CN111254128A

CN111254128A - Method for modifying gene of protein kinase A catalytic subunit TPK1 and application thereof

Info

Publication number: CN111254128A
Application number: CN201811452075.8A
Authority: CN
Inventors: 郭敏; 王静; 许乃庆; 姜灵轩; 赵玉莲; 杨旭; 占魁; 邓蜜妮; 范万巧; 娄旭; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-06-09

Abstract

The invention provides a gene modification method of eukaryote, which carries out base mutation on TPK1 encoding gene of 3 '5' -cyclic adenosine monophosphate-dependent protein kinase, namely protein kinase A catalytic subunit through gene editing, thereby mutating serine corresponding to 179 site of Saccharomyces cerevisiae TPK1 amino acid sequence in TPK1 amino acid sequence of eukaryote into aspartic acid, namely serine at 237 site into aspartic acid. The modification method can not only enhance the translation efficiency of the starting protein of the eukaryotic in vitro biosynthesis system, but also increase the possibility of the in vitro biosynthesis system for synthesizing different proteins. Meanwhile, the invention provides a gene engineering cell which is transformed by the gene transformation method and a eukaryotic cell-free protein synthesis system which comprises a cell extract prepared by the gene engineering cell.

Description

Protein kinase a catalytic subunitTPK1Gene modification method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a method for regulating and controlling the synthesis efficiency of exogenous protein in a cell-free protein synthesis system by amino acid point mutation of a protein kinase A catalytic subunit.

Background

Most proteins undergo post-translational modifications of the protein during synthesis, and some inappropriate modifications are often associated with disease. Protein phosphorylation is a widespread post-translational modification, and approximately 30% of the proteome can be phosphorylated [1 ]. Protein phosphorylation plays a key role in second messenger transmission and enzyme cascades, and many of the most fundamental biological processes such as signal transduction, gene expression, cell division and the like are regulated by protein phosphorylation, and are one of the important regulation and modification forms in prokaryotes and eukaryotes [2 ]. The phosphorylation site of the protein is at an amino acid residue of the peptide chain, such as tyrosine, serine, threonine, etc. These amino acid residues are not charged themselves, and the configuration and activity of the protein are further changed after a phosphate group having a strong negative charge is added to the side chain [3 ]. Protein phosphorylation is accomplished by the catalytic action of a series of protein kinases.

The 3 ', 5' -cyclic adenosine monophosphate-dependent protein kinase (cAMP-dependent protein kinase), Protein Kinase A (PKA), was first identified and purified in 1968 and is an important class of serine/threonine protein kinases in the protein kinase family [4 ]. After binding of the signal molecule to the receptors on the plasma membrane, the activity of Adenylate Cyclase (AC) is modulated by G protein-coupled receptors (Gi or Gs). AC catalyzes ATP to generate cAMP to control cAMP levels, which in turn determines the activity of the protein kinase PKA. PKA is responsible for phosphorylation of many different types of proteins within cells, thereby regulating biological metabolism and gene expression. For example, in a vegetative state, the vegetative growth of cells is promoted by the Ras-cAMP signaling pathway.

The PKA holoenzyme structure is a special tetramer, consisting of two catalytic subunits (C) and two regulatory subunits (R). The catalytic subunit contains an active site for hydrolyzing ATP and a domain that binds to the regulatory subunit. The regulatory subunit contains a domain that binds cAMP [5]. In the absence of cAMP, the activity of the catalytic subunit is inhibited by the regulatory subunit, and thus PKA under the tetrameric holoenzyme structure is inactive. The 4 cAMP molecules can bind to the two regulatory subunits, respectively, causing a conformational change in the PKA kinase, resulting in the dissociation of the two catalytic subunits. Phosphorylation of serine/threonine proteins containing the Arg-Arg-X-Ser/Thr sequence in the presence of ATP can begin without a catalytic subunit that regulates subunit binding inhibition [6 ]]. In Saccharomyces cerevisiae, the regulatory subunit of PKA kinase isBCY1The catalytic subunit is encoded by three genes with repeated functional parts, respectivelyTPK1, TPK2, TPK3. Upon stimulation with glucose, the cAMP/PKA pathway is activated and autophosphorylation of the Ser179 site of TPK1 is increased, resulting in an increased PKA kinase activity. The autophosphorylation mechanism of TPK1 effectively regulates PKA activity in the presence of glucose [7]。

In yeast, the PKA signaling pathway plays an important role in cell growth in response to nutrients. Before the cells were disrupted, PKA activated by autophosphorylation of Ser179 promoted cell growth. And the efficiency of in vitro protein synthesis may be effectively altered after cells in continuous PKA activation state are prepared into lysates by mutating serine to aspartic acid. Therefore, Kluyveromyces as a test strain simulates the autophosphorylation of serine, so as to determine whether it has an influence on the synthesis efficiency of foreign proteins in the cell-free protein synthesis system.

The references are as follows:

1.Ptacek et al., Global analysis of protein phosphorylation in yeast.Nature, 2005. 438: 679-684.

2.Hunter T. Signaling-2000 and beyond. Cell, 2000. 100:113-127.

3.Kim et al., Prediction of phosphorylation sites using SVMs.Bioinformatics, 2004. 20(17):3179-3184.

4.Walsh et al., An adenosine 3’,5’-monophosphate-dependant protein kinasefrom rabbit skeletal muscle. J. Biol. Chem., 1968. 243:3763-3765.

5.Bauman&Scott., Kinase- and phosphatase-anchoring proteins: harnessingthe dynamic duo. Nature Cell Biology, 2002. 4(8):E203-6.

6.Voet et al., Fundamentals of Biochemistry. Wiley. 2006, Pg492

Solari et al., Regulation of PKA activity by an autophosphorylationmechanism in Saccharomyces cerevisiae. Biochem. J., 2014. 462: 567–579。

disclosure of Invention

In order to solve the problems, the corresponding serine locus in the kluyveromyces is found through sequence comparison, and the corresponding nucleotide sequence is modified, so that the serine at the locus is modified into aspartic acid, and the synthesis efficiency of the foreign protein in the cell-free protein synthesis system is improved.

The invention provides a gene modification method of eukaryote, which carries out base mutation on TPK1 encoding gene of 3 '5' -cyclic adenosine monophosphate dependent protein kinase, namely protein kinase A catalytic subunit through gene editing, thereby mutating serine corresponding to 179 th site of saccharomyces cerevisiae TPK1 amino acid sequence in TPK1 amino acid sequence of eukaryote into aspartic acid.

Further, the corresponding position is position 237.

Further, the gene editing technology is the prior art, and the CRISPR/Cas9 gene editing technology can be selected.

In a second aspect, the present invention provides a genetically engineered cell comprising in its genome a modification made by the method of genetic modification of the first aspect.

Further, the cell is a eukaryotic cell.

Further, the eukaryotic cell is one of a mammalian cell, a plant cell, a yeast cell, an insect cell or any combination thereof.

Further, the yeast cell is selected from one of Pichia pastoris, Kluyveromyces yeast or a combination thereof.

Further, the yeast of the genus Kluyveromyces is selected from one of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces multibuyveri, or any combination thereof.

In a third aspect, the invention provides a eukaryotic cell-free protein synthesis system comprising at least a cellular extract from the genetically engineered cell of any one of the second aspects.

The fourth aspect of the present invention provides the use of the method of gene engineering according to the first aspect and the use of the genetically engineered cell according to the second aspect in increasing the expression level of foreign proteins in a eukaryotic cell-free protein synthesis system.

The fifth aspect of the invention provides a method for improving the expression level of foreign proteins in a eukaryotic cell-free protein synthesis system, which comprises the following steps:

step 1, providing a eukaryotic cell-free protein synthesis system according to the third aspect;

and 2, adding DNA molecules for encoding the foreign protein into the integrated system obtained in the step 1, and incubating under certain conditions to synthesize the foreign protein.

Further, the reaction temperature is 20 to 35 ℃, preferably 20 to 30 ℃, and more preferably 25 ℃.

Further, the reaction time is 0.5-20h, preferably 1-18h, more preferably 2-15 h, more preferably 3-12 h; the reaction time can be determined manually according to specific conditions, and can also be 3-15h, 3-20h, or specific time points, such as 3h, 5h, 10h, 15h, 18h, and 20 h.

The main advantages and positive effects of the invention mainly include:

(1) the invention firstly transforms TPK1 gene in cells by gene transformation technology and with the help of high-efficiency cell transformation platform, thereby changing the protein synthesis efficiency of a translation system;

(2) the invention carries out S237D point mutation on TPK1 for the first time by CRISPR-CAS9 gene editing technology, thereby changing the in vitro protein synthesis capacity;

(3) the PKA channel modification thought provided by the invention increases the selectable modification target spots in host bacteria, can enhance the translation efficiency of the eukaryotic in vitro biosynthesis system initiation protein, and can increase the possibility of the in vitro biosynthesis system aiming at different protein synthesis.

Drawings

Figure 1 protein sequence alignment of TPK1-3 for s.cerevisiae and TPK1-2 for k.lacties. In this figure, it was determined that serine at position 179 of TPK1 in s.cerevisiae is more conserved among other proteins, and that TPK1 of k.lactis is serine at position 237.

FIG. 2 is a schematic map of plasmid pCas9_ KlTPK 1.

FIG. 3 is a plasmid map schematic diagram of pMD18T-TPK 1S 237D.

FIG. 4 is a graph of data of an in vitro translational activity assay of the engineered strain. The green fluorescent protein (eGFP) is used as a foreign protein, and the synthesis capacity of the recombinant protein of the in vitro biosynthesis system is indicated according to the fluorescence intensity of the green fluorescent protein. Wherein A represents Kluyveromyces before modification (unmodified), and B represents Kluyveromyces after TPK1 gene modification.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

The invention uses lactate Kluyveromyces lactis by CRISPR-Cas9 gene editing technologyYeast (C)K. lactis) For example, the gene group of the plant is modifiedTPK1And carrying out mutation transformation on the gene. TPK1 is a catalytic subunit of PKA (protein kinase a) that is mimicked by point mutations to autophosphorylate, thereby activating catalytic activity and increasing PKA pathway activity to influence and regulate the efficiency of cell-free in vitro translation systems. However, the above modification utilizes the CRISPR-Cas9 gene editing technology, but it is not described that only this technology can be selected for gene editing, and the existing gene editing technology can be applied. The modification is verified in a Kluyveromyces lactis system, and the modification is not shown to be only applicable to the Kluyveromyces lactis system but also applicable to other eukaryotic systems.

Based onS. cerevisiaeIn yeastTPK1Gene sequences in the NCBI databaseTPK1BLAST comparison analysis is carried out on the genes to determine that the genes are in the Kluyveromyces lactis,TPK1the gene numbering is KLLA0B07205p (SEQ ID NO. 1). The nucleotide sequence of the code TPK1 is shown as SEQ ID NO.1, and the protein sequence of the code TPK1 is shown as SEQ ID NO. 2.

SEQ ID NO.1

ATGAGACCTTTTGACAACGAATCCAAGGGATTTCTTTCTGGCCGAACAGATCAAATTGAATTACCTGTGGTACAGGCTGTTTCAACAAGTTCAAATTCTCAAGAACCACTTCTTTCTAGCAAAGACCCACTACGAGATGGAAGCAACGTGGGGAATAGGTTGAGTGGAAACAGCGAAACTCTGATAACCAACTCTGATGCTGATCATCCTATGGAGACGGATAATAATAACTCTAATCTTAATAATACTAATTCCTCTACTAATACTAATGATAACAAAGATTCGTCATCCAATTCCAGCAATAATGAAAATGGGGACAGTAGCAACAATTCAAATAGAAGGCATAGCAACCATTGTCACAAGCATAACCATCTAAGCAGCACTTCTACCCTCAAGGCAAGAGTTACTTCTGGAAAATATGCATTATATGATTTTCAGATTCTGAGAACTTTGGGTACAGGTTCGTTTGGTAGAGTTCATTTGGTGAGATCCAATCACAACGGCAGGTTCTATGCAATGAAAGTTTTGAAAAAAAACACCGTTGTCAAATTGAAACAAGTGGAGCATACCAACGATGAAAGAAATATGCTAAGTATAGTATCCCATCCTTTCATAATTAGAATGTGGGGAACTTTCCAGGACTCACAGCAATTGTTTATGATCATGGATTACATCGAAGGTGGTGAACTATTCTCTCTTTTAAGGAAGTCTCAAAGGTTCCCAAACCCGGTCGCGAAGTTTTATGCAGCAGAAGTTTGTCTTGCGTTGGAATACCTACATTCTAAAGGCATCATTTACAGAGATTTGAAACCAGAAAATATCCTTTTAGATAAGAACGGACATATCAAGTTAACTGATTTCGGGTTCGCTAAATATGTTCCTGATGTCACTTACACTCTCTGTGGAACACCGGACTATATCGCACCTGAAGTGGTAAGCACAAAACCTTACAACAAATCTGTGGATTGGTGGAGTTTTGGTGTTTTAATCTACGAAATGTTAGCAGGATATACACCTTTCTACGATTCAAACACAATTAAGACCTATGAGAATATTCTAAATGCTCCAGTAAGATTCCCACCATTTTTCCATTCTGATGCTCAAGATCTAATATCGAAACTCATCACAAGAGACTTAAGTCAACGGCTAGGTAATTTACAAAATGGAAGTGAGGACGTAAAGAATCATCCGTGGTTCAGCGAGGTTGTGTGGGAAAAACTACTCTGCAAAAACATTGAAACTCCATATGAACCTCCTATTCAGGCCGGACAAGGCGATACCTCTCAATACGATAGGTATCCAGAGGAAGAGGTTAATTATGGCATTCAAGGCGAAGATCCGTACCATAGTATTTTCACCAACTTTTAG

SEQ ID NO.2

MRPFDNESKGFLSGRTDQIELPVVQAVSTSSNSQEPLLSSKDPLRDGSNVGNRLSGNSETLITNSDADHPMETDNNNSNLNNTNSSTNTNDNKDSSSNSSNNENGDSSNNSNRRHSNHCHKHNHLSSTSTLKARVTSGKYALYDFQILRTLGTGSFGRVHLVRSNHNGRFYAMKVLKKNTVVKLKQVEHTNDERNMLSIVSHPFIIRMWGTFQDSQQLFMIMDYIEGGELFSLLRKSQRFPNPVAKFYAAEVCLALEYLHSKGIIYRDLKPENILLDKNGHIKLTDFGFAKYVPDVTYTLCGTPDYIAPEVVSTKPYNKSVDWWSFGVLIYEMLAGYTPFYDSNTIKTYENILNAPVRFPPFFHSDAQDLISKLITRDLSQRLGNLQNGSEDVKNHPWFSEVVWEKLLCKNIETPYEPPIQAGQGDTSQYDRYPEEEVNYGIQGEDPYHSIFTNF

The serine at the 179 th site of TPK1 in s.cerevisiae and the corresponding serine in kluyveromyces are found to be 237 th site by sequence alignment (see fig. 1), and the corresponding nucleotide sequence is modified, so that the serine at the site is modified into aspartic acid. The modified sequence is as follows:

the nucleotide sequence of the code TPK 1S 237D is shown in SEQ ID NO.3, and the protein sequence of TPK 1S 237D is shown in SEQ ID NO. 4.

ATGAGACCTTTTGACAACGAATCCAAGGGATTTCTTTCTGGCCGAACAGATCAAATTGAATTACCTGTGGTACAGGCTGTTTCAACAAGTTCAAATTCTCAAGAACCACTTCTTTCTAGCAAAGACCCACTACGAGATGGAAGCAACGTGGGGAATAGGTTGAGTGGAAACAGCGAAACTCTGATAACCAACTCTGATGCTGATCATCCTATGGAGACGGATAATAATAACTCTAATCTTAATAATACTAATTCCTCTACTAATACTAATGATAACAAAGATTCGTCATCCAATTCCAGCAATAATGAAAATGGGGACAGTAGCAACAATTCAAATAGAAGGCATAGCAACCATTGTCACAAGCATAACCATCTAAGCAGCACTTCTACCCTCAAGGCAAGAGTTACTTCTGGAAAATATGCATTATATGATTTTCAGATTCTGAGAACTTTGGGTACAGGTTCGTTTGGTAGAGTTCATTTGGTGAGATCCAATCACAACGGCAGGTTCTATGCAATGAAAGTTTTGAAAAAAAACACCGTTGTCAAATTGAAACAAGTGGAGCATACCAACGATGAAAGAAATATGCTAAGTATAGTATCCCATCCTTTCATAATTAGAATGTGGGGAACTTTCCAGGACTCACAGCAATTGTTTATGATCATGGATTACATCGAAGGTGGTGAACTATTCTCTCTTTTAAGGAAGGACCAAAGGTTCCCAAACCCGGTCGCGAAGTTTTATGCAGCAGAAGTTTGTCTTGCGTTGGAATACCTACATTCTAAAGGCATCATTTACAGAGATTTGAAACCAGAAAATATCCTTTTAGATAAGAACGGACATATCAAGTTAACTGATTTCGGGTTCGCTAAATATGTTCCTGATGTCACTTACACTCTCTGTGGAACACCGGACTATATCGCACCTGAAGTGGTAAGCACAAAACCTTACAACAAATCTGTGGATTGGTGGAGTTTTGGTGTTTTAATCTACGAAATGTTAGCAGGATATACACCTTTCTACGATTCAAACACAATTAAGACCTATGAGAATATTCTAAATGCTCCAGTAAGATTCCCACCATTTTTCCATTCTGATGCTCAAGATCTAATATCGAAACTCATCACAAGAGACTTAAGTCAACGGCTAGGTAATTTACAAAATGGAAGTGAGGACGTAAAGAATCATCCGTGGTTCAGCGAGGTTGTGTGGGAAAAACTACTCTGCAAAAACATTGAAACTCCATATGAACCTCCTATTCAGGCCGGACAAGGCGATACCTCTCAATACGATAGGTATCCAGAGGAAGAGGTTAATTATGGCATTCAAGGCGAAGATCCGTACCATAGTATTTTCACCAACTTTTAG

SEQ ID NO.4

MRPFDNESKGFLSGRTDQIELPVVQAVSTSSNSQEPLLSSKDPLRDGSNVGNRLSGNSETLITNSDADHPMETDNNNSNLNNTNSSTNTNDNKDSSSNSSNNENGDSSNNSNRRHSNHCHKHNHLSSTSTLKARVTSGKYALYDFQILRTLGTGSFGRVHLVRSNHNGRFYAMKVLKKNTVVKLKQVEHTNDERNMLSIVSHPFIIRMWGTFQDSQQLFMIMDYIEGGELFSLLRKDQRFPNPVAKFYAAEVCLALEYLHSKGIIYRDLKPENILLDKNGHIKLTDFGFAKYVPDVTYTLCGTPDYIAPEVVSTKPYNKSVDWWSFGVLIYEMLAGYTPFYDSNTIKTYENILNAPVRFPPFFHSDAQDLISKLITRDLSQRLGNLQNGSEDVKNHPWFSEVVWEKLLCKNIETPYEPPIQAGQGDTSQYDRYPEEEVNYGIQGEDPYHSIFTNF

Example 1 engineering of Yeast TPK1 protein by CRISPR-Cas9

1.1 sequence search and CRISPR gRNA sequence determination of TPK1

According to the alignment of the protein sequences,S.cerevisiaeandK.lactisin cellsTPK1The gene is highly conserved at the S site to be engineered. To achieve point mutation engineering of S237D in TPK1, the corresponding grnas were determined to cleave to cause double strand breaks by searching for the PAM sequence (NGG) near this S237 residue. The principle of gRNA selection in this example is: the GC content is moderate (40% -60%), and the existence of a poly T structure is avoided. Finally, the gRNA sequence of the optimized TPK1 determined by the present invention was GTCTCAAAGGTTCCCAAACC.

1.2 construction of CRISPR plasmid

TPK1 gRNA is constructed on pCas9 plasmid, TPK1-gRNA-PF GTCTCAAAGGTTCCCAAACCGTTTTAGAGCTAGAAATAGC, TPK1-gRNA-PR GGTTTGGGAACCTTGACGACGTCGAAGGAAAC is used as primer, PCR amplification is carried out by taking pCAS plasmid as template, 17 mu L of amplification product is taken, 0.2 mu L Dpn I and 2 mu L10 Xdigestion buffer are added, after mixing, water bath at 37 ℃ is carried out for 3h, 10 mu L of product after treatment of Dpn I is taken and added into 50 mu L DH5 α competent cells, the product is placed on ice for 30min, after heat shock at 42 ℃ for 45 s, 1mL LB liquid culture medium at 37 ℃ is added for oscillation culture for 1 h, the product is coated on Kan resistant LB solid culture, the product is subjected to 37 ℃ for oscillation culture until single clone growth, 2 single clones are selected and named as LB liquid culture medium, PCR 9 is extracted after positive oscillation culture and sequencing confirmation, and the pCAS plasmid is shown in figure 1 (see).

1.3 Donor DNA plasmid construction and amplification

To facilitate the storage and amplification of linear donor DNA, this example inserts the donor DNA into the pMD18T plasmid and amplifies by PCR to obtain a linear donor DNA sequence.

Taking pMD18T plasmid as a template, and primer pMD 18T-PF: ATCGTCGACCTGCAGGCATG and pMD 18T-PR: ATCTCTAGAGGATCCCCGGG, carrying out PCR amplification, taking 17 muL of an amplification product, adding 1 muL DpnI and 2 muL 10 Xdigestion buffer, uniformly mixing, and carrying out water bath at 37 ℃ for 3h to obtain a plasmid skeleton linear fragment pMD 18T-vector.

1.3.1 construction of the Donor plasmid pMD18T-TPK 1S 237D

Taking Kluyveromyces lactis genomic DNA as a template, and carrying out reaction on the DNA by using a primer KlTPK1-HR 1-PF: GAGCTCGGTACCCGGGGATCCTCTAGAGATGAACCGCTATATCTTGCATG and KlTPK 1-mutant-PR: GACTGGATTAGGAAAACGCTGGTCTTTCCTTAAAAGAGAGAATAG PCR amplification is carried out, the product is named as TPK1-HR 1;

taking Kluyveromyces lactis genomic DNA as a template, and carrying out reaction on the DNA by using a primer KlTPK 1-mutant-PF: AAGGAAAGACCAGCGTTTTCCTAATCCAGTCGCGAAGTTTTATGCAGCAG and KlTPK1-HR 2-PR: GCATGCCTGCAGGTCGACGATTAACGGCAGCGTTTCTGAAG PCR amplification is carried out, the product is named as TPK1-HR 2;

1 μ L each of the amplification products TPK1-HR1, TPK1-HR2 and pMD18T-vector was added to 3 μ L of Cloning Mix (Transgene pEASY-Uni Seamless Cloning and Assembly Kit, all-open gold Co., Ltd., the same below), and mixed well in water at 50 ℃ for 1 h. After the water bath is finished, the reaction solution is placed on ice for 2min, 6 microliter of reaction solution is completely added into 50 microliter of Trans-T1 competent cells (the whole formula gold company, the same below), the reaction solution is placed on ice for 30min, after heat shock is carried out for 30 s at 42 ℃, 1mL of LB liquid culture medium is added, shaking culture is carried out at 37 ℃ for 1 h, the mixture is coated on Amp resistance LB solid culture, and inversion culture is carried out at 37 ℃ until single colonies grow out. 6 single clones are picked and cultured in LB liquid culture medium in a shaking way, after PCR detection is positive and sequencing is confirmed, plasmids are extracted and stored, and the name of the plasmids is pMD18T-TPK 1S 237D.

1.4 K. lactis electrotransformation

Taking out competence from a refrigerator at minus 80 ℃, thawing on ice, adding 400 ng of gRNA & Cas9 plasmid (or gRNA/Cas9 fragment) and 1000ng of donor DNA fragment obtained by amplifying pMD18T-TPK 1S 237D through a primer pair, uniformly mixing, transferring all the mixture into an electric shock cup, and carrying out ice bath for 2 min; putting the electric shock cup into an electric rotating instrument for electric shock (the parameters are 1.5kV, 200 omega and 25 muF); immediately adding 700 mu L of YPD after the electric shock is finished, and incubating for 1-3 h by using a shaking table at 30 ℃ and 200 rpm; 2-200. mu.L of the suspension was inoculated onto YPD (containing G418 resistance) plates and cultured at 30 ℃ for 2-3 days until single colonies appeared.

1.5 Positive identification

12-24 monoclonals were picked from the transformed plates, and the cells were used as templates, and the DNA fragments were analyzed by using the identifying primers F: AGTCTCAAAGGTTCCCAAAC and R: ATAAGATTATTGCATCGAGC PCR detection was performed on the samples. The negative strain had no PCR band. The cell strain which is positive in PCR result and identified by sequencing through a primer pair F: GGGTATTTCGAATAAGGGAC, R: ATAAGATTATTGCATCGAGC is determined to be a positive cell strain and is named as TPK 1S 237D.

EXAMPLE 2 in vitro protein Synthesis System

2.1 preparation of cell extracts

The preparation method of the cell extract comprises the following steps:

(i) providing cells, which are the TPK 1S 237D cell line prepared in example 1;

(ii) washing the cells to obtain washed cells;

(iii) subjecting the washed cells to cell disruption treatment, thereby obtaining a crude cell extract;

(iv) and carrying out solid-liquid separation on the cell crude extract to obtain a liquid part, namely the cell extract.

The solid-liquid separation method is not particularly limited, and centrifugation is used as the method selected in the present example. The centrifugation conditions were 30000 Xg; centrifuging for 30 min; centrifugation 4^oAnd C, performing.

The washing treatment method is not particularly limited, and the washing treatment method selected in this example is a method of treating the substrate with a washing solution at a pH of 7.4, the washing solution is not particularly limited, and typically the washing solution is selected from the following group: potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof. Potassium acetate was chosen for this example.

Wherein, the cell disruption treatment is not particularly limited, and a preferable cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.

2.2 preparation of in vitro protein Synthesis System

4-hydroxyethylpiperazine ethanesulfonic acid at a final concentration of 22 mM, pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixtures (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate), 0.08-0.24 mM amino acid mixtures (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25 mM creatine phosphate, 1.7 mM dithiol, 0.027-0.054 mg/mL T7 RNA polymerase, 0.27 mg/mL creatine phosphate kinase, 1-4% polyethylene glycol, 0.5% -2% sucrose, and finally adding 50% cell extract.

2.3 in vitro protein Synthesis reactions

Placing the reaction system in an environment with the temperature of about 25 ℃ for reaction for 3 hours;

enhanced green fluorescent protein (eGFP) activity assay: after the reaction, 10 μ L of the reaction solution was added to a 96-well white plate or a 384-well white plate, and immediately placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), and the enhanced green fluorescent protein activity was detected by reading, and the Relative Fluorescence Unit (RFU) was used as the activity Unit, as shown in fig. 4.

Wherein A is unmodified Kluyveromyces and B is modified Kluyveromyces, wherein RFU value of A is 373.7, and RFU of B is 676. The value of B is 1.8 times that of A. Namely, TPK1 was subjected to S237D point mutation by gene editing technique, whereby the ability to synthesize a foreign protein in a cell-free protein synthesis system could be improved.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Kangma (Shanghai) Biotech Co., Ltd

<120> gene modification method of protein kinase A catalytic subunit TPK1 and application thereof

<130>2018

<141>2018-11-30

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<213> Kluyveromyces lactis (Kluyveromyces lactis)

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atgagacctt ttgacaacga atccaaggga tttctttctg gccgaacaga tcaaattgaa 60

ttacctgtgg tacaggctgt ttcaacaagt tcaaattctc aagaaccact tctttctagc 120

aaagacccac tacgagatgg aagcaacgtg gggaataggt tgagtggaaa cagcgaaact 180

ctgataacca actctgatgc tgatcatcct atggagacgg ataataataa ctctaatctt 240

aataatacta attcctctac taatactaat gataacaaag attcgtcatc caattccagc 300

aataatgaaa atggggacag tagcaacaat tcaaatagaa ggcatagcaa ccattgtcac 360

aagcataacc atctaagcag cacttctacc ctcaaggcaa gagttacttc tggaaaatat 420

gcattatatg attttcagat tctgagaact ttgggtacag gttcgtttgg tagagttcat 480

ttggtgagat ccaatcacaa cggcaggttc tatgcaatga aagttttgaa aaaaaacacc 540

gttgtcaaat tgaaacaagt ggagcatacc aacgatgaaa gaaatatgct aagtatagta 600

tcccatcctt tcataattag aatgtgggga actttccagg actcacagca attgtttatg 660

atcatggatt acatcgaagg tggtgaacta ttctctcttt taaggaagtc tcaaaggttc 720

ccaaacccgg tcgcgaagtt ttatgcagca gaagtttgtc ttgcgttgga atacctacat 780

tctaaaggca tcatttacag agatttgaaa ccagaaaata tccttttaga taagaacgga 840

catatcaagt taactgattt cgggttcgct aaatatgttc ctgatgtcac ttacactctc 900

tgtggaacac cggactatat cgcacctgaa gtggtaagca caaaacctta caacaaatct 960

gtggattggt ggagttttgg tgttttaatc tacgaaatgt tagcaggata tacacctttc 1020

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caacggctag gtaatttaca aaatggaagt gaggacgtaa agaatcatcc gtggttcagc 1200

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Met Arg Pro Phe Asp Asn Glu Ser Lys Gly Phe Leu Ser Gly Arg Thr

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Asp Gln Ile Glu Leu Pro Val Val Gln Ala Val Ser Thr Ser Ser Asn

20 25 30

Ser Gln Glu Pro Leu Leu Ser Ser Lys Asp Pro Leu Arg Asp Gly Ser

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Asn Val Gly Asn Arg Leu Ser Gly Asn Ser Glu Thr Leu Ile Thr Asn

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Ser Asp Ala Asp His Pro Met Glu Thr Asp Asn Asn Asn Ser Asn Leu

65 70 75 80

Asn Asn Thr Asn Ser Ser Thr Asn Thr Asn Asp Asn Lys Asp Ser Ser

85 90 95

Ser Asn Ser Ser Asn Asn Glu Asn Gly Asp Ser Ser Asn Asn Ser Asn

100 105 110

Arg Arg His Ser Asn His Cys His Lys His Asn His Leu SerSer Thr

115 120 125

Ser Thr Leu Lys Ala Arg Val Thr Ser Gly Lys Tyr Ala Leu Tyr Asp

130 135 140

Phe Gln Ile Leu Arg Thr Leu Gly Thr Gly Ser Phe Gly Arg Val His

145 150 155 160

Leu Val Arg Ser Asn His Asn Gly Arg Phe Tyr Ala Met Lys Val Leu

165 170 175

Lys Lys Asn Thr Val Val Lys Leu Lys Gln Val Glu His Thr Asn Asp

180 185 190

Glu Arg Asn Met Leu Ser Ile Val Ser His Pro Phe Ile Ile Arg Met

195 200 205

Trp Gly Thr Phe Gln Asp Ser Gln Gln Leu Phe Met Ile Met Asp Tyr

210 215 220

Ile Glu Gly Gly Glu Leu Phe Ser Leu Leu Arg Lys Ser Gln Arg Phe

225 230 235 240

Pro Asn Pro Val Ala Lys Phe Tyr Ala Ala Glu Val Cys Leu Ala Leu

245 250 255

Glu Tyr Leu His Ser Lys Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu

260 265 270

Asn Ile Leu Leu Asp Lys Asn Gly His Ile Lys Leu Thr Asp Phe Gly

275 280 285

Phe Ala Lys Tyr Val Pro Asp Val Thr Tyr Thr Leu Cys Gly Thr Pro

290 295 300

Asp Tyr Ile Ala Pro Glu Val Val Ser Thr Lys Pro Tyr Asn Lys Ser

305 310 315 320

Val Asp Trp Trp Ser Phe Gly Val Leu Ile Tyr Glu Met Leu Ala Gly

325 330 335

Tyr Thr Pro Phe Tyr Asp Ser Asn Thr Ile Lys Thr Tyr Glu Asn Ile

340 345 350

Leu Asn Ala Pro Val Arg Phe Pro Pro Phe Phe His Ser Asp Ala Gln

355 360 365

Asp Leu Ile Ser Lys Leu Ile Thr Arg Asp Leu Ser Gln Arg Leu Gly

370 375 380

Asn Leu Gln Asn Gly Ser Glu Asp Val Lys Asn His Pro Trp Phe Ser

385 390 395 400

Glu Val Val Trp Glu Lys Leu Leu Cys Lys Asn Ile Glu Thr Pro Tyr

405 410 415

Glu Pro Pro Ile Gln Ala Gly Gln Gly Asp Thr Ser Gln Tyr Asp Arg

420 425 430

Tyr Pro Glu Glu Glu Val Asn Tyr Gly Ile Gln Gly Glu Asp Pro Tyr

435 440 445

His Ser Ile Phe Thr Asn Phe

450 455

<210>3

<211>1368

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atgagacctt ttgacaacga atccaaggga tttctttctg gccgaacaga tcaaattgaa 60

ttacctgtgg tacaggctgt ttcaacaagt tcaaattctc aagaaccact tctttctagc 120

aaagacccac tacgagatgg aagcaacgtg gggaataggt tgagtggaaa cagcgaaact 180

ctgataacca actctgatgc tgatcatcct atggagacgg ataataataa ctctaatctt 240

aataatacta attcctctac taatactaat gataacaaag attcgtcatc caattccagc 300

aataatgaaa atggggacag tagcaacaat tcaaatagaa ggcatagcaa ccattgtcac 360

aagcataacc atctaagcag cacttctacc ctcaaggcaa gagttacttc tggaaaatat 420

gcattatatg attttcagat tctgagaact ttgggtacag gttcgtttgg tagagttcat 480

ttggtgagat ccaatcacaa cggcaggttc tatgcaatga aagttttgaa aaaaaacacc 540

gttgtcaaat tgaaacaagt ggagcatacc aacgatgaaa gaaatatgct aagtatagta 600

tcccatcctt tcataattag aatgtgggga actttccagg actcacagca attgtttatg 660

atcatggatt acatcgaagg tggtgaacta ttctctcttt taaggaagga ccaaaggttc 720

ccaaacccgg tcgcgaagtt ttatgcagca gaagtttgtc ttgcgttgga atacctacat 780

tctaaaggca tcatttacag agatttgaaa ccagaaaata tccttttaga taagaacgga 840

catatcaagt taactgattt cgggttcgct aaatatgttc ctgatgtcac ttacactctc 900

tgtggaacac cggactatat cgcacctgaa gtggtaagca caaaacctta caacaaatct 960

gtggattggt ggagttttgg tgttttaatc tacgaaatgt tagcaggata tacacctttc 1020

tacgattcaa acacaattaa gacctatgag aatattctaa atgctccagt aagattccca 1080

ccatttttcc attctgatgc tcaagatcta atatcgaaac tcatcacaag agacttaagt 1140

caacggctag gtaatttaca aaatggaagt gaggacgtaa agaatcatcc gtggttcagc 1200

gaggttgtgt gggaaaaact actctgcaaa aacattgaaa ctccatatga acctcctatt 1260

caggccggac aaggcgatac ctctcaatac gataggtatc cagaggaaga ggttaattat 1320

ggcattcaag gcgaagatcc gtaccatagt attttcacca acttttag 1368

<210>4

<211>455

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Met Arg Pro Phe Asp Asn Glu Ser Lys Gly Phe Leu Ser Gly Arg Thr

1 5 10 15

Asp Gln Ile Glu Leu Pro Val Val Gln Ala Val Ser Thr Ser Ser Asn

20 25 30

Ser Gln Glu Pro Leu Leu Ser Ser Lys Asp Pro Leu Arg Asp Gly Ser

35 40 45

Asn Val Gly Asn Arg Leu Ser Gly Asn Ser Glu Thr Leu Ile Thr Asn

50 55 60

Ser Asp Ala Asp His Pro Met Glu Thr Asp Asn Asn Asn Ser Asn Leu

65 70 75 80

Asn Asn Thr Asn Ser Ser Thr Asn Thr Asn Asp Asn Lys Asp Ser Ser

85 90 95

Ser Asn Ser Ser Asn Asn Glu Asn Gly Asp Ser Ser Asn Asn Ser Asn

100 105 110

Arg Arg His Ser Asn His Cys His Lys His Asn His Leu Ser Ser Thr

115 120 125

Ser Thr Leu Lys Ala Arg Val Thr Ser Gly Lys Tyr Ala Leu Tyr Asp

130 135 140

Phe Gln Ile Leu Arg Thr Leu Gly Thr Gly Ser Phe Gly Arg Val His

145 150 155 160

Leu Val Arg Ser Asn His Asn Gly Arg Phe Tyr Ala Met Lys Val Leu

165 170 175

Lys Lys Asn Thr Val Val Lys Leu Lys Gln Val Glu His Thr Asn Asp

180 185 190

Glu Arg Asn Met Leu Ser Ile Val Ser His Pro Phe Ile Ile Arg Met

195 200 205

Trp Gly Thr Phe Gln Asp SerGln Gln Leu Phe Met Ile Met Asp Tyr

210 215 220

Ile Glu Gly Gly Glu Leu Phe Ser Leu Leu Arg Lys Asp Gln Arg Phe

225 230 235 240

Pro Asn Pro Val Ala Lys Phe Tyr Ala Ala Glu Val Cys Leu Ala Leu

245 250 255

Glu Tyr Leu His Ser Lys Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu

260 265 270

Asn Ile Leu Leu Asp Lys Asn Gly His Ile Lys Leu Thr Asp Phe Gly

275 280 285

Phe Ala Lys Tyr Val Pro Asp Val Thr Tyr Thr Leu Cys Gly Thr Pro

290 295 300

Asp Tyr Ile Ala Pro Glu Val Val Ser Thr Lys Pro Tyr Asn Lys Ser

305 310 315 320

Val Asp Trp Trp Ser Phe Gly Val Leu Ile Tyr Glu Met Leu Ala Gly

325 330 335

Tyr Thr Pro Phe Tyr Asp Ser Asn Thr Ile Lys Thr Tyr Glu Asn Ile

340 345 350

Leu Asn Ala Pro Val Arg Phe Pro Pro Phe Phe His Ser Asp Ala Gln

355 360 365

Asp Leu Ile Ser Lys Leu Ile Thr ArgAsp Leu Ser Gln Arg Leu Gly

370 375 380

Asn Leu Gln Asn Gly Ser Glu Asp Val Lys Asn His Pro Trp Phe Ser

385 390 395 400

Glu Val Val Trp Glu Lys Leu Leu Cys Lys Asn Ile Glu Thr Pro Tyr

405 410 415

Glu Pro Pro Ile Gln Ala Gly Gln Gly Asp Thr Ser Gln Tyr Asp Arg

420 425 430

Tyr Pro Glu Glu Glu Val Asn Tyr Gly Ile Gln Gly Glu Asp Pro Tyr

435 440 445

His Ser Ile Phe Thr Asn Phe

450 455

Claims

1. A method for genetically modifying a eukaryote, comprising: base mutation is carried out on a TPK1 encoding gene of 3 '5' -cyclic adenosine monophosphate-dependent protein kinase, namely a protein kinase A catalytic subunit through gene editing, so that serine corresponding to the 179 th site of a saccharomyces cerevisiae TPK1 amino acid sequence in a TPK1 amino acid sequence of the eukaryote is mutated into aspartic acid.

2. A genetically engineered cell, characterized by: the genetically engineered cell comprises in its genome a modification made by the genetic modification method of claim 1.

3. The genetically engineered cell of claim 2, wherein: the cell is a eukaryotic cell.

4. The genetically engineered cell of claim 3, wherein: the eukaryotic cell is one of mammalian cell, plant cell, yeast cell, insect cell or any combination thereof.

5. The genetically engineered cell of claim 4, wherein: the yeast cell is selected from one of Pichia pastoris and Kluyveromyces yeast or a combination thereof.

6. The genetically engineered cell of claim 5, wherein: the yeast of the genus Kluyveromyces is selected from one of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybracteus, or any combination thereof.

7. A eukaryotic cell-free protein synthesis system, characterized by: the synthesis system at least comprises a cell extract from the genetically engineered cell of any one of claims 2 to 6.

8. The use of the genetic modification method of claim 1 to increase the amount of foreign protein expressed in a eukaryotic cell-free protein synthesis system.

9. Use of the genetically engineered cell of any one of claims 2 to 6 for increasing the expression level of a foreign protein in a eukaryotic cell-free protein synthesis system.

10. A method for improving the expression level of foreign proteins in a eukaryotic cell-free protein synthesis system comprises the following steps:

step 1, providing a eukaryotic cell-free protein synthesis system according to claim 7;

and 2, adding DNA molecules for encoding the foreign protein into the synthesis system in the step 1, reacting for a period of time under a certain temperature condition, and incubating to synthesize the foreign protein.