid sequence with an inducer to express the target protein. Step (a),
The variant nucleic acid sequences encoding mRNA interference enzyme polypeptide by further mutated and replaced with preferred codons rare codons, encoding mRNA interference enzyme having the amino acid sequence identical to the amino acid sequence of the mRNA interfering enzymatic polypeptides double mutant 18. The method of claim 17 , further comprising the step of generating a nucleic acid sequence. Step (b),
Further mutated variant nucleic acid sequences encoding the target protein was replaced with preferred codons rare codons, further comprising the step of producing double mutant nucleic acid sequence encoding a protein having an amino acid sequence identical to the amino acid sequence of the target protein The method of claim 17 . Step (a),
The variant nucleic acid sequences encoding mRNA interference enzyme polypeptide by further mutated and replaced with preferred codons rare codons, encoding mRNA interference enzyme having the amino acid sequence identical to the amino acid sequence of the mRNA interfering enzymatic polypeptides double mutant Further comprising generating a nucleic acid sequence;
Step (b),
Further mutated variant nucleic acid sequences encoding the target protein was replaced with preferred codons rare codons, further comprising the step of producing double mutant nucleic acid sequence encoding a protein having an amino acid sequence identical to the amino acid sequence of the target protein ,
The method of claim 17 . The method according to claim 17 , wherein the mRNA interference enzyme recognition sequence is adenine-cytosine-adenine. 18. The method of claim 17 , wherein the transformable live prokaryotic cell is an E. coli cell. 18. The method of claim 17 , wherein the mRNA interference enzyme polypeptide is MazF. The method of claim 17 , wherein the target protein is a mammalian protein. 27. The method of claim 26 , wherein the target protein is a human protein. The method of claim 17 , wherein the target protein is a yeast protein. The method of claim 17 , wherein the target protein is a trace bacterial protein. 30. The method of claim 29 , wherein the target protein is a toxic minor protein. 18. The method of claim 17 , further comprising the step of incubating the transformed cells in a medium comprising at least one radiolabeled isotope during step (g). 18. The method of claim 17 , further comprising amplifying the isolated nucleic acid sequence encoding the target protein in step (b) by polymerase chain reaction.

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