WO2007137144A2 - Production de protéines simples dans des cellules vivantes par une interférase d'arn messager - Google Patents

Production de protéines simples dans des cellules vivantes par une interférase d'arn messager Download PDF

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WO2007137144A2
WO2007137144A2 PCT/US2007/069194 US2007069194W WO2007137144A2 WO 2007137144 A2 WO2007137144 A2 WO 2007137144A2 US 2007069194 W US2007069194 W US 2007069194W WO 2007137144 A2 WO2007137144 A2 WO 2007137144A2
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mutated
mrna interferase
nucleic acid
acid sequence
protein
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WO2007137144A3 (fr
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Masayori Inouye
Junjie Zhang
Motoo Suzuki
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University Of Medicine And Dentistry Of New Jersey
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • 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.
  • 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.
  • MazF expression causes nearly complete degradation of mRNA, leading to severe reduction of protein synthesis and ultimately, to cell death (Zhang et al., MoI. Cell 12: 913-23 (2003)).
  • MazF is found in selected bacteria, and recently the E. coli protein PemK (encoded by plasmid RlOO) 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.
  • 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)).
  • SPP single protein production
  • Mature eotaxm (A) or HR91(B) and EnvZV (C) were expressed from pColdI(SP-2) (1 ACA in 3'-UTR) or pColdI(SP-4) (No ACA in 3'-UTR) along with MazF from pACYCmazK
  • ACA-less EnvZB was expressed from pColdl(SP-4) along with MazF from pACYCm ⁇ zF. Cultures were grown to an OD 600 of 0.5, shifted to 15°C for 45 min, concentrated to the levels shown and then induced with IPTG for 0 or 21 hr in M9 medium. Samples were subjected to SDS-PAGE followed by Coomassie Blue staining. Molecular weight markers on the left; the position of EnvZB is designated by an arrow to the right. Figure 8. cSPP facilitates high efficiency selenomethionine substitution without toxicity
  • Both panels represent mass spectroscopy of a seven residue YGNG WIK tryptic fragment derived from 20-fold concentrated cultures of ACA-less EnvZB expressed from pColdI(SP-2) along with MazF from pACYCm ⁇ zF grown in either M9 medium (A) or 15 N ⁇ M9 medium (B).
  • 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 niRNAs in vivo, resulting in a precipitous drop in total protein synthesis.
  • SPP single-protein production
  • 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
  • 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 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
  • SPP Single Protein Production
  • Escherichia coli cells exploits the unique properties of MazF, a ssRNA- and ACA-specific endoribonuclease 2 .
  • MazF is a bacterial toxin that selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis leading to cell growth arrest.
  • ACA-less mRNA when a gene encoding a target protein is engineered to transcribe an ACA-less mRNA, its transcript is stably co- expressed and translated in MazF-induced cells. Codon degeneracy enables alteration of an ACA triplet to a cleavage-resistant sequence without changing the protein amino acid sequence, regardless of its position in the reading frame.
  • cSPP and SPP systems call for the construction of an ACA-less target gene (thus encoding an ACA-less mRNA)
  • the target gene contains only a few ACA sequences, these sequences can be altered by oligonucleotide-directed site-specific mutagenesis so that they are no longer cleavable by MazF.
  • the best approach is to chemically synthesize the entire gene using one of the commercially available gene synthesis technologies (e.g. Codon Devices, Cambridge, MA 10 ) This general approach has several advantages.
  • the gene sequences are guaranteed for accuracy so the subsequent sequence verification step that typically follows PCR mutagenesis is no longer necessary.
  • the synthetic gene can be directly inserted into a vector of choice, eliminating the need for a cloning step.
  • the gene sequence can be optimized to the codon usage preferences of the particular organism used for expression.
  • the time and manpower savings more than offset the modest cost of whole gene synthesis. As with new technologies in general, the cost should continue to fall as the methods used for gene synthesis become further improved. In fact, we expect that this technology will completely replace PCR mediated site-directed mutagenesis for creation of ACA-less genes in the next few years.
  • ACA refers to the sequence Adenine-Cytosine-Adenine.
  • encode 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.
  • the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
  • codon 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.
  • 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.
  • Codon selection therefore may be made to optimize expression in a selected 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.
  • 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.
  • 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.
  • the codons UUA, UUG, CUU, CUC, CUA, and CUG all encode the amino acid leucine.
  • 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.
  • each codon in a nucleic acid 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.
  • eotaxin 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.
  • 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 functional 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 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.
  • 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.
  • induce 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.
  • inducing agent refers 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.
  • 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.
  • 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, plasmicl, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • 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 intervention.
  • 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.
  • 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.
  • 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.
  • 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.
  • MazF 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.
  • ispA refers to the gene responsible for signal peptidase ⁇ I activity in E. coli.
  • LspA refers to the gene responsible for Lipoprotein Signal Peptidase activity in E. coli.
  • mRNA interferases 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.
  • 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.
  • 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.
  • oligonucleotide refers to a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three, joined by phosphodiester bonds,
  • 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)
  • 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.
  • 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, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • 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.
  • 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.
  • 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.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • 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.
  • 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.
  • 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.
  • 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.
  • site-directed mutagenesis refers to an in vitro technique whereby base changes i.e., mutations, are introduced into a piece of DNA at a specific site, using recombinant DNA methods.
  • UTR untranslated region
  • variants 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.
  • 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.
  • 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, 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.
  • vector refers 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.
  • 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 pColdl vectors, which induce protein production at low temperatures.
  • 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; SEQ ID NO:1) and cloned into the Ndel-Hindlll sites of pColdl(SP-l) to create plasmid pColdI(SP-l)eotaxin.
  • pColdI(SP-l)eotaxin was constructed as described in the text by site-directed mutagenesis using pColdl(eotaxin) as template. Mutagenesis was carried out 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-l)eotaxin as template. pCold ⁇ (SP-l)eotaxin(+ACA) was constructed by site-directed mutagenesis using pCold ⁇ (SP-l)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 pColdI(SP-2) to create plasmid pColdI(SP-2)HsplO.
  • the ACA-less HsplO gene was amplified by two-step PCR with Yeast chromosome as template and cloned into the Ndel-BamHI sites of pColdI(SP-2) to create plasmid pColdI(SP-2)HsplO( ⁇ ACA),
  • the wild-type and ACA-less Rpbl2 gene was amplified by PCR with wild type Rpbl2 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 pCold ⁇ (SP-2)Rpbl2 and pColdI(SP-2)Rpbl2(-ACA), respectively.
  • E. coli BL21(DE3) carrying plasmids was grown in M9-glucose medium.
  • the culture was shifted to 15 0 C for 45 min and 1 mM of IPTG was added to the culture.
  • 1 ml of culture was added to a test tube containing 10 mCi [ 35 S]-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 ⁇ of SDS-PAGE loading buffer. 10 ⁇ l of each sample was analyzed by SDS-PAGE followed by autoradiography.
  • Example 1 Effects of MazF Induction of Cellular Protein Synthesis
  • E. coli BL21(DE3) carrying pACYCmazF was transformed either with pColdI(SP-l)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 OD 600 of 0.5, the cultures were shifted to 15°C and after incubation at 15 0 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 S-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.
  • SAGE SDS-polyacrylaminde gel electrophoresis
  • 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).
  • Figure 1 shows the expression of Human Eotaxin with Use of pColdl(SP-l) and pColdI(SP-2) with and without MazF coexpression by SDS-PAGE.
  • Figure IB shows the results for cells transformed with pColdI(SP-l)eotaxin (left panel); and transformed with pColdI(SP-2)eotaxin (right panel).
  • Figure 1C shows the results for cells transformed with pACYCmazF and pColdI(SP-2)eotaxin were incubated in LB (left panel) or M9 medium(right panel).
  • 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-l)eotaxin
  • Panel A right panel shows the results for cells transformed with both plasmids.
  • Example 2 Expression of an ACA-less mRNA 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.
  • 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 64 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.
  • the eotaxin gene shown in Figure 2A (SEQ ID NO: 1 ) 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.
  • Example 3 The Negative Effect of ACA Sequences on Protein Production
  • the five native ACA sequences were added to the eotaxin gene without altering its amino acid sequence as shown in Figure 2A (SEQ ID NO: 1).
  • the eotaxin genes were expressed with use of pColdI(SP-2) and cells were treated and labeled with [ 35 S] -methionine in the same manner as described in Figure 1.
  • the 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.
  • 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 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
  • 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 pColdI(SP-2)eotaxin using the wild-type mazF gene
  • Rpbl2 an RNA polymerase subunit.
  • the ORFs for HsplO and Rpbl2 contain 3 and 1
  • ACAs were converted to MazF-uncleavable sequences without altering their amino acid sequences ( Figure 4A). They, together with the wild-type sequences, then were inserted into pColdI(SP-2). The resulting plasmids were termed pColdI(SP-2)HsplO for the wild-type HsplO, pColdI(SP-2)HsplO(- IACA) for the mutant Hspl O, pColcll(SP-2)Rpbl2 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.
  • FIG. 4 shows the expression of HsplO using the wild-type and ACA- less HsplO genes.
  • the hsplO 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).
  • Figure 4 B shows the expression of Rpbl2 using the wild-type and ACA-less genes.
  • the rpbl2 ORF consisting of 70 codons contains one ACA for TlO 5 which was converted to ACC for threonine.
  • Figure 4A shows that HsplO can be expressed with its native 3 ACA sequences (WT) at a reasonably high level. However when all the ACA sequences were removed, HsplO synthesis significantly enhanced a few fold. Noticeably, the background was also significantly reduced with the ACA-less HsplO, likely because more ribosomes were dedicated for the production of HsplO.
  • FIG. 4B shows that although Rpbl2 contains only one ACA, it causes a devastating effect on its production in the SPP system, as little 35 S- methionine incorporation was observed in the WT panel while reasonable incorporation was seen in the ACA-less Rpbl2.
  • 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 mRNA as well as the effective translation of an mRNA by ribosomes, as ribosomes are assumed to protect the mRNA from its cleavage by MazF.
  • Example 5 Application of the SPP System to an Integral Membrane Protein
  • SPP system was applied to a minor integral membrane protein.
  • 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. colt (Wolfe et a!., J. Biol. Chem.
  • LspA Lipoprotein Signal Peptidase
  • IspA ORF 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 pColdI(SP-2) in the SPP system using mazF(-9ACA).
  • 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.
  • LspA a very low abundant inner membrane protein
  • Some proteins may be folded only in living cells, whose structural study may be achieved only by the use of the SPP system.
  • 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 niRNA interferases may be applied for eukaryotic cells to create the SPP systems in yeast and mammalian cells.
  • Example 6 pCoIdI(SP-2) and pCoIdl (SP-4) vectors support sustained, high level target protein expression
  • All SPP plasmids exploit a pColci vector backbone designed for high yield protein expression under cold shock conditions 3 .
  • pColdl features include a cspA promoter and translation-enhancing element to drive high levels of target protein transcription and translation upon cold shock, a Hisg tag sequence, a factor Xa cleavage site and an amino terminal His-Met encoding sequence comprising an Nde I site that enables the in-frame insertion of any target gene.
  • a second generation plasmid pColdI(SP-4) was created from the SPP plasmid pCold ⁇ (SP-2).
  • the pColdI(SP-2) vector was modified such that all of the ACAs were removed from the relevant 5' sequences ! ; subsequent removal of a single ACA from the 3' untranslated region (UTR) of pColdI(SP-2) resulted in the pCoHI(SP-4) SPP vector.
  • Eotaxin synthesis rates from pColdI(SP-4) gradually increased from day one to three, peaked between day three and four, and were sustained at that level through the final seven day time point.
  • eotaxin expression levels from pColdI(SP-2) were relatively modest initially, increased only marginally, peaked later (day five) and sustained peak expression through the seven day time point. Therefore, pColdI(SP-4) supports higher levels of eotaxin expression, suggesting that removal of ACAs at the 3' UTR of the vector would generally enhance expression levels.
  • these results demonstrated that quasi-dormant E. coli cells are able to translate proteins for at least seven days despite being growth arrested.
  • the eotaxin results did not reflect a general trend, instead, the target protein appears to dictate the expression characteristics and protein synthesis rates from either the original pCold ⁇ (SP-2) or the modified pColdI(SP-4) SPP vector.
  • the target protein appears to dictate the expression characteristics and protein synthesis rates from either the original pCold ⁇ (SP-2) or the modified pColdI(SP-4) SPP vector.
  • synthesis rates of new target proteins are never worse, and sometimes even better using pColdI(SP-4), we have selected this modified vector for the remainder of the SPP expression studies described in this study.
  • HR969 139 amino acids
  • HR969 protein synthesis rates measured by [ 35 S]-meth ⁇ onine incorporation were compared to cellular protein levels assessed by Coomassiae staining (Fig. ID).
  • the levels of HR969 protein accumulated steadily to the 45 and 92.5 hour time points when peak expression was observed, representing 21% and 24% of the total protein, respectively.
  • EnvZB and HR91 were obtained comparable results for both EnvZB and HR91 (data not shown), indicating that the pColdI(SP-4) SPP expression vector supports enhanced, sustained and stable recombinant protein production.
  • Example 7 SPP cultures can be highly condensed without significant reduction in target protein yields [0096] Since cell growth is completely inhibited upon MazF induction, we examined if cell cultures could tolerate substantial condensation for protein production by the SPP system without affecting the protein yield. E. coli BL21 cultures harboring pACYCm ⁇ zF and pColdI(SP-4)e « vZZ? were subjected to normal SPP induction conditions except that the cells were first pelleted and resuspended in medium to achieve cell concentrations 10-, 20-, 30-, 40-, 50- and 100-fold higher than normal inducing conditions (OD ⁇ oo of 0.5, corresponding to approximately 3 x 10 8 cells/ml).
  • Example 8 Condensed SPP cultures incorporate selenomethionine with high efficiency and without toxicity
  • the ability to produce high levels of a single protein in condensed cultures may have applications for structural analysis of proteins by X-ray crystallography or NMR. Heavy atoms are used as phasing centers for multiwavelength anomalous diffraction (MAD) in synchrotron crystallography 4 .
  • MAD multiwavelength anomalous diffraction
  • Selenium addition to proteins through in vivo 5 ' 6 or in vitro 7 incorporation of precursor selenomethionine is the most common approach to enable MAD phasing.
  • in vivo selenomethionine incorporation into E. coli proteins is cytotoxic and leads to growth inhibition; the in vitro system is technically difficult to construct in the laboratory and expensive if purchased commercially.
  • cSPP condensed SPP
  • the target protein alone accounts for virtually all of the new protein synthesis so that the other cellular proteins that typically contribute to cytotoxicity upon selenomethionine labeling are virtually absent. Therefore, cSPP should offer considerable advantages for the incorporation of selenomethionine, other analogs or isotopes used for structural determination.
  • Example 9 Effective fluorophenylalanine substitution without toxicity using cSPP
  • Example 10 cSPP cultures incorporate 15 N with very high efficiency [00100]
  • the signal-to-noise ratio of [ 35 S]methionine labeling of a protein of interest was very high in the SPP system since background protein synthesis is virtually absent ! . Therefore, the use of the SPP system may be ideal for ls N and 13 C isotope labeling of proteins for NMR structural analysis.
  • EnvZB was expressed using 20-fold condensed culture containing [ 15 N] NB 4 Cl.
  • a control culture was also prepared in the identical manner except that the medium instead contained [ 14 N] NH 4 Cl.
  • Peak 4 is the mixture of four peptides: peptide one 10 !5 N, 1 14 N 1 O 13 C and 40 12 C, peptide two 9 15 N, 2 14 N,1°C and 39 i2 C, peptide three 9 !5 N, I 14 N 5 O 13 C and 40 12 C, peptide four 8 15 N, 2 ]4 N,1 13 C and 39 12 C.
  • peptide one containing 10 15 N represents 6% of the total peptides analyzed; peptide two (9 15 N), 38%; peptide three (8 15 N), 31% and peptide four (7 15 N), 15%.
  • E. coli BL21(DE3) transformed with pACYCmazF and pCold(SP-4)envZB(- ACA) was grown in 1000 ml of M9-glucose medium at 37 0 C.
  • the culture was chilled in an ice water bath to quickly reach 15 0 C and incubated at 15 0 C for 45 min to acclimate the cells to cold shock conditions.
  • Cells were harvested and resuspended in 10 ml of chilled M9-glucose medium containing 1 mM IPTG (100 times concentrated). Five ml of this 100-fold condensed culture was transferred into a 25-ml culture flask.
  • the remaining 5 ml was appropriately diluted with chilled M9 medium containing 1 mM IPTG to make 50-, 40-, 30-, 20-, 10- and 1-fold condensed cultures.
  • Each culture was transferred into a 25-ml culture flask and incubated in a water bath shaker at 15 0 C for 21 additional hr to induce both MazF and EnvZB.
  • Cells were collected by centrifugation and resuspended such that each sample comprised an extract from an equal number of cells/ml and equal volumes were loaded and subjected to SDS-PAGE followed by Coomassie Blue staining.
  • EnvZB and CspA were expressed in order to demonstrate the efficiency of incorporation of selenomethionine and F-Phe, respectively.
  • E. coli BL21(DE3) transformed with both pACYCw azF and pCold(SP ⁇ 4)envZB( ⁇ ACA) was grown in 200 ml of M9-glucose medium at 37°C. When the OD 600 reached 0.5, the culture was shifted to 15°C for 45 min to acclimate the cells to cold shock conditions.
  • Cells were harvested and suspended with 5 ml of M9-glucose medium containing Lys (100 ⁇ g/ml), Phe (100 ⁇ g/ml), Thr (100 ⁇ g/ml), He (50 ⁇ g/ml), Leu (50 ⁇ g/ml) and VaI (50 ⁇ g/ml) (40-times concentrated culture). Cultures were incubated at 15°C for 15 min to inhibit endogenous methionine biosynthesis and then 25 ⁇ l of 12 mg/rnl seleno-L-methionine (the final concentration of 60 ⁇ g/ml) and 5 ⁇ l of 1 M IPTG (the final concentration of 1 mM) was added to the culture.
  • M9-glucose medium containing Lys (100 ⁇ g/ml), Phe (100 ⁇ g/ml), Thr (100 ⁇ g/ml), He (50 ⁇ g/ml), Leu (50 ⁇ g/ml) and VaI (50
  • E. coli BL21 (DE3) transformed with pACYCmazF and pCold(SP ⁇ 4)envZ£(- ACA) was grown in 1000 ml of M9-glucose medium at 37°C. When the OD 600 reached 0.5, the culture was shifted to 15°C for 45 min to acclimate the cells to cold shock conditions. 1 mM IPTG was added to induce expression of both MazF and EnvZB followed by a 15 0 C for 3 hr incubation at to eliminate isotope incorporation into background cellular proteins.

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Abstract

La présente invention concerne un système de production de protéines simples dans des cellules vivantes d'E.coli qui utilise les propriétés uniques d'une interférase d'ARNm, par exemple, MazF, une toxine bactérienne qui est une endoribonucléase spécifique d'ARN monocaténaire de d'ACA, qui dégrade de manière efficace et sélective tous la ARNm cellulaires in vivo, entraînant une chute abrupte dans la synthèse protéinique totale. Une expression concomitante de MazF et d'un gène cible génétiquement modifié pour coder pour un ARNm exempt d'ACA entraîne une expression cible soutenue et de niveau très élevée (jusqu'à 90%) en l'absence virtuelle de synthèse protéinique cellulaire d'arrière-plan. De manière caractéristique, la synthèse de cible se poursuit pour au moins quatre jours, indiquant que les cellules retiennent une compétence transcriptionnelle et translationnelle malgré leur interruption de croissance. La technologie de production de protéines simples fonctionne bien pour des protéines de levure et humaines, voire une protéine membranaire intégrale bactérienne. Le nouveau système permet des rapports signal/bruit inédit qui devraient simplifier de manière dramatique des études structurelles et fonctionnelles de protéines précédemment réfractaires mais d'importance biologique. La présente invention concerne également un système de production de protéines simples condensées optimisées.
PCT/US2007/069194 2006-05-17 2007-05-17 Production de protéines simples dans des cellules vivantes par une interférase d'arn messager WO2007137144A2 (fr)

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US8916358B2 (en) 2010-08-31 2014-12-23 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through protease manipulation
US8956833B2 (en) 2010-05-07 2015-02-17 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through enzyme relocation
US9469861B2 (en) 2011-09-09 2016-10-18 Greenlight Biosciences, Inc. Cell-free preparation of carbapenems
US9637746B2 (en) 2008-12-15 2017-05-02 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways
US9688977B2 (en) 2013-08-05 2017-06-27 Greenlight Biosciences, Inc. Engineered phosphoglucose isomerase proteins with a protease cleavage site
US10316342B2 (en) 2017-01-06 2019-06-11 Greenlight Biosciences, Inc. Cell-free production of sugars
US10858385B2 (en) 2017-10-11 2020-12-08 Greenlight Biosciences, Inc. Methods and compositions for nucleoside triphosphate and ribonucleic acid production
US10954541B2 (en) 2016-04-06 2021-03-23 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid
US11274284B2 (en) 2015-03-30 2022-03-15 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid

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SUZUKI ET AL.: 'Single protein production in living cells facilitated by an mRNA interferase' MOLECULAR CELL vol. 18, April 2005, pages 253 - 261 *

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US9637746B2 (en) 2008-12-15 2017-05-02 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways
US8956833B2 (en) 2010-05-07 2015-02-17 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through enzyme relocation
US10006062B2 (en) 2010-05-07 2018-06-26 The Board Of Trustees Of The Leland Stanford Junior University Methods for control of flux in metabolic pathways through enzyme relocation
US10036001B2 (en) 2010-08-31 2018-07-31 The Board Of Trustees Of The Leland Stanford Junior University Recombinant cellular iysate system for producing a product of interest
US8916358B2 (en) 2010-08-31 2014-12-23 Greenlight Biosciences, Inc. Methods for control of flux in metabolic pathways through protease manipulation
US9469861B2 (en) 2011-09-09 2016-10-18 Greenlight Biosciences, Inc. Cell-free preparation of carbapenems
US9688977B2 (en) 2013-08-05 2017-06-27 Greenlight Biosciences, Inc. Engineered phosphoglucose isomerase proteins with a protease cleavage site
US10421953B2 (en) 2013-08-05 2019-09-24 Greenlight Biosciences, Inc. Engineered proteins with a protease cleavage site
US11274284B2 (en) 2015-03-30 2022-03-15 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid
US10954541B2 (en) 2016-04-06 2021-03-23 Greenlight Biosciences, Inc. Cell-free production of ribonucleic acid
US10316342B2 (en) 2017-01-06 2019-06-11 Greenlight Biosciences, Inc. Cell-free production of sugars
US10577635B2 (en) 2017-01-06 2020-03-03 Greenlight Biosciences, Inc. Cell-free production of sugars
US10704067B2 (en) 2017-01-06 2020-07-07 Greenlight Biosciences, Inc. Cell-free production of sugars
US10858385B2 (en) 2017-10-11 2020-12-08 Greenlight Biosciences, Inc. Methods and compositions for nucleoside triphosphate and ribonucleic acid production

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