Genetically engineered cell as well as preparation method and application thereof

Abstract

The invention provides a genetically engineered cell as well as a preparation method and application thereof, and the cell-free protein synthesis efficiency is improved by constructing the genetically engineered cell for fusion expression of a His tag. According to the main method, a His tag is introduced to the tail end of a histone coding gene, it is verified that the cell extract prepared from the genetic engineering cells serves as a raw material of a cell-free protein synthesis system, the cell-free protein synthesis efficiency can be improved, and the cell extract has wide application prospects.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a genetically engineered cell, its preparation method and use, and more specifically to a method for improving the efficiency of cell-free protein synthesis by fusion expression of His-tag. Background Art

[0002] Cell-free protein synthesis (CFPS) is a technique that uses exogenous mRNA or DNA as a template and synthesizes proteins in vitro using enzymes, amino acid substrates, and energy. The cell-free protein expression system is an important supplement to the cell-based protein expression system. Compared with the traditional in vivo recombinant expression system, the in vitro cell-free synthesis system has many advantages, such as being able to directly use PCR products as templates to simultaneously and parallelly synthesize multiple proteins, enabling high-throughput screening of polypeptides or protein drugs, having a fast synthesis speed and high efficiency, etc. In addition, the in vitro cell-free synthesis system also has unique advantages in the application of expressing polypeptides or proteins that are toxic to cells.

[0003] Existing cell-free protein synthesis systems can be mainly divided into two categories: One is to combine and reconstitute the purified ribosomes, enzymes, tRNAs, etc. required for protein synthesis in a certain proportion to endow it with the ability to express proteins in vitro, that is, the purified cell-free protein expression system (PURE). This system has clear component information and can conveniently and customarily adjust the formula to obtain different protein expression characteristics, but the disadvantage is that the preparation process is complex and the cost is high. The other category is to obtain the main active substances from the extracts of living cells with protein expression activity and appropriately add the required enzymes, substrates, energy substances, and other components to endow it with the ability to express proteins in vitro, that is, the cell-free protein expression system based on cell extracts. Compared with the PURE system, the latter has the advantages of simple operation process, short cycle, and low cost, but the latter also has disadvantages such as complex components and many uncontrollable factors in the system, so there are higher requirements for the cell characteristics of the source of the cell extract.

[0004] Currently, how to further improve the activity of cell-free protein expression by starting from the improvement of cell extracts remains a research topic worthy of study. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and provide a method for improving the efficiency of cell-free protein synthesis. By genetically modifying to improve the protein expression activity of the cell-free protein expression system of cell extracts, the production efficiency is greatly enhanced.

[0006] In the first aspect of the present invention, a genetically engineered cell is provided, in which a His tag is introduced at the end of the histone-encoding gene, and the end refers to the N-terminus or the C-terminus.

[0007] In another preferred embodiment, the histone-encoding gene refers to the HTZ1 gene, the HTA1 gene, the HTTA2 gene, or the histone HHT1 gene.

[0008] In another preferred embodiment, the genetically engineered cell is selected from prokaryotic cells or eukaryotic cells; the prokaryotic cell refers to bacteria, such as Escherichia coli; the eukaryotic cell is one or any combination of mammalian cells (such as rabbit reticulocytes, Chinese hamster ovary cells, etc.), plant cells (such as wheat germ cells), yeast cells, and insect cells (such as arthropod cells).

[0009] In another preferred embodiment, the yeast cell is selected from one or more of Saccharomyces cerevisiae, Saccharomyces pastorianus, Pichia pastoris, Kluyveromyces lactis, Candida utilis, Saccharomyces sp. of molasses, methanol-induced yeast, Hansenula anomala; preferably Kluyveromyces lactis.

[0010] More preferably, the Kluyveromyces lactis is selected from: the Kluyveromyces lactis includes: Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof.

[0011] In another preferred embodiment, the His tag is composed of 6-10 histidines, such as 6×His tag or 8×His tag, preferably 6×His tag.

[0012] The second aspect of the present invention provides a method for preparing the genetically engineered cell described in the first aspect, specifically achieved by means of CRISPR-Cas9-mediated gene editing.

[0013] In another preferred example, the preparation method specifically includes the following steps:

[0014] Step 1), design the gRNA targeting sequence; design the gRNA for the selected gene and synthesize the corresponding primers;

[0015] Step 2), construct the gRNA_Cas9 co-expression vector; perform PCR using the primers obtained in Step 1), then react the PCR product with the pCas vector in a T4 ligase system, transform it into Escherichia coli cells, perform plate screening and sequencing to obtain the gRNA_Cas9 co-expression vector;

[0016] Step 3), construct the double-stranded DNA donor; adopt the overlap PCR method, and construct the DNA donor containing the target gene by designing forward and reverse primers;

[0017] Step 4), cell transformation and screening, prepare competent cells, add the gRNA_Cas9 expression vector and the DNA donor, perform electrotransformation, and perform plate screening and verification after transformation. After two rounds of plate screening and verification, obtain the cells containing the modified gene.

[0018] The primer design in Steps 1)-3) needs to be adjusted according to the type of genetically engineered cells actually used, especially according to the corresponding gene sequence of histones in the genetically engineered cells. For example, when the cells used in the above method are eukaryotic cells such as yeast cells, it is necessary to design according to the sequences of different copies (or called constituent units) of histones in yeast cells.

[0019] The third aspect of the present invention provides a fusion protein, and the fusion protein has the structure of formula (Ⅰ):

[0020] A-B(Ⅰ);

[0021] Wherein A represents histone, and the coding gene of A is selected from the HTZ1 gene, HTA1 gene, HTTA2 gene or HHT1 gene;

[0022] B represents a His tag, and the His tag refers to (6-10)×His tag;

[0023] “-” represents none or any connection method;

[0024] The connection order of A and B is that the N-terminus of A is connected to the C-terminus of B, or the C-terminus of A is connected to the N-terminus of B.

[0025] The fourth aspect of the present invention provides a cell extract, which is obtained by culturing, lysing and centrifuging the genetically engineered cells described in the first aspect or the cells containing modified genes prepared by the preparation method described in the second aspect.

[0026] The fifth aspect of the present invention provides a cell-free protein synthesis system, wherein the raw materials of the system include the cell extract or the fusion protein described in the fourth aspect.

[0027] The sixth aspect of the present invention provides a method for improving the expression activity of a cell-free protein synthesis system, wherein a His tag is added to the end of the histone gene of a strain used to prepare a cell extract; or protein synthesis is performed using the synthesis system described in claim 10.

[0028] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0029] (1) The genetically engineered cells provided by the present invention are obtained by adding a his tag to the end of a histone gene to obtain a modified genetically engineered cell. The genetically engineered cell is cultured to obtain a cell extract with improved expression activity. The cell extract is used for cell-free protein synthesis, which can significantly improve the expression activity of the protein and enhance the synthesis efficiency.

[0030] (2) The genetic modification of the present invention adopts CRISPR-Cas9-mediated gene editing, which has the advantages of high efficiency, precision, and ease of operation. It can accurately obtain genes with mutations at the target site, is reproducible, and has practicality and scalability.

[0031] (3) The present invention designs primers at each step according to the locus of the target gene, and cooperates with Overlap PCR technology to accurately achieve site-directed mutagenesis and obtain strains with mutations in the target gene, thereby improving operability and repeatability. The genetic traits of the transformed cells are stable and can be used in actual production.

[0032] (4) The present invention also provides an in vitro protein synthesis system for expressing proteins. Since the system contains the cell extract with enhanced expression activity, the protein synthesis efficiency of the system is improved, thereby further improving the production efficiency of in vitro protein synthesis.

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Shows the amino acid sequence alignment results between the histone H2A (including H2A.Z), H2B, H3, and H4 of K. lactis and their homologous proteins in S. cerevisiae. Among them, 1A is the amino acid sequence alignment between K. lactis histone and S. cerevisiae H2A (including H2A.Z); 1B is the amino acid sequence alignment between K. lactis histone H2B and S. cerevisiae H2B; 1C is the amino acid sequence alignment between K. lactis histone H3 and S. cerevisiae H3; 1D is the amino acid sequence alignment between K. lactis histone and S. cerevisiae H4. It shows that the amino acid sequences of the homologous histone core regions are highly conserved in the two yeast species.

[0035] Figure 2 Shows the nucleotide sequence alignment of two copies of histone H2A, H2B, H3, and H4 in the K. lactis genome. To show that more than 90% (391 / 411) of the bases in its ORF region are the same, for ensuring the specificity of editing, the gRNA is designed in the non-coding region (5'-UTR) adjacent to the ORF.

[0036] Among them, 2A is Histone H2A (Query: KLLA0_E17359g vs Sbjct: KLLA0_F13332g);

[0037] 2B is Histone H2B (Query: KLLA0_E17337g vs Sbjct: KLLA0_F13310g);

[0038] 2C is Histone H3 (Query: KLLA0_E08625g vs Sbjct: KLLA0_E17623g);

[0039] 2D is Histone H4 (Query: KLLA0_E08647g vs Sbjct: KLLA0_E17601g).

[0040] Figure 3Shows the gRNA positions and the overlap PCR primer design schemes. Among them, the upper figure shows the design scheme for introducing a 6×His tag at the N-terminus of HTZ1 (KLLA0_C05918g). A gRNA was designed at the junction of its 5'-untranslated region (UTR) and open reading frame (ORF). Therefore, a double-stranded DNA donor carrying a 6×His tag can be assembled by overlap PCR mediated by 4 primers. The other schemes with 4 primers designed are similar. Figure 3 Shows the design scheme for introducing a 6×His tag at the N-terminus of HHT2 (KLLA0_E08625g). Since the ORF region has a very high similarity to that of HHT1, the gRNA was designed in the 5'-UTR. A double-stranded DNA donor carrying a 6×His tag was assembled by overlap PCR mediated by 6 primers. The other schemes with 6 primers designed are similar.

[0041] Figure 4 Shows the IVTT activities of cell extracts from mutant strains and control strains. Among them, Control represents the control group, that is, the original strain without introducing a His tag at the end of the histone gene, and the rest are recombinant strains with a His tag introduced at the N-terminus of the corresponding gene, represented by the gene name. Detailed implementation methods

[0042] Through extensive and in-depth research, and through a large number of screenings and explorations, the present invention first proposes a method for gene modification, specifically a method for improving the efficiency of cell-free protein synthesis through gene modification. This method introduces a His tag at the end of the histone gene in the cell through gene editing to obtain mutant strains or cells, and then cultures and prepares cell extracts, thereby improving the cell-free protein synthesis activity.

[0043] The following further clarifies the present invention in combination with detailed implementation methods and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional substitution selections based on the existing technologies on the basis of the technical ideas of the present invention, not limited to the specific records of the examples of the present invention.

[0044] For the experimental methods without specific conditions noted in the following examples, the conditions described in the specific implementation manners referred to above shall be preferred and referred to first, and then the conventional conditions can be followed, such as the experimental conditions described in documents such as "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-Free Protein Synthesis Experimental Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008", or the conditions recommended by the manufacturer.

[0045] Unless otherwise specified, the percentages and parts mentioned in the present invention are weight percentages and weight parts.

[0046] Unless otherwise specified, the materials and reagents used in the embodiments of the present invention are all commercially available products.

[0047] Unless otherwise specified, the temperature unit in this application is degrees Celsius (°C).

[0048] Term Introduction

[0049] The following are the explanations or descriptions of the meanings of some relevant "nouns" and "terms" adopted in the present invention, so as to better understand the present invention. The corresponding explanations or descriptions apply to the whole text of the present invention, both to the following text and to the above text. When the present invention involves citing documents, the definitions of relevant terms, nouns, and phrases in the cited documents are also cited. However, when there is a conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When there is a conflict between the definitions in the cited documents and the definitions in the present invention, it does not affect the components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc. determined in the cited documents to prevail.

[0050] In the present invention, "cell-free protein synthesis", also known as "in vitro protein synthesis", "in vitro cell-free protein synthesis", etc., refers to the reaction of synthesizing proteins in an in vitro cell-free environment synthesis system, which at least includes the translation process. It includes but is not limited to IVT reaction (in vitro translation reaction), IVTT reaction (in vitro transcription-translation reaction), and IVDTT reaction (in vitro replication-transcription-translation reaction). In the present invention, the IVTT reaction is preferably used. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to such in vitro protein synthesis systems as D2P systems, D-to-P systems, D_to_P systems, DNA-to-Protein systems; the corresponding in vitro protein synthesis methods are also referred to as D2P methods, D-to-P methods, D_to_P methods, DNA-to-Protein methods.

[0051] In the present invention, "cell-free", or "cell-free system", means that when performing in vitro protein synthesis, it is not through the secretion and expression mode of intact cells. It should be noted that in the in vitro cell-free protein synthesis system of the present invention, it is also allowed to add cell components to promote the reaction, but the added cells do not mainly aim at secreting and expressing exogenous target proteins. In addition, in the D2P system without intact cells constructed under the guidance of the present invention, a small amount of intact cells are intentionally added (for example, the protein content provided by them does not exceed 30 wt% compared with the protein content provided by cell extracts). Such an "evasion" method is also included in the protection scope of the present invention.

[0052] In the present invention, one of the specific operation modes of the cell-free protein synthesis further includes, but is not limited to, for example, the Escherichia coli-based cell-free protein synthesis system described in WO2016005982A1. The in vitro cell-free protein synthesis systems including, but not limited to, those based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus, as described in other cited documents of the present invention and their directly and indirectly cited documents, are also incorporated into the present invention as implementation modes of the in vitro protein synthesis system of the present invention. For example, the in vitro cell-free protein synthesis systems (In vitro cell-free protein synthesis system) described in the cited documents on pages 27-28 of the section "2.1 Systems and Advantages" in the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" can all be used as the in vitro protein synthesis system for implementing the present invention. For example (unless it conflicts with the present invention, the following documents and their cited documents are cited for all purposes and in their entirety), the in vitro cell-free protein synthesis systems, DNA template construction and amplification methods described in documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, etc., and their cited documents can all be used as the in vitro protein synthesis system of the present invention and the DNA template construction and amplification method of the present invention.

[0053] In the present invention, "protein" and "protein" have the same meaning and are both translated as "protein" and can be used interchangeably.

[0054] In the present invention, both "system" and "system" are translated as "system" and can be used interchangeably.

[0055] In the present invention, "expression activity", "synthesis activity", "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably, and all represent the ability of the cell-free protein synthesis system to synthesize proteins.

[0056] In the present invention, the terms "cell extract", "cell extract solution", "cell lysate", etc. have the same meaning and can be used interchangeably. They can be described in English as cell extract, cell lysate, etc. They are all substances obtained after cell disruption, and the cell extract does not contain intact cells.

[0057] In the present invention, "allele" refers to genes located at the same position on a pair of homologous chromosomes that control different forms of the same trait.

[0058] In the present invention, "Overlap PCR", "overlapping PCR", or "overlap extension PCR", "SOE PCR", "genesplicing by overlap extension PCR" is a technique that uses primers with complementary ends to form overlapping strands of PCR products, so that in subsequent amplification reactions, different sources of amplified fragments are overlapped and spliced together through the extension of the overlapping strands.

[0059] "CRISPR-Cas9" was originally an adaptive immune defense formed by bacteria and archaea during long-term evolution and can be used to combat invading viruses and foreign DNA. In the present invention, "CRISPR-Cas9" or "CRISPR-Cas9-mediated gene editing" refers to a gene editing technique based on the above principle, that is, a technique for performing specific DNA modifications on target genes, which is a commonly used and technically mature gene editing method in the field of biotechnology in recent years. Except for the steps described in the present invention, the rest of the specific operation methods are all conventional operations recorded in the prior art.

[0060] In the present invention, "His tag", or "His tag", "His-tag monoclonal antibody", "His-tag protein", etc. are tags often used in protein recombination technology. Its sequence is usually 6-10 histidines, that is, a polypeptide composed of 6-10 histidines. The commonly used one is the 6×His tag, and its preparation method is usually to artificially synthesize the number of amino acids. The His tag can be used to identify the expression of His-tagged proteins (such as the relative expression level and molecular weight of the target protein), its localization in cells, and other characteristics. In the present invention, the His tag is used as a modification means for histone genes to enhance the translation regulation ability of histone genes by inserting the His tag at the end of the histone gene.

[0061] In the present invention, the terms "vector", "plasmid", "primer", "target gene", "gene", "transformation", "PCR", etc. all have their conventional meanings in the prior art and will not be elaborated here one by one.

[0062] The present invention will be further illustrated below in conjunction with specific embodiments and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional replacement selections based on the existing technologies on the basis of the technical concept of the present invention, not limited to the specific records of the embodiments of the present invention only.

[0063] The genetically engineered cell of the present invention has a His tag introduced at the end of the histone coding gene in the genetically engineered cell, and the end refers to the N-terminus or C-terminus. Histones are highly conserved in many organisms, especially eukaryotes. Histones are the main protein components of chromatin and act as spools around which DNA is wound, playing a supporting role, but histones also play a role in gene regulation, especially various histone modifications, which are often used as research directions for translational initiation regulation. In the present invention, it is found through experiments that introducing a His tag at the end of the histone coding gene can regulate protein translation. Therefore, a genetically engineered cell with a His tag introduced at the end of the histone coding gene is constructed. Specifically, a His tag can be inserted at the N-terminus or C-terminus, and it is expected that they will have the same effect.

[0064] In one example, the histone coding gene refers to the histone HTZ1 gene, HTA1 gene, HTTA2 gene, or HHT1 gene.

[0065] In one example, the genetically engineered cell is selected from prokaryotic cells or eukaryotic cells; the prokaryotic cell refers to bacteria, such as Escherichia coli; the eukaryotic cell is one or any combination of mammalian cells (such as rabbit reticulocytes, Chinese hamster ovary cells, etc.), plant cells (such as wheat germ cells), yeast cells, and insect cells (such as arthropod cells). The genetic engineering method of the present invention is applicable to all organisms containing histones. Therefore, there is no specific limitation on the source of the genetically engineered cell. Considering that the main use of the genetically engineered cell is cell-free protein synthesis, the biological cells commonly used in cell-free protein synthesis systems are preferably used above.

[0066] In one example, it is preferred that the yeast cell is selected from one or more of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Pichia pastoris, Kluyveromyces, Candida utilis, Saccharomyces mellis, methanol-induced yeast, Hansenula; preferably Kluyveromyces.

[0067] Further preferably, the Kluyveromyces is selected from: the Kluyveromyces includes: Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof.

[0068] Yeast has the advantages of simple culture, efficient protein folding, and post-translational modification, etc., and can be used as a raw material for preparing an in vitro translation system, that is, yeast extract can be used for in vitro protein translation and expression. Pichia pastoris or Saccharomyces cerevisiae are commonly used yeasts for in vitro protein expression. Kluyveromyces is an ascomycetous yeast, and Kluyveromyces marxianus and Kluyveromyces lactis among them are widely used yeasts in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as super secretion ability, better large-scale fermentation characteristics, food safety level, and the ability of post-translational modification of proteins, etc., and has great application potential. In a preferred embodiment of the present invention, an in vitro protein expression system of Kluyveromyces lactis is used.

[0069] Similar to the model fungus Saccharomyces cerevisiae, there are also 9 histone-encoding genes in the Kluyveromyces lactis (taking K. lactis Y1140 as an example) genome, including two copies of Histone H2A, H2B, H3, and H4 each, and one copy of H2A.Z (see Table 1); H2A and H2B are adjacent in the genome, and H3 and H4 are also adjacent in the genome. Multiple post-translational modifications can be introduced at the N-terminus of histones, and these modifications play a crucial regulatory role in gene expression regulation.

[0070] Table 1 List of histone genes in Kluyveromyces lactis

[0071] Gene ID Gene Name Gene Description Information KLLA0_C05918g HTZ1 histone H2A.Z KLLA0_E08625g HHT2 histone H3 KLLA0_E08647g HHF2 histone H4 KLLA0_E17337g HTB1 histone H2B KLLA0_E17359g HTA1 histone H2A KLLA0_E17601g HHF1 histone H4 KLLA0_E17623g HHT1 histone H3 KLLA0_F13310g HTB2 histone H2B KLLA0_F13332g HTA2 histone H2A

[0072] It should be noted that sometimes when expressing gene names, when the gene description information corresponds one-to-one with the gene name, either the gene name or its gene description can be used. For example, HTZ1 and H2A.Z express the same gene, so they can be used interchangeably.

[0073] In one example, the His tag is a His tag composed of 6 - 10 histidines, preferably 6×His tag or 8×His tag, and more preferably 6×His tag. It is known that His tags composed of 6 - 10 histidines are all common His tag types. Among them, 6×His tag or 8×His tag are the most commonly used His tag types. In the specific embodiments of the present invention, 6×His tag is used as the insertion sequence to modify the histone gene.

[0074] In one example, a method for preparing a genetically engineered cell is also provided, specifically achieved by CRISPR-Cas9-mediated gene editing means.

[0075] In a preferred example, the preparation method specifically includes the following steps:

[0076] Step 1), design the gRNA targeting sequence; design gRNA for the selected gene and synthesize the corresponding primers;

[0077] Step 2), construct the gRNA_Cas9 co-expression vector; perform PCR using the primers obtained in Step 1), then react the PCR product with the pCas vector in a T4 ligase system, transform it into Escherichia coli cells, screen by plating and sequence to obtain the gRNA_Cas9 co-expression vector;

[0078] Step 3), construct the double-stranded DNA donor; use the overlap PCR method to construct a DNA donor containing the target gene by designing forward and reverse primers;

[0079] Step 4), cell transformation and screening, prepare competent cells, add the gRNA_Cas9 expression vector and the DNA donor, perform electrotransformation, screen by plating after transformation and verify. After two rounds of plating screening and verification, cells containing the modified gene are obtained.

[0080] The method described above is a summary of the main steps of gene editing. In fact, the operating steps and parameters of each step need to be adjusted according to the type of cells targeted, and creative work is also required to finally obtain the successfully transformed cells containing the target gene. In the above method, an important content is to design the primers required for each step. The primer design in steps 1)-3) needs to be adjusted according to the actual type of genetically engineered cells, especially according to the corresponding gene sequences of histones in the genetically engineered cells. For example, when the cells used in the above method are eukaryotic cells such as yeast cells, the design needs to be based on the sequences of different copies (or called constituent units) of histones in yeast cells. In addition, the conditions of each PCR are also important parameters. Conditions such as the annealing temperature, time, and the use of enzymes determine the results of PCR and require inventors to continuously explore and try.

[0081] In one example, a fusion protein is also provided. The fusion protein has the structure of formula (Ⅰ):

[0082] C-B(Ⅰ);

[0083] Wherein A represents a histone, and the coding gene of A is selected from the HTZ1 gene, HTA1 gene, HTTA2 gene, or HHT1 gene;

[0084] B represents a His tag, and the His tag refers to (6-10)×His tag;

[0085] "-" represents none or any connection mode;

[0086] The connection order of A and B is that the N-terminus of A is connected to the C-terminus of B, or the C-terminus of A is connected to the N-terminus of B. The above fusion protein is the fusion protein expressed after introducing a His tag at the end of the aforementioned histone gene. It is expressed by the aforementioned genetically engineered bacteria, and the mixture containing the fusion protein after expression can be used in a cell-free protein synthesis reaction to improve the reaction efficiency.

[0087] In one example, a cell extract is provided. The cell extract is obtained by culturing, centrifuging, lysing, etc. the cells containing the modified gene prepared by using the genetically engineered cells described in the first aspect or the preparation method described in the second aspect.

[0088] For example, when yeast cells are used as the cells, the yeast cell extract is prepared as follows: A histone gene-modified strain based on a yeast strain is used to ferment and culture yeast cells. An appropriate amount of yeast cells are used as raw materials, and the cells are quickly frozen with liquid nitrogen, broken, and the supernatant is collected by centrifugation to obtain the cell extract; alternatively, other conventional means are used to lyse and separate the cultured yeast cells to obtain the yeast cell extract. The obtained yeast cell extract contains a certain concentration of protein, for example, about 10 - 100 mg / mL.

[0089] In one example, a use of the cell extract is also provided, specifically for an in vitro cell-free protein synthesis system. Since the cell extract contains a modified gene and a protein fused with a his tag, which has the effect of enhancing the translation ability and increasing the yield of protein synthesis, the cell extract can be used in the in vitro cell-free protein synthesis system to improve its synthesis efficiency.

[0090] In one example, an in vitro protein synthesis system for expressing proteins is also provided, which comprises the aforementioned cell extract or the aforementioned fusion protein. Since the aforementioned cell extract containing site-directed mutations is used in this system, it has the advantages of efficient translation and expression and increased protein yield. There is no strict limitation on the protein to be synthesized in the system, as long as it is applicable to the in vitro cell-free synthesis system. The cell-free protein synthesis system of the present invention further comprises one or more components selected from the following groups: substrates for protein synthesis, substrates for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, buffer, energy regeneration system, polyethylene glycol (PEG) or its analogs, dithiothreitol (DTT), and an optional solvent, and the solvent is water or an aqueous solvent.

[0091] Furthermore, the cell extract does not contain yeast endogenous long-chain nucleic acid molecules.

[0092] Furthermore, the substrate for synthesizing RNA includes: one of nucleoside monophosphates, nucleoside triphosphates, or a combination thereof.

[0093] Furthermore, the substrate for synthesizing proteins includes: 20 natural amino acids and non-natural amino acids.

[0094] Furthermore, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following groups: magnesium acetate, magnesium glutamate, or a combination thereof.

[0095] Furthermore, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following groups: potassium acetate, potassium glutamate, or a combination thereof.

[0096] Further, the energy regeneration system is selected from the group consisting of: creatine phosphate / creatine phosphokinase system, one of the glycolytic pathway intermediate energy systems, sucrose, or a combination thereof.

[0097] Further, the buffer is selected from the group consisting of: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0098] Further, the protein synthesis system contains polyethylene glycol (PEG) or its analog. The concentration of polyethylene glycol or its analog is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analog is 0.1-8%, preferably 0.5-4%, more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of: PEG3000, PEG3350, PEG6000, PEG8000, or a combination thereof.

[0099] Further, 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., preferably polyethylene glycol with a molecular weight of 3000-10000.

[0100] An alternative solution is that the protein synthesis system provided by the present invention includes: cell extract, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphokinase, RNA polymerase, polyethylene glycol, sucrose.

[0101] The cell extract does not contain intact cells. Typical cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl-tRNA synthetase, initiation factors and elongation factors required for protein synthesis, and termination release factors. In addition, the cell extract also contains some other proteins derived from the cytoplasm of the cells, especially soluble proteins.

[0102] In the present invention, the protein content in the cell extract is 20-100 mg / ml, preferably 50-100 mg / ml.

[0103] In one example, a method for improving the expression activity of a cell-free protein synthesis system is also provided. A His tag is added to the end of the histone gene of the strain used to prepare the cell extract; or the aforementioned synthesis system is used for protein synthesis. As described above, since the cell-free protein synthesis system contains the aforementioned cell extract, which can enhance the translation expression level and increase the protein yield, therefore, using this cell extract in the cell-free protein synthesis system can improve the expression activity of the cell-free protein synthesis system. The specific method of the cell-free protein synthesis is carried out according to the conventional operation methods in the art. For example, it includes the following steps:

[0104] (i) Provide various raw material components of the in vitro cell-free protein synthesis system as described above;

[0105] (ii) Add a DNA molecular template encoding an exogenous protein and carry out an incubation reaction under suitable conditions to synthesize the exogenous protein;

[0106] (iii) Optionally, isolate or detect the exogenous protein from the in vitro cell-free protein synthesis system.

[0107] In one example, the suitable conditions for carrying out the incubation reaction in the above method are 20 - 30 °C.

[0108] The following uses specific examples to explain the detailed method of the present invention.

[0109] Example 1 Design of gRNA Target Sequences

[0110] Select the region (100 - 2000 bp) of the gene for which the gRNA needs to be designed, complete the design of the gRNA on the CRISPOR (tefor.net) (http: / / crispor.tefor.net / ) website, and select the gRNAs with higher scores as alternative sequences. For each selected gene, select 1 sequence with a relatively high comprehensive score as the final gRNA sequence. The specific information of the gRNAs designed for each gene is shown in Table 2. Then, according to the designed gRNA target sequences, synthesize the corresponding 24-base primer pairs, that is, add 4-base sequences of AATC and AAAC to the 5'-ends of the forward sequence and the reverse complementary sequence of each gRNA respectively. The finally obtained pair of 24-base primers constitutes a 24-base primer pair.

[0111] Table 2 gRNA List

[0112]

[0113] Example 2 Construction of gRNA_Cas9 Expression Vector

[0114] The paired 24-base primer pairs synthesized in Example 1 were respectively diluted to 10 μM. 10 μL of each was taken and added to a PCR tube. After mixing, the annealing process was completed on a PCR instrument according to the following program: 95°C for 3 min; 72°C for 2 min; 65°C for 2 min; 60°C for 2 min; 55°C for 2 min; 50°C for 2 min; 16°C for 2 min. 0.5 μL of the annealed primer pair and 20 ng of the pCasMFR vector (obtained by the inventors' self-modification, for the specific method, see Patent Application CN2023116023642) digested with BsaI enzyme and purified were mixed in a 10 μL T4 ligase system and ligated at 16°C for 1 h. The ligation product was completely transformed into Escherichia coli DH5α competent cells and screened on an LB plate containing 50 μg / mL kanamycin. 2 single colonies were randomly selected for sequencing verification. Clones with correct sequencing were selected to extract the gRNA_Cas9 co-expression plasmid, and after measuring the concentration, it was used for subsequent electroporation experiments.

[0115] Construction of Double-stranded DNA Donor in Example 3

[0116] The DNA donor used in this example was obtained by overlap PCR method.

[0117] In order to introduce a 6×His tag at the N-terminus of the histone HTZ1 (KLLA0_C05918g) gene, a gRNA was designed at the junction of its 5'-untranslated region (UTR) and open reading frame (ORF). Therefore, a double-stranded DNA donor carrying a 6×His tag could be assembled by overlap PCR mediated by 4 primers (see Table 3 for details, the same below). Other schemes with 4 primers designed were similar.

[0118] When introducing a 6×His tag at the N-terminus of HHT2 (KLLA0_E08625g), since its ORF region has a very high similarity with that of HHT1, the gRNA was designed in the 5'-UTR. A double-stranded DNA donor carrying a 6×His tag was assembled by overlap PCR mediated by 6 primers. Other schemes with 6 primers designed were similar.

[0119] Therefore, when constructing the DNA donor, a total of 4 primers were designed for the assembly of some gene donors. First, using the genomic DNA of K. lactis Y1140 as a template, xxxN-p1 + xxxN-p2 and xxxN-p3 + xxxN-p4 were used as forward and reverse primer pairs (xxxN is the corresponding gene name in Table 3, see Table 3 for details), and Phanta Super Fidelity DNA polymerase was used for PCR amplification to obtain PCR products H1 and H2. Then, using xxxN-p1 + xxxN-p4 as forward and reverse primers, and the products H1 and H2 from the previous PCR as templates, a second round of PCR was performed. The products of the second round of PCR were precipitated with 70% ethanol and dissolved in 50 μL of ddH2O and stored at -20 °C for later use.

[0120] For the assembly of another part of some gene donors, a total of 6 primers were designed. First, using the genomic DNA of K. lactis Y1140 as a template, xxxN-p1 + xxxN-p2, xxxN-p3 + xxxN-p4, and xxxN-p5 + xxxN-p6 were used as forward and reverse primer pairs (xxxN is the corresponding gene name, see Table 3 for details), and Phanta Super Fidelity DNA polymerase was used for PCR amplification to obtain PCR products H1, H2, and H3. Then, using xxxN-p1 + xxxN-p4 as forward and reverse primers, and the products H1, H2, and H3 from the previous PCR as templates, a second round of PCR was performed. The products of the second round of PCR were precipitated with 70% ethanol and dissolved in 50 μL of ddH2O and stored at -20 °C for later use.

[0121] Thus, DNA double-stranded donors of different histone genes were prepared.

[0122] Table 3 Primer List

[0123]

[0124]

[0125]

[0126] The bases represented by capital characters in the sequence indicate the positions where mutations are introduced.

[0127] Example 4 Transformation and Screening of Yeast Strains

[0128] The transformation of Kluyveromyces lactis strains was completed by electroporation. According to the requirements of the electroporation method, competent cells were prepared with Kluyveromyces lactis as the starting strain. Before electroporation, 40 μL of the competent cell mixture was fully mixed with 500 ng of the corresponding gRNA_Cas9 expression plasmid prepared in Example 2 and 1-2 μg of the DNA Donor prepared in Example 3. The electroporation process was completed according to the standard operation process. The yeast cells after electroporation were screened on a YPD plate containing 250 μg / mL G418. Single colonies were picked for PCR verification, and the positive PCR products were sent to the sequencing company for sequencing; the single colonies with correct sequencing were streaked on the plate, and single colonies were picked for the second round of PCR verification. Only strains with correct two rounds of verification will be prepared with glycerol cryopreservation tubes and stored in a -80 ° C refrigerator. Thus, mutant strains with 6×His tags inserted at the ends of different histone genes were prepared, and the mutant strains were named after their corresponding histone genes, as shown in Table 1.

[0129] Example 5 Lysate Preparation and IVTT Activity Detection

[0130] After streaking the YPD plate of each test strain frozen at -80°C, pick a single colony and inoculate it into a 250mL Erlenmeyer flask containing 100mL of seed culture medium, and shake culture at 200rpm in a 30°C shaker for 24h; the seed liquid was transferred to a 1000mL Erlenmeyer flask containing 400mL of fermentation medium, and shake culture at 200rpm in a 30°C shaker until the harvest period, the cells were collected by high-speed centrifugation, and the cell lysate was prepared under liquid nitrogen protection for subsequent IVTT activity test.

[0131] Take a certain amount of lysate prepared according to the standard process, and add and perform cell-free protein synthesis in the same way to the cell lysate obtained from each mutant strain according to the operating procedure of in vitro cell-free protein synthesis. Use mEGFP (green fluorescent protein) as the reporter protein to complete the detection and analysis of D2P expression activity in the lysate, and judge the IVTT activity of the corresponding lysate based on the detected reporter fluorescence intensity (RFU value). The results are as follows: Figure 4 The corresponding data are shown in Table 4.

[0132] Table 4 IVTT activity results

[0133]

[0134]

[0135] In Table 4, the results of each strain after transformation were analyzed for percentage difference with the control group (untransformed strain).

[0136] from Figure 4From the experimental results in Table 4, it can be seen that fusing a 6×His tag at the N-terminus of some histones can enhance the IVTT activity of the cell extracts of the strain. Among them, fusing a 6×His tag at the N-terminus of HTZ1 has the most significant effect, showing prominent advantages both at 3 hours and 20 hours of synthesis. Fusing a 6×His tag at the N-terminus of HHT1 also has a certain enhancing effect, showing higher activity at 3 hours and 20 hours. If considering the short-term synthesis effect, HTA1 and HTA2 also have certain advantages, with activities higher than those of the control group. Therefore, the cell extracts of the above 4 mutant strains all have certain application potential.

[0137] The above are only partial embodiments of the present invention, and the present invention is not limited to the content of the above embodiments.

[0138] All the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference. In addition, it should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A genetically engineered cell, characterized in that, An His tag is introduced at the end of the histone-encoding gene in the genetically engineered cell, and the end refers to the N-terminus or the C-terminus.

2. The genetically engineered cell according to claim 1, characterized in that: The histone-encoding gene refers to the histone HTZ1 gene, HTA1 gene, HTTA2 gene, or HHT1 gene.

3. The genetically engineered cell according to claim 1 or 2, characterized in that: The genetically engineered cell is selected from prokaryotic cells or eukaryotic cells; the prokaryotic cell refers to bacteria; the eukaryotic cell is one or any combination of mammalian cells, plant cells, yeast cells, and insect cells.

4. The genetically engineered cell according to claim 3, wherein: The yeast cell is selected from one or more of Saccharomyces cerevisiae, Saccharomyces pastorianus, Pichia pastoris, Kluyveromyces, Candida utilis, Saccharomyces molasses, methanol-induced yeast, Hansenula; preferably Kluyveromyces.

5. The genetically engineered cell according to any one of claims 1-4, characterized in that: The His tag is a His tag composed of 6-10 histidines; preferably 6×His tag.

6. A method for preparing a genetically engineered cell according to any one of claims 1-5, characterized in that, Through CRISPR-Cas9-mediated gene editing means.

7. The method for preparing the genetically engineered cell according to claim 6 specifically includes the following steps: Step 1), designing the gRNA targeting sequence; Step 2), constructing the gRNA_Cas9 co-expression vector; Step 3), constructing the double-stranded DNA donor; Step 4), cell transformation and screening.

8. A fusion protein, the fusion protein having the structure of formula (Ⅰ): A-B (Ⅰ); wherein A represents a histone, and the encoding gene of A is selected from the HTZ1 gene, HTA1 gene, HTTA2 gene, or HHT1 gene; B represents an His tag, and the His tag refers to (6-10)×His tag; "-" represents none or any connection mode; The connection order of A and B is that the N-terminus of A is connected to the C-terminus of B, or the C-terminus of A is connected to the N-terminus of B.

9. A cell extract, characterized in that, The cell extract is obtained from the genetically engineered cell according to any one of claims 1-5 or the cell obtained by the preparation method according to claims 6 and 7 through the steps of culturing, lysing, and centrifuging.

10. A cell-free protein synthesis system, characterized in that, The raw materials of the system include the cell extract according to claim 9 or the fusion protein according to claim 8.

11. A method for improving the expression activity of a cell-free protein synthesis system, characterized in that, Adding an His tag to the end of the histone gene of the strain for preparing the cell extract; or performing protein synthesis using the synthesis system according to claim 10.

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