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Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system

A technology of unnatural amino acids and amino acids, which is applied in the field of single-loading system of introducing unnatural amino acids into proteins by using in vitro protein synthesis system, which can solve the problems of high efficiency, poor feasibility, low efficiency, etc.

Inactive Publication Date: 2012-02-08
SUTRO BIOPHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these purely reconstituted translation systems require purified translation components, which are poorly feasible, expensive and not proven to be efficient except for research purposes
[0021] There is a need in the art for the incorporation of unnatural amino acids into elongated polypeptide chains in which an orthogonal tRNA / aminoacyl tRNA synthetase pair can be avoided, the natural isofunctional tRNA aminoacylated with the unnatural amino acid recognizes the sense codon and subsequently Incorporation of unnatural amino acids into extended polypeptide chains at positions defined by sense codons enables the incorporation of many unnatural amino acids at defined positions and enables the use of natural cell-free protein synthesis systems, which avoid purely reconstituted in vitro translation System is poor, expensive and inefficient

Method used

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  • Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system
  • Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system
  • Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system

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Embodiment 1 1

[0207] Example 1. General method

[0208] Standard methods in molecular biology have been described (Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning Cloning, 3yd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif). The standard method is also published in Bindereif, & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany, which describes detailed methods for RNA manipulation and analysis.

[0209] Methods for protein purification, chromatography, electrophoresis, centrifugation, and crystallization have been described (Coligan et al. (2000) Current Protocols in Protein Science, Vol.1, John Wiley and Sons, Inc., New York ). Methods for cell-free synthesis are described in Spirin & Swartz (2008) Cell-free Pr...

Embodiment 2

[0210] Example 2. In vitro transcription and isolation of tRNA 2', 3'-cyclic phosphoric acid

[0211] Isofunctional tRNAs aminoacylated with unnatural amino acids can be generated by in vitro transcription from the designed tRNA-HDV ribozyme template DNA shown in the example in Figure 1, followed by purification by size exclusion chromatography (SEC) to enzymatically remove 2′,3′-cyclic phosphate, and use engineered tRNA synthetase to load tRNA with unnatural amino acid (nnAA), the process as figure 2 shown. To optimize transcription yield, 4 different in vitro transcription protocols were tested image 3 and Figure 4 tRNA transcripts indicated. All 4 different in vitro transcription protocols gave similar tRNA yields. Transcription optimization is usually performed at 37°C in a 50 μL reaction for 2-3 h. The reaction conditions are as follows: (1) 40mM HEPES (pH 7.9), 10mM DTT, 10mM MgCl 2 , 2.5mM spermidine, 4U / ml pyrophosphatase, 0.4U / ml superRNAse-in (Ambion), 20mM N...

Embodiment 3

[0216] Example 3. Dephosphorylation of tRNA 2',3'-cyclic phosphates to release active tRNA

[0217] T4 polynucleotide kinase (PNK), which is required for the removal of 2′,3′-cyclic phosphates from HDV ribozyme-cleaved tRNAs, was produced as follows (cf. figure 2 and 5 ). The PNK gene (DNA 2.0, Menlo Park., CA) with an N-terminal 6-histidine tag was genetically synthesized and cloned into plasmid pYD317. BL21(DE3) cells were transformed with plasmid T4PNK_pYD317. These cells were grown for 18 hours in a Braun 10L fermentor to a final OD of 21 on autoinduction medium (Studier F.W. (2005) Protein Expr. Purif., 41:207-234). Cells were harvested by centrifugation and a 240 g cell pellet was obtained. A 40 g cell pellet was resuspended in 500 ml buffer A (50 mM Tris (pH 7.8), 300 mM KCl, 10 mM imidazole), lysed by homogenization, clarified by centrifugation, and loaded onto a 35 mL Ni-IMAC column. Wash the column with 5 column volumes of buffer A. Buffer A plus 0.5 mM BME, 30...

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Abstract

This invention provides for a novel means of incorporating non-native amino acids into preselected positions of a protein using a cell-free synthesis system. The methods involve the use of non-orthogonal, native isoaccepting sense tRNAs that are encoded by the genetic code. Such methods allow for numerous non-native amino acids to be incorporated through the use of sense codons without having to rely upon orthogonal tRNA-synthetase pairs.

Description

[0001] Cross References to Related Applications [0002] Pursuant to 35 U.S.C. §1.119(e), this application claims the benefit of U.S. Application Nos. 61 / 144,097, 61 / 144,083, and 61 / 144,030, all filed January 12, 2009, and for all purposes will Each application is incorporated by reference in its entirety. [0003] Statement Concerning Rights to Inventions Made Under Federally Sponsored Research and Development [0004] Not applicable [0005] Appendices involving "sequence listings", tables or computer program listings submitted on CD-ROM [0006] Not applicable Background of the invention [0007] Protein synthesis is a fundamental biological process that is the basis for the development of peptide therapeutics, vaccines, diagnostics, and industrial enzymes. With the advent of recombinant DNA (rDNA) technology, it has become possible to use the catalytic machinery of cells to produce desired proteins. This can be achieved within a cellular environment or in vitro using ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/04C12P19/34
CPCC07K1/02C12P21/02
Inventor 阿列克谢·M·沃洛申詹姆士·F·扎瓦达丹尼尔·戈德克里斯托弗·詹姆士·默里詹姆士·爱德华·罗泽拉纳森·与特尔殷钢
Owner SUTRO BIOPHARMA INC
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