WO2020135623A1 - 通过Edc3基因敲除改变体外蛋白合成能力的方法及其应用 - Google Patents

通过Edc3基因敲除改变体外蛋白合成能力的方法及其应用 Download PDF

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WO2020135623A1
WO2020135623A1 PCT/CN2019/128833 CN2019128833W WO2020135623A1 WO 2020135623 A1 WO2020135623 A1 WO 2020135623A1 CN 2019128833 W CN2019128833 W CN 2019128833W WO 2020135623 A1 WO2020135623 A1 WO 2020135623A1
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cell
protein synthesis
synthesis system
edc3
cells
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郭敏
姜灵轩
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康码（上海）生物科技有限公司
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to the knockout of Edc3 gene can enhance the protein synthesis capacity of eukaryotic cell-free protein reaction system.
  • the in vitro cell-free protein synthesis system refers to an in vitro system that uses exogenous mRNA or DNA as a template to synthesize proteins using an enzyme system of cell extracts.
  • the cell-free protein reaction system Compared with the traditional in vivo recombinant expression system, the cell-free protein reaction system has many advantages, such as the ability to express cytotoxic proteins or special proteins containing unnatural amino acids, and the use of its simple operation can be used for high-throughput drug screening and Proteomics research.
  • the currently used cell-free protein synthesis systems include prokaryotic E. coli system, eukaryotic wheat germ extract, rabbit reticulocyte lysate and yeast cell extract system.
  • yeast cells can be obtained in large quantities through fermentation, which has the advantages of eukaryotic system protein modification and large-scale industrial application.
  • yeast cells can be obtained in large quantities through fermentation, which has the advantages of eukaryotic system protein modification and large-scale industrial application.
  • the Edc3 (Enhancer of mRNA-decapping protein 3) gene is relatively conserved in eukaryotes, and its encoded protein product can promote the function of the mRNA 5'decapping enzyme complex, and the mRNA with the 5'cap structure removed is more likely to be used by cells Exonuclease degrades from 5'to 3'. In yeast cells, Edc3 and Pat1 bind to mRNA whose translation is inhibited.
  • Edc3 can bind to RNA and recruit Dhh1 and Scd6 proteins that inhibit translation and the exonuclease Xrn1 that degrades mRNA. Therefore, it is speculated that Edc3 protein may have an inhibitory effect on the translation of exogenous mRNA template and cause its degradation. There is the ability to inhibit RPS28B gene transcription resulting in decreased ribosome function. Therefore, in the present invention, by knocking out Edc3, it is tested whether the knockout has an effect on the protein synthesis ability of the cell-free protein synthesis system in vitro.
  • the present invention provides a method for improving the protein synthesis ability of an in vitro cell-free protein synthesis system, which improves the protein synthesis ability of the cell-free protein synthesis system from the molecular level, thereby enabling further cost-saving and simple operation of the cell-free protein synthesis system the goal of.
  • the present invention mainly includes the following aspects:
  • a method for changing the protein synthesis capacity of an in vitro cell-free protein synthesis system includes the following steps:
  • step (3) The cell lysate or cell extract obtained in step (2) is used in a cell-free protein synthesis system in vitro.
  • the eukaryotic cell is one of mammalian cells, plant cells, yeast cells, insect cells, or any combination thereof.
  • yeast cell is selected from one of Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces or any combination thereof.
  • yeast of the genus Kluyveromyces is selected from one of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces doublyi or any combination thereof.
  • an in vitro cell-free protein synthesis system comprising at least the following components: a cell lysate or a cell extract, the cell lysate or a cell extract prepared from a modified strain of ⁇ Edc3, said The ⁇ Edc3 modified strain is obtained by removing the original Edc3 gene in the eukaryotic cells to be modified by gene editing technology.
  • the synthesis system of the second aspect further includes one or more components selected from the group consisting of a substrate for synthesizing RNA, a substrate for synthesizing proteins, polyethylene glycol or the like , Magnesium ion, potassium ion, buffer, RNA polymerase, energy regeneration system, dithiothreitol (DTT), optional water or aqueous solvent.
  • a substrate for synthesizing RNA a substrate for synthesizing proteins
  • polyethylene glycol or the like Magnesium ion, potassium ion, buffer, RNA polymerase, energy regeneration system, dithiothreitol (DTT), optional water or aqueous solvent.
  • DTT dithiothreitol
  • a method for synthesizing a foreign protein includes the following steps:
  • the method of the third aspect further includes: (iii) isolating or detecting the foreign protein.
  • suitable conditions include a reaction temperature of 20-35°C, preferably 20-30°C, and more preferably 25°C.
  • the incubation period is specifically 0.5-20h, preferably 1-18h, more preferably 2-15h, more preferably 3-12h; the above reaction time can be determined artificially according to the specific situation, and can also be 3- 15h can also be 3-20h, or it can be a specific time point, such as 3h, 5h, 10h, 15h, 18h, 20h.
  • kits comprising a container and components in the in vitro cell-free protein synthesis system according to the second aspect located in the container.
  • the present invention verifies for the first time that the knockout of Edc3 gene can improve the protein synthesis capacity of the cell-free protein synthesis system in vitro through gene directed transformation and activity measurement;
  • the present invention uses CRISPR-Cas9 gene editing technology to knock out Edc3 gene, thereby changing the in vitro protein synthesis ability;
  • the formed in vitro cell-free protein synthesis system can further achieve the goals of cost reduction and simple operation.
  • Figure 1 is a schematic diagram of pCas9_Edc3_gRNA1 map.
  • the plasmid carries the K.lactis SNR52 promoter and SNR52 terminator, and carries a kana selection marker.
  • Figure 2 is a schematic diagram of pCas9_Edc3_gRNA2 map.
  • Figure 3 is a schematic diagram of the plasmid map of pKMD1- ⁇ Edc3.
  • the start codon 879 bp upstream of KlEdc3 is HR1, and the 888 bp downstream of the stop codon is HR2.
  • the plasmid carries the Amp selection marker.
  • Figure 4 is a comparison of the amount of green fluorescent protein synthesis in the cell-free protein synthesis system of the ⁇ Edc3 strain and the wild-type strain in vitro.
  • the inventors found that the cells knocked out of the Edc3 gene can significantly improve the protein synthesis capacity of the cell-free protein synthesis system in vitro and increase the expression yield of foreign proteins compared with wild-type unmodified cells.
  • the term “about” may refer to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the 5'capping complex of mRNA is a protease complex that removes the 5'cap structure of mRNA in cells.
  • the complex mainly includes the catalytic subunits Dcp1/Dcp2 and the activating factors Pat1, Edc3, Dhh1, Scd6, and the exonuclease Xrn1 responsible for the 5'to 3'degradation of mRNA.
  • the mRNA decapping and degradation processes of the complex are mainly as follows: first, the translation factors including eIF4E and eIF4G bound to the cap are detached from the mRNA, thereby exposing the 5'cap structure; second, the decapping enzyme complex passes and Skeletal proteins such as Edc3 and Pat1 bound by mRNA are recruited to the mRNA; finally, the Dcp2 catalyzed decap reaction, the mRNA with the cap structure removed is rapidly degraded by Xrn1.
  • the present invention provides an in vitro cell-free protein synthesis system expressing foreign proteins.
  • the synthesis system mainly includes at least: cell lysate or cell extract; the cell lysate or cell extract is derived from the Edc3 knockout Except for engineered cells, the engineered cell extract does not contain the expression product of Edc3 gene, that is, the cell lysate or cell extract is prepared for ⁇ Edc3 modified strain.
  • the synthesis system further includes one or more components selected from the group consisting of substrates for protein synthesis, substrates for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, and buffers , Energy regeneration system, polyethylene glycol (PEG) or its analogs, dithiothreitol (DTT) and optional solvent, the solvent is water or aqueous solvent.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is one of mammalian cells, plant cells, yeast cells, insect cells, or any combination thereof.
  • the yeast cell is selected from one or a combination of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces; Kluyveromyces yeast is selected from Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces doubly One of yeasts or any combination thereof; preferably, the yeast cell is Kluyveromyces cerevisiae, more preferably Kluyveromyces lactis.
  • the cell extract is an aqueous extract of yeast cells.
  • the cell extract does not contain yeast endogenous long-chain nucleic acid molecules.
  • the substrate for synthesizing RNA includes one or a combination of nucleoside monophosphate and nucleoside triphosphate.
  • the substrate for the synthetic protein includes: 20 kinds of natural amino acids and unnatural amino acids.
  • the magnesium ion is derived from a magnesium ion source, and the magnesium ion source is selected from the group consisting of one of magnesium acetate and magnesium glutamate or a combination thereof.
  • the potassium ion is derived from a potassium ion source, and the potassium ion source is selected from the group consisting of one of potassium acetate and potassium glutamate or a combination thereof.
  • the energy regeneration system is selected from the group consisting of phosphocreatine/phosphocreatine enzyme system, one of glycolysis pathway and its intermediate product energy system, sucrose or a combination thereof.
  • the buffering agent is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the protein synthesis system contains polyethylene glycol (PEG) or its analogs.
  • concentration of polyethylene glycol or its analogue is not particularly limited. Generally, the concentration (w/v) of polyethylene glycol or its analogue is 0.1-8%, preferably, 0.5-4%, more preferably, 1-2%, based on the total weight of the protein synthesis system.
  • a representative PEG is selected from the group consisting of one of PEG3000, PEG3350, PEG6000, PEG8000, or a combination thereof.
  • the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.
  • the molecular weight is 3000- 10,000 polyethylene glycol.
  • the protein synthesis system includes: yeast cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenine nucleoside triphosphate (ATP), guanine nucleus Glycoside triphosphate (GTP), Cytosine nucleoside triphosphate (CTP), Thymine nucleotide triphosphate (TTP), Amino acid mixtures, Phosphocreatine, Dithiothreitol (DTT), Phosphocreatine kinase, RNA polymerization Enzymes, polyethylene glycol, sucrose.
  • yeast cell extract 4-hydroxyethylpiperazineethanesulfonic acid
  • potassium acetate magnesium acetate
  • adenine nucleoside triphosphate ATP
  • GTP guanine nucleus Glycoside triphosphate
  • CTP Cytosine nucleoside triphosphate
  • TTP Thymine nucleotide triphosphate
  • the cell extract does not contain intact cells.
  • Typical cell extracts include ribosomes for protein translation, transport RNA, aminoacyl tRNA synthetase, initiation factors and elongation factors required for protein synthesis And the termination release factor.
  • the cell extract contains some other proteins in the cytoplasm of the cell, especially soluble proteins.
  • the protein content of the cell extract is 20-100 mg/ml, preferably 50-100 mg/ml.
  • the method for determining the protein content is the Coomassie brilliant blue determination method.
  • the preparation method of the cell extract is not limited, and a preferred preparation method includes the following steps:
  • the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.
  • the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000g, preferably 8000-30000g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is 0.5min-2h, preferably 20min-50min.
  • the temperature of the centrifugation is not particularly limited.
  • the centrifugation is performed at 1-10°C, preferably at 2-6°C.
  • the washing treatment method is not particularly limited.
  • a preferred washing treatment method is to use a washing liquid to perform treatment at a pH of 7-8 (preferably, 7.4).
  • the washing liquid is not particularly limited.
  • the typical washing solution is selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.
  • the manner of the cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (eg, liquid nitrogen low temperature) disruption.
  • a preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (eg, liquid nitrogen low temperature) disruption.
  • the nucleoside triphosphate mixture in the protein synthesis system is adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate.
  • concentration of various single nucleotides is not particularly limited, and the concentration of each single nucleotide is generally 0.5-5 mM, preferably 1.0-2.0 mM.
  • the amino acid mixture in the protein synthesis system may include natural or unnatural amino acids, and may include D-type or L-type amino acids.
  • Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the concentration of each amino acid is usually 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.
  • the in vitro cell-free protein synthesis system further contains sucrose, and the concentration of the sucrose is 0.03-40 wt%, preferably, 0.08-10 wt%, more preferably, 0.1-5 wt%, based on the protein The total weight of the synthetic system.
  • a particularly preferred in vitro cell-free protein synthesis system in addition to yeast cell extracts, contains the following components: 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid with a pH of 7.4, 30-150 mM potassium acetate, 1.0- 5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture, 0.08-0.24mM amino acid mixture, 25mM creatine phosphate, 1.7mM dithiothreitol, 0.27mg/mL phosphocreatine kinase, 1%-4% poly Ethylene glycol, 0.5%-2% sucrose, 0.027-0.054mg/mL T7 RNA polymerase.
  • the reagents and materials in the embodiments of the present invention are commercially available products.
  • the present invention takes Kluyveromyces lactis (K. lactis) as an example, but the same design, analysis, and experimental methods are also applicable to lower eukaryotic cells such as other yeast and higher animal cells.
  • the genetic modification method in the present invention is CRISPR-Cas9 technology, but it is not limited thereto, and may be any known and existing genetic modification method.
  • the gene number of Edc3 in K. lactis cells was determined to be KLLA0A11308g.
  • the principle of gRNA selection in this embodiment is that the GC content is moderate (40%-60%), and the existence of polyT structure is avoided.
  • the sequence of the K1Edc3 gene gRNA1 is TCAAATTGAGATCGAATTGA
  • the sequence of the K1Edc3 gene gRNA2 is GGACATATACCCGGGTTTCT (the nucleic acid coding sequence of the K1Edc3 gene is SEQ No. 1; the corresponding amino acid sequence is SEQ No. 2).
  • KlEdc3gRNA1 The plasmid construction and transformation methods of KlEdc3gRNA1 are as follows: using primers pCas9-Edc3-gRNA1-PF: TCAAATTGAGATCGAATTGAGTTTTAGAGCTAGAAATAGC and pCas9-Edc3-gRNA1-PR: TCAATTCGATCTCAATTTGAAAAGTCCCATTCGCCACCCG, using pCAS plasmid as a template for PCR amplification. Take 17 ⁇ L of the amplified product, add 0.2 ⁇ L Dpn I, 2 ⁇ L 10 ⁇ digestion buffer, mix well and mix in a 37°C water bath for 3 hours.
  • KlEdc3gRNA2 The construction method of KlEdc3gRNA2 is as follows: using primers pCas9-Edc3-gRNA2-PF: GGACATATACCCGGGTTTCTGTTTTAGAGCTAGAAATAGC and pCas9-Edc3-gRNA2-PR: AGAAACCCGGGTATATGTCCAAAGTCCCATTCGCCACCCG, using pCAS plasmid as a template for PCR amplification.
  • the transformation and identification methods are the same as above, and the positive plasmid is named pCas9_Edc3_gRNA2.
  • primers pMD18T-PF ATCGTCGACCTGCAGGCATG and pMD18T-PR: ATCTCTAGAGGATCCCCGGG for PCR amplification, take the amplified product 17 ⁇ L, add 1 ⁇ L DpnI, 2 ⁇ L 10 ⁇ digestion buffer, after mixing, 37°C water bath for 3h to obtain the plasmid Skeleton linear fragment pMD18T-vector.
  • KlEdc3-HR1-PF CAGGAAACAGCTATGACTACCCGGGGATCCTCTAGAGATGGTAGCTCCAATAATCCAAG
  • KlEdc3-HR1-PR ACACATCTATCTTTACAGATATAAGTCAAAAGTGACTTGTTCTATAAACT PCR amplification
  • Primer KlEdc3-HR2-PF AGAAGCTATAAGTTTATAGAACAAGTCACTTTTGACTTATATCTGTAAAG and KlEdc3-HR2-PR: GTAAAACGACGGCCAGTTGCATGCCTGCAGGTCGACGATAACTCCAACAACGACGTCAC for PCR amplification, the product name is Edc3-HR2;
  • the preparation method of the cell extract includes the following steps:
  • the solid-liquid separation method is not particularly limited, and the method selected in this embodiment is centrifugation.
  • the centrifugation condition is 30,000 ⁇ g; the centrifugation time is 30 min; centrifugation is carried out at 4°C.
  • the washing treatment method is not particularly limited.
  • the washing treatment method selected in this embodiment is to use a washing solution for processing at a pH of 7.4.
  • the washing solution is not particularly limited.
  • the typical washing solution is selected from the following group: 4 -Hydroxyethylpiperazine potassium sulfonate, potassium acetate, magnesium acetate, or a combination thereof. In this embodiment, potassium acetate is selected.
  • a preferred cell disruption process includes high-pressure disruption and freeze-thaw (eg, liquid nitrogen low temperature) disruption.
  • 15ng/ ⁇ L enhanced green fluorescent protein DNA was added to the above system, mixed and placed in an environment of 20-30°C for 3h.
  • Enhanced green fluorescent protein (eGFP) activity measurement After the reaction is completed, add 10 ⁇ L of the reaction solution to a 96-well white plate or a 384-well white plate and immediately place it on the Envision 2120 multi-functional microplate reader (Perkin Elmer) to read and detect enhanced green Fluorescent protein activity, relative fluorescence unit value (Relative Fluorescence Unit, RFU) as the activity unit, as shown in Figure 4.
  • eGFP Enhanced green fluorescent protein activity measurement
  • ⁇ Edc3 represents Edc3 knockout Kluyveromyces lactis cell line
  • WT represents wild-type Kluyveromyces lactis cell line.
  • the reaction time is 3h
  • the average RFU of ⁇ Edc3 is 1386
  • the average RFU of WT is 946
  • ⁇ Edc3 is about 1.47 times that of WT.
  • the comparative data shows that knocking out the Edc3 gene through gene editing technology can significantly improve the protein synthesis capacity of the cell-free protein synthesis system in vitro.
  • the Edc3 gene knockout cell line of the present invention has an increased protein synthesis capacity of about 47% in a cell-free protein synthesis system in vitro.

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Abstract

提供一种改变体外无细胞蛋白合成体系的蛋白合成能力的方法，其在真核生物的基因组中通过基因编辑技术将Edc3基因敲除，并将其用于体外无细胞蛋白合成体系。经试验验证，Edc3基因敲除能显著提高无细胞蛋白合成体系的蛋白表达能力。同时提供包含利用该基因改造方法进行改造的基因工程细胞的无细胞蛋白质合成体系及其蛋白合成方法及相应的试剂盒。

Description

通过Edc3基因敲除改变体外蛋白合成能力的方法及其应用 技术领域
本发明涉及生物技术领域，具体涉及Edc3基因敲除可以增强真核生物无细胞蛋白反应体系的蛋白合成能力。
背景技术
体外无细胞蛋白合成体系是指以外源的mRNA或者DNA为模板，利用细胞抽提物的酶系中来合成蛋白质的体外系统。与传统的体内重组表达系统相比，无细胞蛋白反应体系有很多优点，如可以表达具有细胞毒性的蛋白或者含有非天然氨基酸的特殊蛋白质，而且利用其操作简便性可以进行高通量药物筛选和蛋白质组学研究。目前常用的无细胞蛋白质合成系统包括原核的大肠杆菌系统，真核的麦胚提取物、兔网织红细胞裂解物以及酵母细胞抽提物系统。其中酵母细胞可以通过发酵途径大量获取，兼具真核系统的蛋白修饰功能和大规模工业化应用的优势。但是目前市场上还没有真正可产业化的体外无细胞蛋白合成系统，不高的蛋白合成能力，昂贵的合成成本是制约这一技术的主要因素之一。
Edc3(Enhancer of mRNA-decapping protein 3)基因在真核生物中相对保守，其编码的蛋白产物能促进mRNA 5’去帽酶复合体的功能，去除5’帽子结构的mRNA更容易被细胞中的核酸外切酶进行5’到3’的降解。在酵母细胞中，Edc3和Pat1与翻译被抑制的mRNA结合，它们作为骨架蛋白进一步招募其他蛋白，比如Dhh1，Scd6，Dcp1/Dcp2,Lsm1-7和Xrn1等；这些蛋白形成的复合体进一步抑制mRNA的翻译，同时使得mRNA上结合的翻译因子进一步与mRNA脱离；然后蛋白复合体对mRNA 5’进行去帽，并进一步降解。除了作为mRNA 5’去帽的激活因子以外，在酵母中将Edc3基因敲除能上调YRA1和RPS28B两个基因的mRNA，其中YRA1参与到mRNA出核的早期过程，RPS28B是核糖体40S亚基的一个组成成分。
综上所述，Edc3能和RNA结合并且招募抑制翻译的Dhh1和Scd6蛋白以及降解mRNA的外切酶Xrn1，故推测Edc3蛋白可能对外源的mRNA模板翻 译有抑制作用并导致其降解，同时Edc3的存在能抑制RPS28B基因转录从而导致核糖体的功能下降。故本发明通过敲除Edc3，测试其敲除是否对体外无细胞蛋白合成体系的蛋白合成能力产生影响。
参考文献
1.Parker R.RNA degradation in Saccharomyces cerevisae.Genetics，2012.191:671-702.
2.Feng He,Chunfang Li,Bijoyita Roy,Allan Jacobson.Yeast Edc3 targets RPS28B mRNA for decapping by binding to a 3’untranslated region decay-inducing regulatory element.Molecular and Cellular Biology,2014.34:1438–1451.
3.Nissan T.,Rajyaguru P.,She M.,Song H.,Parker R.Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.Mol.Cell,2010.39:773-783.
4.Harigaya Y.,Jones B.N.,Muhlrad D.,Gross J.D.,Parker R.Identification and analysis of the interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae.Mol.Cell.Biol,2010.30:1446-1456.
5.Sharif H.,Ozgur S.,Sharma K.,Basquin C.,Urlaub H.,Conti E.Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1,Edc3 and RNA in mutually exclusive interactions.Nucleic Acids Res,2013.41:8377-8390.
6.Kshirsagar M.,Parker R.Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae.Genetics,2004.166:729-739.
7.Dong S.,Li C.,Zenklusen D.,Singer R.H.,Jacobson A.,He F.YRA1 autoregulation requires nuclear export and cytoplasmic Edc3p-mediated degradation of its pre-mRNA.Mol.Cell,2007.25:559-573.
8.Kolesnikova O.,Back R.,Graille M.,Seraphin B.Identification of the Rps28 binding motif from yeast Edc3 involved in the autoregulatory feedback loop controlling RPS28B mRNA decay.Nucleic Acids Res,2013.41:9514-9523.
9.He F.,Li C.,Roy B.,Jacobson A.Yeast Edc3 targets RPS28B mRNA for decapping by binding to a 3'untranslated region decay-inducing regulatory element.Mol.Cell.Biol,2014.34:1438-1451.
发明内容
鉴于此，本发明提供一种提高体外无细胞蛋白合成体系的蛋白合成能力的方法，从分子水平提高无细胞蛋白合成体系的蛋白合成能力，从而能够进一步实现无细胞蛋白合成体系节约成本、操作简单的目的。
本发明主要包括以下几个方面：
第一方面，提供一种改变体外无细胞蛋白合成体系的蛋白合成能力的方法，所述方法包括以下步骤：
(1)将待改造的真核细胞中原始存在的Edc3基因通过基因编辑技术去除，获得ΔEdc3改造菌株；
(2)以上述ΔEdc3改造菌株制备细胞裂解液或细胞提取物；
(3)将步骤(2)获得的细胞裂解液或细胞提取物用于体外无细胞蛋白质合成体系。
进一步的，所述真核细胞为哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞之一或其任意组合。
进一步的，所述酵母细胞选自酿酒酵母、毕氏酵母、克鲁维酵母之一或其任意组合。
进一步的，所述克鲁维酵母属酵母选自乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母之一或其任意组合。
第二方面，提供一种体外无细胞蛋白合成体系，所述合成体系至少包括以下组分：细胞裂解液或细胞提取物，所述细胞裂解液或细胞提取物由ΔEdc3改造菌株制备获得，所述ΔEdc3改造菌株为将待改造的真核细胞中原始存在的Edc3基因通过基因编辑技术去除获得。
进一步的，第二方面的所述合成体系还包括选自下组的一种或多种组分：用于合成RNA的底物，用于合成蛋白的底物，聚乙二醇或其类似物，镁离子，钾离子，缓冲剂，RNA聚合酶，能量再生系统，二硫苏糖醇(DTT)，任选的水或水性溶剂。
第三方面，提供一种合成外源蛋白的方法，所述方法包括以下步骤：
(i)提供第二方面所述的体外无细胞蛋白合成体系；
(ii)添加编码外源蛋白的DNA分子模板，在适合的条件下，孵育一段时间，合成所述的外源蛋白。
进一步的，第三方面的所述方法还包括：(iii)分离或检测所述外源蛋白。
进一步的，合适的条件包括反应温度为20-35℃，优选的为20-30℃，更优选的为25℃。
进一步的，孵育一段时间具体为0.5-20h，优选的为1-18h，更优选的为2-15h，更优选的为3-12h；上述反应时间可根据具体情况人为确定，也可以为3-15h，也可以为3-20h，也可以为具体的时间点，如3h、5h、10h、15h、18h、20h。
第四方面，提供一种试剂盒，所述的试剂盒包括容器以及位于所述容器中的第二方面所述的体外无细胞蛋白合成体系中的组分。
本发明的主要优点包括：
(1)本发明通过基因定向改造以及活性测定，首次验证敲除Edc3基因可以提高体外无细胞蛋白合成体系的蛋白合成能力；
(2)本发明通过CRISPR-Cas9基因编辑技术对Edc3进行基因敲除改造，从而改变体外蛋白质合成能力；
(3)通过体外无细胞蛋白合成体系验证基因敲除对蛋白表达的影响，并依托无细胞蛋白体系的低成本、试验方便等优点，为基因定向改造提供试验研究平台。
(4)所形成的体外无细胞蛋白合成体系能够进一步实现降低成本、操作简单的目的。
附图说明
图1是pCas9_Edc3_gRNA1图谱示意图。该质粒带有K.lactis SNR52启动子和SNR52终止子，并带有kana筛选标记。
图2是pCas9_Edc3_gRNA2图谱示意图。
图3是pKMD1-ΔEdc3的质粒图谱示意图。KlEdc3的起始密码子上游879bp是HR1，终止密码子下游的888bp是HR2，质粒带有Amp筛选标记。
图4是ΔEdc3菌株和野生型菌株体外无细胞蛋白合成体系绿色荧光蛋白合成量的比较。
具体实施方式
本发明人经过广泛而深入的研究，发现Edc3基因敲除的细胞与野生型未改造细胞相比，能显著提高体外无细胞蛋白合成体系的蛋白合成能力，提高外源蛋白的表达产量。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
5’去帽复合体(decapping complex)
mRNA的5’去帽复合体是细胞中对mRNA的5’帽子结构进行去除的蛋白酶复合体。在酵母细胞中，该复合体主要包括催化亚基Dcp1/Dcp2和激活因子Pat1，Edc3，Dhh1，Scd6，以及负责对mRNA进行5’到3’降解的外切酶Xrn1。目前对该复合体参与的mRNA去帽以及降解过程主要为：首先，和帽子结合的翻译因子包括eIF4E和eIF4G从mRNA上脱离，从而暴露出5’帽子结构；其次，去帽酶复合体通过和mRNA结合的Edc3和Pat1等骨架蛋白被招募到mRNA上；最后就是具有Dcp2催化去帽反应，去掉帽子结构的mRNA迅速被Xrn1进行降解。
体外无细胞蛋白合成体系
本发明提供了一种表达外源蛋白的体外无细胞蛋白合成体系，所述合成体系主要至少包括：细胞裂解液或细胞提取物；所述细胞裂解液或细胞提取物来自于所述的Edc3敲除工程细胞，该工程细胞提取物中不含有Edc3基因的表达产物，即为ΔEdc3改造菌株制备细胞裂解液或细胞提取物。
进一步的，所述合成体系还包括选自下组的一种或多种组分：用于合成蛋白质的底物、用于合成RNA的底物、RNA聚合酶、镁离子、钾离子、缓冲剂、能量再生系统、聚乙二醇(PEG)或其类似物、二硫苏糖醇(DTT)和任选的溶剂，所述溶剂为水或水性溶剂。
进一步的，所述细胞为真核细胞。所述真核细胞为哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞之一或其任意组合。其中，所述酵母细胞选自酿酒酵母、毕氏酵母、克鲁维酵母之一或其组合；克鲁维酵母属酵母选自乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母之一或其任意组合；较佳地，所述的酵母细胞为克鲁维酵母，更佳地为乳酸克鲁维酵母。
进一步的，所述细胞提取物为对酵母细胞的水性提取物。
进一步的，所述细胞提取物不含酵母内源性的长链核酸分子。
进一步的，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸之一或其组合。
进一步的，所述的合成蛋白质的底物包括：20种天然氨基酸以及非天然氨基酸。
进一步的，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁之一或其组合。
进一步的，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾之一或其组合。
进一步的，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统之一、蔗糖或其组合。
进一步的，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷之一或其组合。
进一步的，所述蛋白合成体系含有聚乙二醇(PEG)或其类似物。聚乙二醇或其类似物的浓度没有特别限制，通常，聚乙二醇或其类似物的浓度(w/v)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％，以所述蛋白合成体系的总重量计。代表性的PEG选自下组：PEG3000、PEG3350、PEG6000、PEG8000之一或其组合。
进一步的，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，如PEG200、400、1500、2000、4000、6000、8000、10000等，较佳地，分子量为3000-10000的聚乙二醇。
可选的方案为，本发明提供的蛋白合成体系包括：酵母细胞提取物，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA聚合酶，聚乙二醇，蔗糖。
在本发明中，所述的细胞提取物不含完整的细胞，典型的细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外，细胞提取物中还含有一些源自细胞的细胞质中的其他蛋白，尤其是可溶性蛋白。
在本发明中，所述的细胞提取物所含蛋白含量为20-100mg/ml,较佳为50-100mg/ml。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中，所述的细胞提取物的制备方法不受限制，一种优选的制备方法包括以下步骤：
(i)提供细胞；
(ii)对细胞进行洗涤处理，获得经洗涤的细胞；
(iii)对经洗涤的细胞进行细胞破碎处理，从而获得细胞粗提物；
(iv)对所述细胞粗提物进行固液分离，获得液体部分，即为细胞提取物。
在本发明中，所述的固液分离方式不受特别限制，一种优选的方式为离心。
在本发明中，所述离心条件不受特别限制，一种优选的离心条件为 5000-100000g，较佳地，8000-30000g。
在本发明中，所述离心时间不受特别限制，一种优选的离心时间为0.5min-2h，较佳地，20min-50min。
在本发明中，所述离心的温度不受特别限制，优选的，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在本发明中，所述的洗涤处理方式不受特别限制，一种优选的洗涤处理方式为采用洗涤液在pH为7-8(较佳地，7.4)下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中，所述细胞破碎处理的方式不受特别限制，一种优选的所述的细胞破碎处理包括高压破碎、冻融(如液氮低温)破碎。
所述蛋白合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.5-5mM，较佳地为1.0-2.0mM。
所述蛋白合成体系中的氨基酸混合物可包括天然或非天然氨基酸，可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于)20种天然氨基酸：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM，较佳地0.02-0.2mM，如0.05、0.06、0.07、0.08mM。
在优选例中，所述体外无细胞蛋白合成体系还含有蔗糖，所述蔗糖的浓度为0.03-40wt％，较佳地，0.08-10wt％，更佳地，0.1-5wt％，以所述蛋白合成体系的总重量计。
一种特别优选的体外无细胞蛋白合成体系，除了酵母细胞提取物之外，还含有以下组分：22mM pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物，0.08-0.24mM的氨基酸混合物，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，1％-4％聚乙二醇，0.5％-2％蔗糖，0.027-0.054mg/mL T7 RNA聚合酶。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York: Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
本发明以乳酸克鲁维酵母(Kluyveromyces lactis，K.lactis)为实施例，但同样的设计和分析、实验方法也适用于其他酵母等低等真核细胞以及高等动物细胞。本发明中的基因改造方法为CRISPR-Cas9技术，但并不限于此，可以为已知的、现有的任意基因改造方法。
实施例1 通过CRISPR-Cas9敲除Edc3基因
1.1 Edc3序列检索及CRISPR gRNA质粒的构建
根据酿酒酵母(S.cerevisiae)的Edc3蛋白序列比对，确定K.lactis细胞中Edc3的基因编号为KLLA0A11308g。在Edc3基因编码序列靠近5’和3’分别选择PAM序列(NGG)，并确定对应的gRNA序列。本实施例中gRNA选择的原则为：GC含量适中(40％-60％)，避免poly T结构的存在。在本实施例中，KlEdc3基因gRNA1的序列为TCAAATTGAGATCGAATTGA，KlEdc3基因gRNA2的序列为GGACATATACCCGGGTTTCT(KlEdc3基因的核酸编码序列为SEQ No.1；相应的氨基酸序列为SEQ No.2)。
KlEdc3 gRNA1的质粒构建及转化方法如下：使用引物pCas9-Edc3-gRNA1-PF：TCAAATTGAGATCGAATTGAGTTTTAGAGCTAGAAATAGC和pCas9-Edc3-gRNA1-PR：TCAATTCGATCTCAATTTGAAAAGTCCCATTCGCCACCCG，以pCAS质粒为模板，进行PCR扩增。取扩增产物17μL，加入0.2μL Dpn I，2μL 10×digestion buffer，混匀后37℃水浴3h。取Dpn I处理后产物10μL加入50μL DH5α感受态细胞中，冰上放置30min，42℃热激45s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Kan抗性LB固体培养，37℃倒置培养至单克隆长出。挑取2个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pCas9_Edc3_gRNA1。
KlEdc3 gRNA2的构建方法如下：使用引物pCas9-Edc3-gRNA2-PF： GGACATATACCCGGGTTTCTGTTTTAGAGCTAGAAATAGC和pCas9-Edc3-gRNA2-PR：AGAAACCCGGGTATATGTCCAAAGTCCCATTCGCCACCCG，以pCAS质粒为模板，进行PCR扩增。转化及鉴定方法同上，阳性质粒命名为pCas9_Edc3_gRNA2。
1.2 Edc3敲除供体DNA质粒构建及扩增
以pMD18T质粒为模板，以引物pMD18T-PF：ATCGTCGACCTGCAGGCATG和pMD18T-PR：ATCTCTAGAGGATCCCCGGG进行PCR扩增，取扩增产物17μL，加入1μL DpnI，2μL 10×digestion buffer，混匀后37℃水浴3h，获得质粒骨架线性片段pMD18T-vector。
1.2.1构建供体质粒pMD18T-ΔEdc3
以乳酸克鲁维酵母基因组DNA为模板，用引物KlEdc3-HR1-PF：CAGGAAACAGCTATGACTACCCGGGGATCCTCTAGAGATGGTAGCTCCAATAATCCAAG和KlEdc3-HR1-PR：ACACATCTATCTTTACAGATATAAGTCAAAAGTGACTTGTTCTATAAACT进行PCR扩增，产物名为Edc3-HR1；以乳酸克鲁维酵母基因组DNA为模板，利用引物KlEdc3-HR2-PF：AGAAGCTATAAGTTTATAGAACAAGTCACTTTTGACTTATATCTGTAAAG和KlEdc3-HR2-PR：GTAAAACGACGGCCAGTTGCATGCCTGCAGGTCGACGATAACTCCAACAACGACGTCAC进行PCR扩增，产物名为Edc3-HR2；
将扩增产物Edc3-HR1、Edc3-HR2和pMD18T-vector各1μL，加入3μL Cloning Mix(Transgene pEASY-Uni Seamless Cloning and Assembly Kit，全式金公司，下同)，混匀后50℃水浴1h。水浴结束后置于冰上2min，将6μL反应液全部加入50μL Trans-T1感受态细胞(全式金公司，下同)中，冰上放置30min，42℃热激30s后，加入1mL LB液体培养基37℃振荡培养1h，涂布于Amp抗性LB固体培养，37℃倒置培养至单克隆长出。挑取6个单克隆在LB液体培养基中振荡培养，PCR检测阳性并测序确认后，提取质粒保存，命名为pMD18T-ΔEdc3。
1.3 K.lactis电转化
从-80℃冰箱取出感受态，冰上融化，加入200ng质粒pCas9_Edc3_gRNA1，200ng质粒pCas9_Edc3_gRNA2和通过引物对pMD18T-ΔEdc3扩增得到的1000ng供体DNA片段，混匀后全部转入电击杯中，冰浴2min；将电击杯放入电转仪中进行电击(参数为1.5kV，200Ω，25μF)；电击结束后立即加入700μL YPD，30℃，200rpm摇床孵育1～3h；取2～200μL接种于YPD(含G418抗性)平板，30℃培养2～3天至单菌落出现。
1.4阳性鉴定
在细胞转化后的平板上挑取12-24个单克隆，以细胞基因组为模板，利用鉴定引物ΔEdc3-CF:CGCATTTTAACCGTTTAGCC和ΔEdc3-CR:TCTGCTTACCGGACTAACAT对样品进行PCR检测。阳性菌株条带2004bp，阴性菌株条带3411bp。经测序鉴定的阳性细胞株，命名为ΔEdc3。
实施例2 体外无细胞蛋白合成体系
2.1细胞提取物(细胞裂解液)的制备
细胞提取液的制备方法包括以下步骤：
(i)提供细胞，细胞为实施例1制备的ΔEdc3细胞株；
(ii)对细胞进行洗涤处理，获得经洗涤的细胞；
(iii)对经洗涤的细胞进行细胞破碎处理，从而获得细胞粗提物；
(iv)对所述细胞粗提物进行固液分离，获得液体部分，即为细胞提取物(细胞裂解液)。
其中，固液分离方式不受特别限制，本实施例选择的方式为离心。离心条件为30000×g；离心时间为30min；离心4℃下进行。
其中，洗涤处理方式不受特别限制，本实施例选择的洗涤处理方式为采用洗涤液在pH为7.4下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。本实施例选择醋酸钾。
其中，细胞破碎处理的方式不受特别限制，一种优选的所述的细胞破碎处理包括高压破碎、冻融(如液氮低温)破碎。
2.2体外无细胞蛋白合成体系的配制
终浓度为22mM pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾， 1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物(腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸)，0.08-0.24mM的氨基酸混合物(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸)，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.027-0.054mg/mL T7 RNA聚合酶，0.27mg/mL磷酸肌酸激酶，1％-4％的聚乙二醇，0.5％-2％的蔗糖，最后加入50％体积的细胞提取物。
2.3体外无细胞蛋白合成反应
上述体系中加入15ng/μL增强型绿色荧光蛋白DNA，混匀后放置在20-30℃的环境中反应3h。
增强型绿色荧光蛋白(eGFP)活性测定：反应结束后，在96孔白板或384孔白板中加入10μL反应液，立即放置于Envision 2120多功能酶标仪(Perkin Elmer),读数，检测增强型绿色荧光蛋白活性，相对荧光单位值(Relative Fluorescence Unit，RFU)作为活性单位，如图4所示。
其中ΔEdc3表示Edc3敲除的乳酸克鲁维酵母细胞株，WT表示野生型的乳酸克鲁维酵母细胞株。反应时间为3h时，ΔEdc3的RFU平均值为1386，WT的RFU平均值是946，ΔEdc3是WT的约1.47倍。该对比数据表明通过基因编辑技术敲除Edc3基因，能够显著提高体外无细胞蛋白合成体系的蛋白合成能力。
本发明实施例结果表明：
与野生型细胞株相比，本发明的Edc3基因敲除细胞株体外无细胞蛋白合成体系的蛋白合成能力提高了约47％。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
SEQ No.1
Figure PCTCN2019128833-appb-000001
SEQ No.2
Figure PCTCN2019128833-appb-000002

Claims (10)

  1. 一种改变体外无细胞蛋白合成体系的蛋白合成能力的方法，其特征在于：所述方法包括以下步骤：
    (1)将待改造的真核细胞中原始存在的Edc3基因通过基因编辑技术去除，获得ΔEdc3改造菌株；
    (2)以上述ΔEdc3改造菌株制备细胞裂解液或细胞提取物；
    (3)将步骤(2)获得的细胞裂解液或细胞提取物用于体外无细胞蛋白质合成体系。
  2. 根据权利要求1所述的方法，其特征在于：所述真核细胞为哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞之一或其任意组合。
  3. 根据权利要求2所述的方法，其特征在于：所述酵母细胞选自酿酒酵母、毕氏酵母、克鲁维酵母之一或其任意组合。
  4. 根据权利要求3所述的方法，其特征在于：所述克鲁维酵母属酵母选自乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母之一或其任意组合。
  5. 一种体外无细胞蛋白合成体系，其特征在于：所述合成体系至少包括以下组分：细胞裂解液或细胞提取物，所述细胞裂解液或细胞提取物由ΔEdc3改造菌株制备获得，所述ΔEdc3改造菌株为将待改造的真核细胞中原始存在的Edc3基因通过基因编辑技术去除获得。
  6. 根据权利要求5所述的合成体系，其特征在于：所述合成体系还包括选自下组的一种或多种组分：用于合成RNA的底物，用于合成蛋白的底物，聚乙二醇或其类似物，镁离子，钾离子，缓冲剂，RNA聚合酶，能量再生系统，二硫苏糖醇(DTT)，任选的水或水性溶剂。
  7. 一种合成外源蛋白的方法，其特征在于，所述方法包括以下步骤：
    (i)提供权利要求5-6任一项所述的体外无细胞蛋白合成体系；
    (ii)添加编码外源蛋白的DNA分子模板，在适合的条件下，孵育一段时间，合成所述的外源蛋白。
  8. 根据权利要求7所述的方法，其特征在于，合适的条件包括反应温度为20-35℃。
  9. 根据权利要求7或8任一项所述的方法，其特征在于，所述方法还包括：(iii)分离或检测所述外源蛋白。
  10. 一种试剂盒，其特征在于，所述的试剂盒包括容器以及位于所述容器中 的权利要求5-6任一项所述的体外无细胞蛋白合成体系的组分。
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