Single protein production in living cells facilitated by a messenger RNA interferase

A cell protein and living cell technology, applied in the field of endoribonuclease, can solve the problems of cell death, severe reduction of protein synthesis, etc.

Inactive Publication Date: 2007-10-10
UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Thus, MazF expression causes almost complete degradation of mRNA, leading to a severe reduction in protein synthesis and ultimately cell death (Zhang et al, Mol. Cell 12:913-23(2003))

Method used

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  • Single protein production in living cells facilitated by a messenger RNA interferase
  • Single protein production in living cells facilitated by a messenger RNA interferase
  • Single protein production in living cells facilitated by a messenger RNA interferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Effect of MazF on Inducing Cellular Protein Synthesis

[0057] Large intestine harboring pACYCmazF was transformed with pColdI(SP-1) eotaxin (left panel of A and B) or pColdI(SP-2) eotaxin (right panel of B and C) Bacillus BL21 (DE3). Cells were grown in M9 glucose medium at 37°C. When OD 600 When 0.5 was reached, the culture was transferred to 15°C, and after acclimatization of the cells to the low temperature by incubation at 15°C for 45 minutes, IPTG (1 mM) was added to induce eotaxin and MazF expression (0 time). At the time points indicated above each gel, use 35 Cells were pulse-labeled with S-methionine for 15 minutes, and total cellular protein was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.

[0058] The mazF gene was cloned into pACYC (a low copy number plasmid containing the IPTG-inducible phage T7 promoter) to generate pACYCmazF. Cloning techniques can generally be found in J. Sambrook and D.W. Russel...

Embodiment 2

[0061] Example 2: Expression of ACA-free mRNA in MazF-induced cells

[0062] We speculate that if an mRNA engineered not to contain the ACA sequence is expressed in MazF-induced cells, this mRNA may be stably maintained in the cell such that the protein encoded by the mRNA can be produced without any other cellular protein. To test this possibility, we synthesized the gene for human eotaxin by eliminating all ACA sequences within the gene without altering the amino acid sequence. Figure 2A shows the amino acid sequence of human eotaxin and the nucleotide sequence of its gene. In the following experiments, nucleotide sequences were designed using preferred E. coli codons, and those triplets underlined were changed to ACA. Among the 64 possible triplet sequences, the ACA sequence is unique in that it can be altered to other MazF non-cleavable sequences without changing the amino acid sequence of the protein, regardless of the in-frame position of the ACA sequence.

[0063] The...

Embodiment 3

[0069] Example 3: Negative effect of ACA sequence on protein production

[0070] To verify that MazF-induced intracellular eotaxin production observed in Figure 1 was due to the ACA-free mRNA of eotaxin, into the eotaxin gene Five natural ACA sequences were added without changing their amino acid sequence as shown in Figure 2A. Utilize pColdI(SP-2) to express the eotaxin gene, and process and use [ 35 S]-methionine labeled cells. The left panel shows the results for the eotaxin gene without ACA (same as the left panel of Figure IB) and the right panel shows the results for the eotaxin gene with 5 ACA sequences.

[0071] When the gene was expressed together with pColdI(SP-1) and pACYCmazF under the same conditions as described in Figure 1, only the first 2 hours were observed compared to mRNA expression without ACA (Figure 2B, left panel). Low levels of eotaxin production were reached, after which levels further decreased to background levels (Fig. 2B, right panel).

[0072...

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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

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 60 / 624,976, filed November 4, 2004, entitled "Facilitation of Single Protein Production in Living Cells by mRNA Interferases," filed by Inouye et al. The entire contents of which are hereby incorporated into this application by reference. technical field [0003] The present invention relates to a system for promoting the production of single proteins in living cells by mRNA interferases, which are single-stranded RNAs, and sequence-specific endoribonucleases. Background technique [0004] Most bacteria contain suicide genes whose expression after exposure to cellular stress causes growth arrest and eventual death (reviewed in Elenberg-Kulka and Gerdes, Ann. Rev. Microbiol. 53:43-70 (1999 ); Engelberg-Kulka et al., Trends Microbiol. 12:66-71 (2004)). These toxin genes are often co-expressed with their cognate antitoxin genes in the same operon (termed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12P21/00
CPCC12P21/02C12N9/22C12N5/00C12N5/10C12P21/06
Inventor I·政赖J·张M·铃木
Owner UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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