CA2577180A1 - Single protein production in living cells facilitated by a messenger rna interferase - Google Patents

Abstract

The present invention describes a single-protein production (SPP) system in living E. coli cells that exploits the unique properties of an mRNA
interferase, for example, MazF, a bacterial toxin that is a single stranded RNA- and ACA-specific endoribonuclease, which efficiently and selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis. Concomitant expression of MazF and a target gene engineered to encode an ACA-less mRNA results in sustained and high-level (up to 90%) target expression in the virtual absence of background cellular protein synthesis. Remarkably, target synthesis continues for at least 4 days, indicating that cells retain transcriptional and translational competence despite their growth arrest. SPP technology works well for yeast and human proteins, even a bacterial integral membrane protein. This novel system enables unparalleled signal to noise ratios that should dramatically simplify structural and functional studies of previously intractable but biologically important proteins.

Description

DEMANDE OU BREVET VOLUMINEUX

LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:

NOTE POUR LE TOME / VOLUME NOTE:

SINGLE PROTEIN PRODUCTION IN LIVING CELLS FACILITATED BY A
MESSENGER RNA INTERFERASE

CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No.
60/624,976, entitled "Single Protein in Living Cells Facilitated by an mRNA Interferase"
by Inouye et al., filed on November 4, 2004. The entire disclosure of this application is incorporated herein by reference.

FIELD OF THE INVENTION
[0002] The present invention relates to a system for producing a single-protein in living cells facilitated by an mRNA interferase that is a single-stranded RNA- and sequence-specific endoribonuclease.

BACKGROUND OF THE INVENTION

[0003] Most bacteria contain suicidal genes whose expression leads to growth arrest and eventual death upon exposure to cellular stress (reviewed by Elenberg-Kulka and Gerdes, Ann. Rev. Microbiol. 53: 43-70 (1999); Engelberg-Kullca et al., Trends Microbiol. 12: 66-71 (2004)). These toxin genes are usually co-expressed with their cognate antitoxin genes in the same operon (referred to as an addiction module or antitoxin-toxin system). E.
coli has five addiction modules (Christensen et al., J. Mol. Biol. 332: 809-19 (2003)) among which the MazE/MazF module has been most extensively 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 been recently characterized (Zhang et al, J.
Biol. Chem. 278:
32300-306 (2003)).

[0004] MazF is a sequence-specific endoribonuclease that specifically cleaves single-stranded RNAs (ssRNAs) at ACA sequences. An endonuclease is one of a large group of enzymes that cleave nucleic acids at positions within a nucleic acid chain.
Endoribonucleases or ribonucleases are specific for RNA. MazF is referred to as an mRNA
interferase since its primary target is messenger RNA (mRNA) in vivo. Transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) appear to be protected from cleavage because of either their secondary structure or association with ribosomal proteins, respectively. Therefore, MazF expression causes nearly complete degradation of mRNA, leading to severe reduction of protein synthesis and ultimately, to cell death (Zhang et al., Mol. Cell 12: 913-23 (2003)). MazF is found in selected bacteria, and recently the E. coli protein PemK (encoded by plasmid R100) was also shown to be a sequence-specific endoribonuclease (Zhang et al., J.
Biol. Chem.
279: 20678-20684 (2004)). PemK cleaves RNA with high specificity at a specific nucleic acid sequence, i.e., UAX, wherein X is C, A or U. See PCT/US2004/018571, which is incorporated herein by reference. These sequence-specific endoribonucleases are conserved, underscoring their essential roles in physiology and evolution. We refer to this family of sequence-specific endoribonuclease toxins as "mRNA interferases" (Zhang et al., J. Biol.
Chem. 279: 20678-20684 (2004)).

[0005] In the present study, we have exploited the unique cleavage properties of MazF to design a single-protein production (SPP) system in living E. coli cells. Upon expression of a gene engineered to express an ACA-less mRNA without altering its amino acid sequence, high levels of individual target protein synthesis were sustained for at least for 96 hours while background cellular protein synthesis was virtually absent. Therefore, the toxic effect of MazF is directed at mRNA with minimal side effects on cellular physiology. In fact, despite their state of growth arrest, these cells retain essential metabolic and biosynthetic activities for energy metabolism (ATP production), amino acid and nucleotide biosynthesis and transcription and translation. In addition to demonstrating the efficacy of the SPP system for human and yeast proteins, the technology was also effective for overexpression of an integral inner membrane protein whose natural levels of expression are relatively low.
The SPP
system yields unprecedented signal to noise ratios that both preclude any protein purification steps for experiments that require recovery of proteins in isolation, and, more importantly, enable structural and functional studies of proteins in intact, living cells.

BRIEF DESCRIPTION OF THE FIGURES

[0006] Figure 1. Expression of Human Eotaxin with Use of pColdI(SP-1) and pCo1dI(SP-2) with and without MazF Coexpression [0007] Figure 2. Effect of ACA Sequences on Eotaxin Expression [0008] Figure 3. Effect of Removal of All ACA Sequences in the MazF ORF on Eotaxin Expression [0009] Figure 4. Expression of Yeast Proteins in the SPP System Page 2 of 30 [0010] Figure 5. Expression of LspA, an Inner Membrane Protein in the SPP
System Using pColdIV(SP-2).

SUMMARY OF THE INVENTION

[0011] The present invention describes a single-protein production (SPP) system in living E. coli cells that exploits the unique properties of an mRNA interferase, for example, MazF, a bacterial toxin that is a single stranded RNA- and ACA-specific endoribonuclease, which efficiently and selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis. In one embodiment of the present invention, a system for expressing a single target protein in a transformable living cell while reducing non-target cellular protein synthesis includes: (a) an isolated transformable living cell comprising cellular mRNA having at least one first mRNA interferase recognition sequence;
(b) a first expression vector comprising an isolated nucleic acid sequence encoding an mRNA
interferase polypeptide, wherein the isolated nucleic acid sequence encoding the mRNA
interferase polypeptide is mutated by replacing at least one second mRNA
interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated mRNA interferase polypeptide; and (c) optionally, a second expression vector comprising an isolated nucleic acid sequence encoding a target protein, wherein the isolated nucleic acid sequence encoding the target protein is mutated by replacing at least one third mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated target protein; wherein the isolated cell is transformed with the first expression vector and the second expression vector; and wherein the isolated cell is maintained under conditions permitting expression of the mutant target protein in the cell.

[0012] In another embodiment, the present invention provides a method of increasing expression of a target protein in an isolated living cell including the steps:
(a) mutating an isolated nucleic acid sequence encoding an mRNA interferase polypeptide to replace at least one first mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated mRNA interferase polypeptide, (b) mutating an isolated nucleic acid sequence encoding the target protein to replace at least one second mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated target protein;
(c) providing a Page 3 of 30 first expression vector comprising the mutated nucleic acid sequence of step (a) and a second expression vector comprising the mutated nucleic acid sequence of step (b);
(d) providing an isolated living transformable cell having cellular messenger RNA sequences comprising at least one of a third mRNA interferase recognition sequence, (e) introducing the first expression vector and the second expression vector into the isolated living transformable cell;
(f) expressing the mutated mRNA interferase polypeptide, and (g) maintaining the isolated cell under conditions permitting expression of the mutant target protein in the cell.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The following definitions set forth the parameters of the present invention.

[0014] The abbreviation "ACA" refers to the sequence Adenine-Cytosine-Adenine.

[0015] As used herein, the terms "encode", "encoding" or "encoded", with respect to a specified nucleic acid, refers to information stored in a nucleic acid for translation into a specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.

[0016] The term "codon" as used herein refers to triplets of nucleotides that together specify an amino acid residue in a polypeptide chain. Most organisms use 20 or 21 amino acids to make their polypeptides, which are proteins or protein precursors.
Because there are four possible nucleotides, adenine (A), guanine (G), cytosine (C) and thymine (T) in DNA, there are 64 possible triplets to recognize only 20 amino acids plus the termination signal.
Due to this redundancy, most amino acids are coded by more than one triplet.
The codons that specify a single amino acid are not used with equal frequency. Different organisms often show particular "preferences" for one of the several codons that encode the same given amino acids. If the coding region contains a high level or a cluster of rare codons, removal of the rare codons by resynthesis of the gene or by mutagenesis can increase expression. See J.
Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), at 15.12; which is incorporated herein by reference. "Codon selection" therefore may be made to optimize expression in a selected Page 4 of 30 host. The most preferred codons are those which are frequently found in highly expressed genes. For "codon preferences" in E. coli, see Konigsberg, et al., Proc.
Nat'l. Acad. Sci.
U.S.A. 80:687-91 (1983), which is incorporated herein by reference.

[0017] One of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. The term "conservatively modified variants"
applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons UUA, UUG, CUU, CUC, CUA, and CUG all encode the amino acid leucine. Thus, at every position where a leucine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein which encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG , which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.
Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is within the scope of the present invention.

[0018] The term "eotaxin" as used herein refers to a chemotactic factor consisting of 74 amino acid residues that belongs to the C-C (or beta) chemokine family and has been implicated in animal and human eosinophilic inflammatory states.

[0019] The present invention includes active portions, fragments, derivatives, mutants, and functional variants of mRNA interferase polypeptides to the extent such active portions, fragments, derivatives, and fiulctional variants retain any of the biological properties of the mRNA interferase. An "active portion" of an mRNA interferase polypeptide means a peptide that is shorter than the full length polypeptide, but which retains measurable biological activity. A "fragment" of an mRNA interferase means a stretch of amino acid residues of at Page 5 of 30 least five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. A
"derivative" of an mRNA interferase or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g.., by manipulating the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion, or substitution of one or more amino acids, and may or may not alter the essential activity of the original mRNA interferase.

[0020] The term "gene" refers to an ordered sequence of nucleotides located in a particular position on a segment of DNA that encodes a specific functional product (i.e, a protein or RNA molecule). It can include regions preceding and following the coding DNA
as well as introns between the exons.

[0021] The term "induce" or inducible" refers to a gene or gene product whose transcription or synthesis is increased by exposure of the cells to an inducer or to a condition, e.g., heat.

[0022] The terms "inducer" or "inducing agent" refer to a low molecular weight compound or a physical agent that associates with a repressor protein to produce a complex that no longer can bind to the operator.

[0023] The term "induction" refers to the act or process of causing some specific effect, for example, the transcription of a specific gene or operon, or the production of a protein by an organism after it is exposed to a specific stimulus.

[0024] The terms "introduced", "transfection", "transformation", "transduction" in the context of inserting a nucleic acid into a cell, include reference to the incorporation of a nucleic acid into a prokaryotic cell or eukaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0025] The term "isolated" refers to material, such as a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment; or, if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human Page 6 of 30 intervention. For example, an "isolated nucleic acid" may comprise a DNA
molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA
of a prokaryotic or eukaryotic cell or host organism. When 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 has been sufficiently separated from other nucleic acids with which it is generally associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0026] The abbreviation "IPTG" refers to isopropyl-beta-D-thiogalactopyranoside, which is a synthetic inducer of beta-galactosidase, an enzyme that promotes lactose utilization, by binding and inhibiting the lac repressor. For example, IPTG is used in combination with the synthetic chromogenic substrate Xgal to differentiate recombinant from non-recombinant bacterial colonies in cloning strategies using plasmid vectors containing the lacZ gene.

[0027] The term "MazF" as used herein refers to the general class of endoribonucleases, to the particular enzyme bearing the particular name, and active fragments and derivatives thereof having structural and sequence homology thereto consistent with the role of MazF
polypeptides in the present invention.

[0028] The abbreviation "1spA" refers to the gene responsible for signal peptidase II
activity in E. coli.

[0029] The abbreviation "LspA" refers to the gene responsible for Lipoprotein Signal Peptidase activity in E. coli.

[0030] The family of enzymes encompassed by the present invention is referred to as "mRNA interferases". It is intended that the invention extend to molecules having structural and functional similarity consistent with the role of this family of enzymes in the present invention.

[0031] As used herein, the term "nucleic acid" or "nucleic acid molecule"
includes any DNA or RNA molecule, either single or double stranded, and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
Unless otherwise limited, the term encompasses known analogues.

Page 7 of 30 [0032] The term "oligonucleotide" refers to a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three, joined by phosphodiester bonds.

[0033] The term "operator" refers to the region of DNA that is upstream (5') from a gene(s) and to which one or more regulatory proteins (repressor or activator) bind to control the expression of the gene(s) [0034] As used herein, the term "operon" refers to a functionally integrated genetic unit for the control of gene expression. It consists of one or more genes that encode one or more polypeptide(s) and the adjacent site (promoter and operator) that controls their expression by regulating the transcription of the structural genes. The term "expression operon" refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals, polyadenylation signals, tenninators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.

[0035] The phrase "operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0036] The abbreviation "ORF" stands for "open reading frame, a portion of a gene's sequence that contains a sequence of bases, uninterrupted by internal stop sequences, and which has the potential to encode a peptide or protein. Open reading frames start with a start codon, and end with a termination codon. A termination or stop codon determines the end of a polypeptide.

[0037] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0038] The abbreviation "PCR" refers to polymerase chain reaction, which is a technique for amplifying the quantity of DNA, thus making the DNA easier to isolate, clone and sequence. See, e.g., U.S. Pat. No. 5,656,493, 5,33,675, 5,234,824, and 5,187,083, each of which is incorporated herein by reference.

Page 8 of 30 [0039] As used herein the term "promoter" includes reference to a region of DNA
upstream (5') from the start of transcription and involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. The term "inducible promoter" refers to the activation of a promoter in response to either the presence of a particular compound, i.e., the inducer or inducing agent, or to a defined external condition, e.g., elevated temperature.

[0040] The phrase "site-directed mutagenesis" refers to an in vitro technique wlzereby base changes i.e., mutations, are introduced into a piece of DNA at a specific site, using recombinant DNA methods.

[0041] The term "untranslated region" or UTR, as used herein refers to a portion of DNA
whose bases are not involved in protein synthesis.

[0042] The terms "variants", "inutants" and "derivatives" of particular sequences of nucleic acids refer to nucleic acid sequences that are closely related to a particular sequence but which may possess, either naturally or by design, changes in sequence or structure. By "closely related", it is meant that at least about 60%, but often, more than 85%, of the nucleotides of the sequence match over the defined length of the nucleic acid sequence.
Changes or differences in nucleotide sequence between closely related nucleic acid sequences may represent nucleotide changes in the sequence that arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence.
Other changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as "mutants" or "derivatives" of the original sequence.

[0043] A skilled artisan likewise can produce protein variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions;
and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, Page 9 of 30 such as, for example, an epitope for an antibody. The techniques for obtaining such variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known to the skilled artisan.

[0044] As used herein, the terms "vector" and "expression vector" refer to a replicon, i.e., any agent that acts as a carrier or transporter, such as a phage, plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element and so that sequence or element can be conveyed into a host cell. The E. coli SPP system described herein utilizes pColdI vectors, which induce protein production at low temperatures.

[0045] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise.
All technical and scientific terms used herein have the same meaning.

[0046] 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 herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

EXAMPLES

[0047] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g.
amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0048] Strains and Plasmids Page 10 of 30 [0049] E. coli BL21(DE3) cells were used in the experiments described below.
The mazF
gene was cloned into the Ndel-Xhol sites of pACYCDuet (Novagen) to create plasmid pACYCmazF. pACYCmazF(-9ACA) was constructed by site-directed mutagenesis using pACYCmazF as template. The eotaxin gene was synthesized on the basis of the optimal E.
coli codon usage (See Figure 2A) and cloned into the Ndel-Hindlll sites of pColdI(SP-1) to create plasmid pColdl(SP-1)eotaxin. pCo1dI(SP-1)eotaxin was constructed as described in the text by site-directed mutagenesis using pCo1dl(eotaxin) as template.
Mutagenesis was carried out using Pfu DNA polymerase (Stratagene) according to the instructions for the QuickChange Site-Directed Mutagenesis Kit (Stratagene). pCo1dl(SP-2)eotaxin was also constructed by site-directed mutagenesis using pCo1dl(SP-1)eotaxin as template. pCo1dl(SP-1)eotaxin(+ACA) was constructed by site-directed mutagenesis using pColdl(SP-1)eotaxin as template. The wild-type HsplO gene was amplified by PCR with Yeast chromosome as template and cloned into the Ndel-BamHI sites of pCo1dI(SP-2) to create plasmid pCo1dI(SP-2)Hsp10. The ACA-less HsplO gene was amplified by two-step PCR with Yeast chromosome as template and cloned into the Ndel-Ban1HI sites of pCo1dl(SP-2) to create plasmid pColdI(SP-2)Hsp10(-ACA). The wild-type and ACA-less Rpbl2 gene was amplified by PCR with wild type Rpb 12 plasmid as template and 5' and 3' oligonucleotides containing the altered sequence cloned into the Ndel-BamHI sites of pColdI(SP-2) to create plasmid pCo1dI(SP-2)Rpb12 and pCo1dI(SP-2)Rpb12(-ACA), respectively. The ACA-less LspA
gene was amplified by two-step PCR and cloned into the NdeI-BamHI sites of pColdIV(SP-2) to create plasmid pCo1dIV(SP-2)1spA(-ACA).

[0050] Assays of Protein Synthesis in Vivo [0051] E. coli BL21(DE3) carrying plasmids was grown in M9-glucose medium.
When the OD600 of the culture reached 0.5, the culture was shifted to 15 C for 45 min and 1 mM of IPTG was added to the culture. At the indicated time intervals, 1 ml of culture was added to a test tube containing 10 mCi [35S]-methionine. After incubation for 15 min (pulse), 0.2 ml of 40 mg/ml methionine was added and incubated for another 5 min (chase). The labeled cells were washed with M9-glucose medium and suspended in 100 l of SDS-PAGE
loading buffer. 10 l of each sample was analyzed by SDS-PAGE followed by autoradiography.

[0052] Preparation of the Membrane Fraction [0053] The cells harvested from 1 ml of culture by centrifugation (10,000 x g for 5min) were suspended in the 10 mM Tris-HCI (pH 7.5) and disrupted by sonication. The total Page 11 of 30 membrane fraction was obtained by centrifugation (100,000 x g, for 60 min) after the removal of unbroken cells.

[0054] Examnle 1. Effects of MazF Induction of Cellular Protein Synthesis [0055] E. coli BL21(DE3) carrying pACYCmazF was transformed either with pCo1dI(SP-1)eotaxin (A and left panel in B) or pColdI(SP-2)eotaxin (right panel in B and C).
Cells were grown in M9 medium at 37 C. At OD6o of 0.5, the cultures were shifted to 15 C
and after incubation at 15 C for 45 min to make cells acclimate low temperature, IPTG (1 mM) was added to induce both eotaxin and MazF expression (0 time). Cells were pulse-labeled with 35S-methionine for 15 min at the time points indicated on top of each gel and total cellular proteins were analyzed by SDS-polyacrylaminde gel electrophoresis (PAGE) followed by autoradiography.

[0056] The mazF gene was cloned into pACYC, a low copy number plasmid containing an IPTG inducible phage T7 promoter, yielding pACYCmazF. Cloning techniques generally may be found in J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), which is incorporated herein by reference. E. colt BL21 (DE3) transformed with pACYCmazF was sensitive to IPTG, a lac inducer, as no colonies were formed on agar plates containing IPTG
(not shown).

[0057] Figure 1 shows the expression of Human Eotaxin with Use of pColdI(SP-1) and pColdI(SP-2) with and without MazF coexpression by SDS-PAGE. Figure 1B shows the results for cells transformed with pCo1dI(SP-1)eotaxin (left panel); and transformed with pColdI(SP-2)eotaxin (right panel). Figure 1C shows the results for cells transformed with pACYCmazF and pCo1dI(SP-2)eotaxin were incubated in LB (left panel) or M9 medium(right panel). Cells were treated in the same manner as in Figure lA and Figure 1B, and, at the time points indicated, total cellular proteins were analyzed by SDS-PAGE
followed by Coomassie Blue staining. Note that the same volumes of the cultures were taken for the analysis. Positions of molecular weight markers are shown at the left hand side of the gels and the position of eotaxin is indicated by an arrow. As MazF effectively cleaves mRNAs at ACA sequences, cellular protein synthesis was dramatically inhibited at 37 C
upon MazF induction (Zhang et al., Mol. Cell 12: 913-23 (2003)) or at 15 C as shown in Figure 1A. In this cold-shock experiment, cells carrying pACYCmazF were first incubated Page 12 of 30 for 45 min at 15 C to induce cold-shock proteins required for cold-shock acclimation (see Thieringer et al., Bioassays 20(1): 49-57 (1998)). Then IPTG was added to the culture to induce MazF (0 time in Figure lA, left panel). Cells were pulse-labeled with [35S]
methionine for 15 min at the time points indicated on top of the gel. Panel A
left panel shows the results for cells transformed only with pACYCeotaxin; panel A middle panel shows the results for cells transformed only with pCold(SP-1)eotaxin; and Panel A right panel shows the results for cells transformed with both plasmids.

[0058] At 0 time, a very similar protein pattern was observed as that of the cells in the absence of IPTG (control, indicated as C), while cellular protein synthesis was dramatically inhibited at 1 hr after the addition of IPTG. After 6 hr, the synthesis of almost all cellular proteins was ahnost completely blocked.

[0059] Example 2. Expression of an ACA-less mRNA in MazF-induced Cells [0060] We speculated that if an mRNA that is engineered to contain no ACA
sequences is expressed in MazF-induced cells, the mRNA might be stably maintained in the cells so that the protein encoded by the mRNA may be produced without producing any other cellular proteins. To test this possibility, we synthesized the gene for human eotaxin, eliminating all ACA sequences in the gene without altering the amino acid sequence. Fig. 2A
shows the amino acid sequence of human eotaxin and the nucleotide sequences of its gene.
The nucleotide sequence was designed using preferred E. coli codons and those triplets underlined were changed to ACA in the experiment below. The ACA sequence is unique among possible triplet sequences, as it can be altered to other MazF-uncleavable sequences without changing the amino acid sequence of a protein regardless of the position of an ACA sequence in a reading frame.

[0061] The eotaxin gene shown in Figure 2A was fused with a 17-residue sequence consisting of a sequence from a translation enhancing element from the cspA
gene for the major cold-shock protein, CspA (Qing et al, Nat. Biotechnol. 22: 877-882 (2004)), 6 His residues, factor Xa cleavage site and the His-Met sequence derived from the Ndel site for gene insertion. The entire coding region for the fusion protein was inserted into pColdI(SP-1) and pColdI(SP-2) vectors, cold-shock vectors allowing a high protein expression upon cold shock (Qing et al, Nat. Biotechnol. 22: 877-882 (2004)). In pCold(SP-1) two ACA
sequences, one between the Shine-Dalgarno sequence and the initiation codon and the other in the translation enhancing element were converted to AUA. In pColdl(SP-2) in addition to Page 13 of 30 the two ACA sequences in pColdI(SP-1) three other ACA sequences in the 5'-untranslated region (5'-UTR) also were altered to MazF-uncleavable sequences by base substitutions (to GCA, AUA and GCA from the 5' ACA to the 3' ACA, respectively). The resulting constructs, pColdI(SP-1) eotaxin and pColdl(SP-2)eotaxin, respectively, were transformed into E. coli BL21 (DE3) cells.

[0062] After the cells transformed with pColdI(SP-1)eotaxin were cold-shocked at 15 C
and acclimated to the low temperature for 1 hr, IPTG was added to induce eotaxin production. Cells then were pulse-labeled with [35S]methionine for 15 min (0 time; Figure 1A, middle panel). Eotaxin was produced almost at a constant level from 0 time during 72 hr incubation together with other cellular proteins. The production of eotaxin at the 12 hr time point was approximately 11% of total cellular protein synthesis as judged from [35S]methionine incorporation.

[0063] When both eotaxin and mazF genes were coexpressed using E. coli BL21 (DE3) harboring both pACYCmazF and pColdI(SP-1)eotaxin, background cellular protein synthesis was dramatically reduced after 3 hr induction, while eotaxin production continued for 72 hr at an almost constant level (Figure 1A, right panel). Interestingly the level of eotaxin production in this experiment was higher (Figure 1A, right panel; 11% of total protein production at 12 hr) than that in the absence of MazF induction (Figure 1A, middle panel; 47%
at 12 hr). This approximately 5 fold enrichment is likely due to the fact that more ribosomes became available for eotaxin mRNA translation as cellular mRNAs were degraded by MazF. Notably, no specific protein bands were observed after the 12 hr time point.

[0064] When the identical experiment was carried out with the cells harboring both pACYCmazF and pColdI(SP-2)eotaxin, eotaxin was almost exclusively produced(Figure 1B, right panel). Notably, eotaxin production was substantially higher than that with pColdl(SP-1)eotaxin (Figure 1B, left panel). This higher production of eotaxin is likely due to the stabilization of the eotaxin mRNA by further removal of ACA sequences in the 5'-UTR in pColdI(SP-1). Approximately 90% of [35S]methionine was incorporated into eotaxin at 12 hr after MazF induction and notably no distinct cellular protein bands were discernible (Figure 1B, right panel) indicating that the signal-to-noise ratio of eotaxin was dramatically improved by the present SPP system. It is interesting to note that the high level of eotaxin production did not diminish even 96 hr after induction. Furthermore, background cellular protein Page 14 of 30 synthesis diminished sooner (at 3 hr) than that with pColdl(SP-1)eotaxin (at 6 hr) (compare the left panel with the right panel in Figure 1B).

[0065] With both vectors (Figure 1A and B), cell growth was completely blocked upon MazF induction as judged by OD600 and also by [35S]methionine incorporation into cellular proteins. These results indicate that growth-arrested cells by MazF induction are not physiologically dead and instead are fully capable of synthesizing proteins if their mRNAs have no ACA sequences. This in turn indicates that the cellular integrity of the E. coli BL21 (DE3) cells is kept intact for a long period of time so that not only energy metabolism but also biosynthetic functions for amino acids and nucleotides are fully active in the growth-arrested cells. Furthermore, transcriptional and translational machineries are also well maintained including RNA polymerase, ribosomes, tRNA, and all the other factors required for protein synthesis.

[0066] The production of eotaxin with pColdl(SP-2) eotaxin appears as a major band by Coomassie Blue staining after SDS polyacrylamide gel electrophoresis (Figure 1 C). At the 0 hr time point, the eotaxin band was barely discemable while at 12 hr it became the major band and its density increased even more after 24 hr. However, longer incubation did not significantly enhance the level of its production, suggesting that there is a threshold level of eotaxin production in MazF-induced cells. Since the [35S]methionine incorporation was constantly maintained for 96 hr (Figure. 1B), its seems that eotaxin production and degradation in the SPP system may equilibrate after 24 hr. It is important to note that the density of the bands for cellular proteins remained constant as expected from complete growth inhibition upon MazF induction. We examined if eotaxin production is affected by rich media such as LB medium and found that the use of LB medium did not enhance eotaxin production any more than the level obtained with defined M9 medium if pColdI(SP-2) was used.

[0067] Example 3. The Negative Effect of ACA Sequences on Protein Production [0068] In order to confirm that the exclusive eotaxin production in MazF-induced cells observed in Figure 1 is due to the ACA-less mRNA for eotaxin, the five native ACA
sequences were added to the eotaxin gene without altering its amino acid sequence as shown in Figure 2A. The eotaxin genes were expressed with use of pColdI(SP-2) and cells were treated and labeled with [35S] -methionine in the same manner as described in Figure 1. The Page 15 of 30 left panel shows the results for the ACA-less eotaxin gene (same as the left panel of Figure 1 B) and the right panel shows the results for the eotaxin gene with 5 ACA
sequences.

[0069] When this gene was expressed with use of pCo1dl(SP-1) together with pACYCmazF under the same condition as described for Figure 1, only a low level of eotaxin production was observed for the first 2 hours after which point the production was further reduced to a background level (Figure 2B, right panel) in comparison with the expression with the ACA-less mRNA (Figure 2B, left panel).

[0070] Curiously, the mazF gene encodes an mRNA that has an unusually high ACA
content (9 ACA sequences for a 111 residue protein)--in a dramatic contrast to MazE (82 amino acid residues with only 2 ACA sequences)--suggesting that mazF
expression is negatively regulated in cells. Therefore, we constructed the mazF gene with no ACA
[pACYCmazF(-9ACA)] and tested whether the removal of these ACA sequences from the mazF coding region may cause more effective reduction of background cellular protein production.

[0071] Fig. 3 shows the effect of removal of all ACA sequences in the mazF ORF
on eotaxin expression. Panel A shows the amino acid sequence of MazF and the nucleotide sequence of its ORF. The triplet sequences underlined (a total of nine) were originally ACA
in the wild-type mazF gene, which were changed to MazF-uncleavable sequences.
Panel B
shows the expression of eotaxin with pColdl(SP-2)eotaxin using the wild-type mazF gene (left panel) and ACA-less mazF gene (right panel). The experiments were carried as described in Figure 1.

[0072] As shown in Figure 3A, none of the base substitutions alter the amino acid sequence of MazF. Although cells harboring pYCACmazF(-9ACA) grew a little slower than cells harboring pYCACmazF in M9 medium, the background protein synthesis was further reduced without significant effects on the eotaxin production (Figure 3B).
These results clearly demonstrate that ACA sequences in mRNAs play the crucial role in protein production in MazF-induced cells.

[0073] Example 4. Application of the SPP System to Yeast Proteins [0074] We applied the SPP system to two yeast proteins: HsplO, a heat-shock factor and Rpbl2, an RNA polymerase subunit. The ORFs for HsplO and Rpbl2 contain 3 and 1 ACAs, respectively, which were converted to MazF-uncleavable sequences without altering their amino acid sequences (Figure 4A). They, together with the wild-type sequences, then Page 16 of 30 were inserted into pColdI(SP-2). The resulting plasmids were termed pCo1dI(SP-2)Hsp10 for the wild-type HsplO, pColdl(SP-2)Hsp10(-lACA) for the mutant Hsp10, pCo1dl(SP-2)Rpb12 for the wild-type Rpbl2 and pColdI(SP-2)Rpbl2(-3ACA), respectively. These plasmids were individually transformed into E. coli BL21(DE3) harboring pACYCmazF.
Protein expression patterns then were examined for 48 hours at 15 C.

[0075] The expression of Yeast Proteins in the SPP System is shown in Figure 4. Using pCo1dI(SP-2), yeast Hsp10 and Rpb12 were expressed in the SPP system in the presence and the absence of ACA sequences in their genes. Experiments were carried out as described supra for Figure 1. Figure 4A shows the expression of HsplO using the wild-type and ACA-less HsplO genes. The hsp 10 ORF consisting of 106 codons contains 3 ACA
sequences;
GCA-CAA for A25-Q26, ACA for T29 and CCA-CAG for P76-Q77, which were converted to GCC-CAA, ACC and CCC-CAG, respectively (altered bases are in bold). These base substitutions do not alter the amino acid sequence of Hsp 10. Figure 4B shows the expression of Rpbl2 using the wild-type and ACA-less genes. The rpbl2 ORF consisting of 70 codons contains one ACA for T10, which was converted to ACC for threonine.

[0076] Figure 4A shows that Hsp10 can be expressed with its native 3 ACA
sequences (WT) at a reasonably high level. However when all the ACA sequences were removed, Hsp10 synthesis significantly enhanced a few fold. Noticeably, the background was also significantly reduced with the ACA-less Hsp10, likely because more ribosomes were dedicated for the production of HsplO. Figure 4B shows that although Rpbl2 contains only one ACA, it causes a devastating effect on its production in the SPP system, as little 355-methionine incorporation was observed in the WT panel while reasonable incorporation was seen in the ACA-less Rpbl2. These results suggest that mRNA sensitivity to MazF may be governed, not only by the number of ACA sequences in an mRNA, but also by effective susceptibility of an ACA sequence to MazF. It is likely that the ACA sequence susceptibility is determined by its location in a single-stranded region of an inRNA as well as the effective translation of an mRNA by ribosomes, as ribosomes are assumed to protect the inRNA from its cleavage by MazF.

[0077] Example 5. Application of the SPP System to an Integral Membrane Protein [0078] We attempted to apply the SPP system to a minor integral membrane protein. We chose the gene 1spA for signal peptidase II in E. coli, which is specifically required for cleavage of the signal peptides of lipoproteins (Tokuda and Matsuyarna, Biochem. Biophys.

Page 17 of 30 Acta 1693: 5-13 (2004)). E. coli contains a total of 96 lipoproteins, which are known to assemble either in the inner membrane or in the outer membrane depending upon the nature of the second amino acid residue (acidic or neutral) of the mature lipoproteins (Yamaguchi and Inouye, Cell 53: 423-432 (1988); Tokuda and Matsuyama, Biochem. Biophys.
Acta 1693: 5-13 (2004)). The signal peptides of all the other secreted proteins are cleaved by signal peptidase I (leader peptidase) , which is estimated to exist only at a level of 500 molecules per cell in E. coli (Wolfe et al., J. Biol. Chem. 257: 7898-7902 (1982)).

[0079] Lipoprotein Signal Peptidase (LspA) also is considered to be a very low abundant protein in the inner membrane. It consists of 164 amino acid residues and contains four presumed transmembrane domains, indicating that LspA is an integral inner membrane protein. Three ACA sequences in the IspA ORF were altered to non-MazF-cleavable sequences without changing its amino acid sequence and the ACA-less LspA was expressed using pColdl(SP-2) in the SPP system using mazF(-9ACA).

[0080] The expression of LspA, an inner membrane protein in the SPP system using pColdL(SP-2) are shown in Fig. 5. LspA, signal peptidase II or lipoprotein signal peptidase was expressed in the SPP system as described in Figure 1. Panel A shows total cellular proteins; and Panel B shows the membrane fraction: The position of LspA is shown by an arrow.

[0081] As shown in Figure 5A, the expression of LspA in the SPP system apparently is toxic to the cells, as 35S-methionine incorporation lasted only 1 hour after IPTG induction.
Nevertheless, as shown in Figure 5B, a reasonable 35S-methionine incorporation into LspA
appears to be achieved as the LspA band densities at 0 and 1 hr time points were the highest comparing them with other cellular protein bands (compare with the C lane in Figure 5A).
The background cellular protein synthesis observed at 0 and 1 hr was easily removed by ultracentrifugation, and 35S-methionine incorporation was highly enriched in the membrane fraction.

[0082] Discussion [0083] The present work demonstrates that complete inhibition of cellular protein synthesis by an mRNA interferase does not cause deteriorating effects on the cellular physiology. As a result of fragmentation of almost all cellular mRNAs by MazF
at ACA
sequences, cellular protein synthesis is completely blocked, which in turn leads to complete cell growth arrest. However, to our surprise, growth arrested cells by MazF
induction were Page 18 of 30 found to be fully capable of synthesizing proteins at a high level for a long period of time (at least 96 hr at 15 C) if their mRNAs are engineered to have no ACA sequences.
In this fashion we have achieved for the first time to establish the single-protein production (SPP) in vivo.

[0084] Our results demonstrate that MazF-induced cells are not dead. MazF
induction does not hamper cellular integrity maintaining energy metabolism producing enough ATP
required various cellular functions including RNA and protein synthesis. In addition biosynthesis of amino acids and nucleotides are also maintained intact. It is quite surprising to find that in the complete absence of new cellular protein synthesis, all the protein factors required for these cellular functions (for example protein factors required for protein synthesis) and cellular metabolisms are stably maintained at least 96 hours at 15 C. It remains to be determined how long these cellular functions could be retained without affecting the SPP capability. Although at a glance they appear to be in a dormant state, they are fully capable of RNA and protein synthesis and distinctly different from the dormancy caused by the stationary phase due to nutritional deprivation. We propose to term the physiological state created by MazF induction "quasi-dormant" state. It remains to be determined if the quasi-dormant cells are dead or undead. Bacterial viability is often determined by the colony forming ability of cells after various treatments.
The viability of E.
coli cells after MazF induction has been examined in this fashion and shown to be resumed during limited time incubation after MazF induction if MazE is induced (Pedersen et al., Mol.
Microbiol. 45: 501-10 (2002); Amitai et al., J. Bacteriol. 186: 8295-8300 (2004)).
Therefore, the effect of MazF is reversible to a certain extent, however it has been argued that there is 'a point of no return', from which point all cells are destined to die (Amitai et al., J.
Bacteriol. 186: 8295-8300 (2004)). Importantly, the MazE gene used by both group contains two ACA sequence in its ORF. The present results clearly indicate that in order for any genes to be expressed in MazF-induced cells, ACA sequences in these genes have to be converted to MazF-uncleavable sequences. Therefore it is highly possible that the quasi-dormant cells expressing MazF cannot express MazE unless all the ACA sequences are eliminated from its ORF.

[0085] The ability to produce only a single protein of interest in living cells or undead cells provides a novel approach for studying the various aspects of proteins in living cells previously unattainable. Since by using the SPP system a protein of interest can be Page 19 of 30 exclusively labeled with isotopes (15N and 13C) in living cells, it may be even possible to examine NMR structures of proteins in living cells. Recently we have shown that NMR
structural determination of a protein can be achieved using cell lysates without protein purification by expressing a protein of interest by high expression cold-shock vectors, pCold (Qing et al., Nat. Biotechnol. 22: 877-882 (2004)). We now demonstrate that the use of MazF together with pCold vectors dramatically reduces the signal-to-noise ratio as the background cellular protein synthesis can be almost completely blocked by MazF
induction.
In these experiments we showed that the removal of ACA sequences from pCo1dI
vector itself is also very important by which 5 fold improvement of eotaxin production was observed. When combined with MazF, the rate of eotaxin synthesis was at the leve190% of the total cellular protein synthesis as judged by 35S-methionine incorporation. The remaining 10% consisted of a general background without incorporation into any specific protein bands.
This in turn enables one to perform the structural study of very low abundant proteins, whose production is limited because of their toxicity when expressed in a large quantity. We indeed demonstrated in the present paper that LspA, a very low abundant inner membrane protein, can be exclusively expressed in the membrane fraction. Some proteins may be folded only in living cells, whose structural study may be achieved only by the use of the SPP system.

[0086] Another unique advantage of the SPP system is that a protein of interest can be produced or labeled with isotopes in a highly concentrated culture as cell growth is completely blocked upon MazF induction. It is possible that the SPP system can be applied for the production of not only proteins but also other non-protein compounds.
Furthermore the SPP system may not be limited only to bacteria, and MazF and other mRNA
interferases may be applied for eukaryotic cells to create the SPP systems in yeast and mammalian cells.

[0087] Where a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

Page 20 of 30 [0088] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0089] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the Invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

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LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS

THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:

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Claims (68)

1. A system for expressing a single target protein in a transformable living cell while reducing non-target cellular protein synthesis, comprising (a) an isolated transformable living cell comprising cellular mRNA having at least one first mRNA interferase recognition sequence;
(b) a first expression vector comprising an isolated nucleic acid sequence encoding an mRNA interferase polypeptide, wherein the isolated nucleic acid sequence encoding the mRNA interferase polypeptide is mutated by replacing at least one second mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated mRNA interferase polypeptide;
(c) optionally, a second expression vector comprising an isolated nucleic acid sequence encoding a target protein, wherein the isolated nucleic acid sequence encoding the target protein is mutated by replacing at least one third mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated target protein;
wherein the isolated cell is transformed with the first expression vector and the second expression vector; and wherein the isolated cell is maintained under conditions permitting expression of the mutant target protein in the cell.

2. The system according to claim 1, wherein the first and second expression vectors each further comprise at least one regulatory sequence.

3. The system according to claim 2, wherein the at least one regulatory sequence is at least one inducible promoter.

4. The system according to claim 3, wherein the at least one inducible promoter in the first expression vector is operably linked to the mutated nucleic acid sequence encoding the mutated mRNA interferase polypeptide.

5. The system according to claim 3, wherein the at least one inducible promoter in the second expression vector is operably linked to the mutated nucleic acid sequence encoding the mutated target protein.

6. The system according to claim 1, wherein the mutated nucleic acid sequence in (b) encodes a mutated mRNA interferase polypeptide having an amino acid sequence identical to the amino acid sequence of a nonmutated mRNA interferase polypeptide.

7. The system according to claim 1, wherein the mutated nucleic acid sequence in (c) encodes a mutant target protein having an amino acid sequence identical to the amino acid sequence of a nonmutated target protein.

8. The system according to claim 1, wherein the mutant mRNA interferase polypeptide when expressed in the cell recognizes the at least one first mRNA
interferase recognition sequence in cellular messenger RNA.

9. The system according to claim 1, wherein cellular messenger RNA is selectively cleaved by the mutant mRNA interferase polypeptide thereby reducing nontarget cellular protein synthesis.

10. The system according to claim 1, wherein the first mRNA interferase recognition sequence, the second mRNA interferase recognition sequence, and the third mRNA interferase recognition sequence are the same mRNA interferase recognition sequence.

11. The system according to claim 10, wherein the mRNA interferase recognition sequence is adenine-cytosine-adenine.

12. The system according to claim 1, wherein an expressed messenger RNA
encoding the mutated target protein is stably maintained in the cell.

13. The system according to claim 1, wherein the mutated nucleic acid sequence encoding the mutated target protein is further mutated to replace rare codons with preferred codons to produce a twice-mutated nucleic acid sequence, wherein the twice-mutated nucleic acid sequence encodes a twice-mutated target protein having an amino acid sequence identical to the amino acid sequence of a nonmutated target protein.

14. The system according to claim 13, wherein the twice-mutated nucleic acid sequence encoding the twice-mutated target protein comprises an inducible promoter operably linked to the twice-mutated nucleic acid sequence encoding the twice-mutated target protein.

15. The system according to claim 13 wherein an expressed messenger RNA
encoding the twice-mutated target protein is stably maintained in the cell.

16. The system according to claim 1, wherein the mutated nucleic acid sequence encoding the mutated mRNA interferase polypeptide is further mutated to replace rare codons with preferred codons to produce a twice-mutated nucleic acid sequence, wherein the twice mutated nucleic acid sequence encodes a twice-mutated mRNA interferase polypeptide having an amino acid sequence identical to the amino acid sequence of a nonmutated mRNA
interferase polypeptide.

17. The system according to claim 16, wherein the twice mutated nucleic acid sequence encoding the twice-mutated target protein comprises an inducible promoter operably linked to the twice-mutated nucleic acid sequence encoding the twice-mutated mRNA interferase polypeptide.

18. The system according to claim 1, wherein the cell is a mammalian cell.

19. The system according to claim 1, wherein the cell is a eukaryotic cell.

20. The system according to claim 1, wherein the cell is a prokaryotic cell.

21. The system according to claim 20, wherein the cell is an E. coli cell.

22. The system according to claim 1, wherein the mutated mRNA interferase polypeptide is MazF.

23. The system according to claim 1, wherein the mutated mRNA interferase polypeptide is a functional fragment of MazF.

24. The system according to claim 1, wherein the mutated mRNA interferase polypeptide is a functional variant of MazF.

25. The system according to claim 1, wherein the target protein is a mammalian protein.

26. The system according to claim 25, wherein the mammalian protein is a human protein.

27. The system according to claim 1, wherein the target protein is a yeast protein.

28. The system according to claim 1, wherein target protein is a minor bacterial protein.

29. The system according to claim 28, wherein the target protein is a toxic low abundant protein.

30. The system according to claim 1, wherein the cell is maintained in media comprising at least one radioactively labeled isotope.

31. The system according to claim 30, wherein the mutant protein when expressed is radiolabeled.

32. The system according to claim 1, wherein the isolated nucleic acid sequence encoding the target protein is amplified by polymerase chain reaction.

33. A method of increasing expression of a target protein in an isolated living cell, the method comprising the steps (a) mutating an isolated nucleic acid sequence encoding an mRNA interferase polypeptide to replace at least one first mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated mRNA interferase polypeptide, (b) mutating an isolated nucleic acid sequence encoding the target protein to replace at least one second mRNA interferase recognition sequence with an alternate triplet codon sequence to produce a mutated nucleic acid sequence encoding a mutated target protein;
(c) providing a first expression vector comprising the mutated nucleic acid sequence of step (a) and a second expression vector comprising the mutated nucleic acid sequence of step (b);
(d) providing an isolated living transformable cell having cellular messenger RNA
sequences comprising at least one of a third mRNA interferase recognition sequence, (e) introducing the first expression vector and the second expression vector into the isolated living transformable cell;
(f) expressing the mutated mRNA interferase polypeptide, and (g) maintaining the isolated cell under conditions permitting expression of the mutant target protein in the cell.

34. The method according to claim 33, wherein the first and second expression vectors each further comprise at least one regulatory sequence.

35. The method according to claim 34, wherein the at least one regulatory sequence is at least one inducible promoter.

36. The method according to claim 35, wherein the inducible promoter in the first expression vector is operably linked to the mutated nucleic acid sequence encoding the mutated mRNA interferase polypeptide.

37. The method according to claim 36, further comprising the step of inducing the inducible promoter operably linked to the mutated nucleic acid sequence encoding the mutated mRNA interferase polypeptide with an inducing agent to express the mutated mRNA
interferase polypeptide.

38. The method according to claim 37, wherein the mutated mRNA interferase polypeptide selectively cleaves the cellular messenger RNA, thereby reducing nontarget cellular protein synthesis.

39. The method according to claim 35, wherein the inducible promoter in the second expression vector is operably linked to the mutated nucleic acid sequence encoding the mutated target protein.

40. The method according to claim 39, further comprising the step of inducing the inducible promoter operably linked to the mutated nucleic acid sequence encoding the -mutated target protein with an inducing agent to express the mutated target protein.

41. The method according to claim 33, wherein the inducible promoter in the first expression vector is operably linked to the mutated nucleic acid sequence encoding the mutated mRNA interferase polypeptide, and the inducible promoter in the second expression vector is operably linked to the mutated nucleic acid sequence encoding the mutated target protein, the method further comprising the steps:
inducing the inducible promoter operably linked to the mutated nucleic acid sequence encoding the mutated mRNA interferase polypeptide with a first inducing agent to express the mutated mRNA interferase polypeptide, and inducing the inducible promoter operably linked to the mutated nucleic acid sequence encoding the mutated target protein with a second inducing agent to express the mutated target protein.

42. The method according to claim 33, wherein the cell is co-tranfected with the first expression vector and the second expression vector.

43. The method according to claim 33, step (a) further comprising the step of further mutating the mutated nucleic acid sequence encoding the mutated mRNA
interferase polypeptide to replace rare codons with preferred codons to produce a twice-mutated nucleic acid sequence encoding a twice-mutated mRNA interferase having an amino acid sequence identical to the amino acid sequence of the nonmutated mRNA
interferase polypeptide.

44. The method according to claim 33, step (b) further comprising the step of further mutating the mutated nucleic acid sequence encoding the mutated target protein to replace rare codons with preferred codons to produce a twice-mutated nucleic acid sequence encoding a twice-mutated target protein having an amino acid sequence identical to the amino acid sequence of the nonmutated target protein.

45. The method according to claim 33, step (a) further comprising the step further mutating the mutated inducible nucleic acid sequence encoding the mutated mRNA interferase polypeptide to replace rare codons with preferred codons to produce a twice-mutated inducible nucleic acid sequence encoding a twice-mutated mRNA
interferase having an amino acid sequence identical to the amino acid sequence of the nonmutated mRNA interferase polypeptide; and step (b) further comprising the step further mutating the mutated nucleic acid sequence encoding the mutated target protein to replace rare codons with preferred codons to produce a twice-mutated nucleic acid sequence encoding a twice-mutated target protein having an amino acid sequence identical to the amino acid sequence of the nonmutated target protein.

46. The method according to claim 45, wherein the twice-mutated nucleic acid sequence encoding the twice-mutated mRNA interferase polypeptide of the first expression vector comprises a first inducible promoter operably linked to the twice-mutated nucleic acid sequence encoding the twice-mutated mRNA interferase polypeptide; and the twice-mutated nucleic acid sequence encoding the twice-mutated target protein of the second expression vector comprises a second inducible promoter operably linked to the twice-mutated nucleic acid sequence encoding the twice-mutated target protein.

47. The method according to claim 46, further comprising the steps of inducing the first inducible promoter operably linked to the twice-mutated nucleic acid sequence encoding the twice-mutated mRNA interferase polypeptide with a first inducing agent to express the twice-mutated mRNA interferase polypeptide; and inducing the second inducible promoter operably linked to the twice-mutated nucleic acid sequence encoding the twice-mutated target protein with a second inducing agent to express the twice-mutated target protein..

48. The method according to claim 33, wherein the at least one first mRNA
interferase recognition sequence in step (a), the at least one second mRNA
interferase recognition sequence in step (b), and the at least one third mRNA interferase recognition sequence in step (d) are the same mRNA interferase recognition sequence.

49. The method according to claim 48, wherein the mRNA interferase recognition sequence in steps (a), (b), and (d) is adenine-cytosine-adenine.

50. The method according to claim 33, wherein in step (g), a messenger RNA
encoding the mutated target protein is stably maintained in the cell.

51. The method according to claim 45, wherein in step (g), a messenger RNA
encoding the twice-mutated target protein is stably maintained in the cell.

52. The method according to claim 33, wherein the cell is a eukaryotic cell.

53. The method according to claim 52, wherein the cell is a mammalian cell.

54. The method according to claim 33, wherein the cell is a prokaryotic cell.

55. The method according to claim 54, wherein the cell is an E. coli cell.

56. The method according to claim 33, wherein the mutated mRNA interferase polypeptide is MazF.

57. The method according to claim 33, wherein the mutated mRNA interferase polypeptide is a functional fragment of MazF.

58. The method according to claim 33, wherein the mutated mRNA interferase polypeptide is a functional variant of MazF.

59. The method according to claim 45 wherein the twice-mutated mRNA
interferase polypeptide is MazF.

60. The method according to claim 45 wherein the twice-mutated mRNA
interferase polypeptide is a functional fragment of MazF.

61. The method according to claim 45, wherein the twice-mutated mRNA
interferase polypeptide is a functional variant of MazF.

62. The method according to claim 33, wherein the target protein is a mammalian protein.

63. The method according to claim 62, wherein the target protein is a human protein.

64. The method according to claim 33, wherein the target protein is a yeast protein.

65. The method according to claim 33, wherein the target protein is a minor bacterial protein.

66. The method according to claim 65, wherein the target protein is a toxic low abundant protein.

67. The method according to claim 33, further comprising the step of incubating the cell during step (g) in media comprising at least one radioactively labeled isotope.

68. The method according to claim 33, further comprising the step of amplifying the isolated nucleic acid sequence encoding the target protein in step (b) by polymerase chain reaction.