Protease translation elongation factor mutant protein as well as preparation method and application thereof

Abstract

The invention provides a protease translation elongation factor mutant protein as well as a preparation method and application thereof, and particularly relates to a method for editing proteasome related genes in a Kluyveromyces lactis strain by virtue of a CRISPR-Cas9 mediated gene editing means, mutating phosphorylation modification sites T57 and T59 of a translation elongation factor eEF2, and carrying out mutation on the phosphorylation modification sites T57 and T59 of the translation elongation factor eEF2 to obtain the protease translation elongation factor mutant protein. The in-vitro protein expression activity of the cell extract can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a protease translation elongation factor mutant protein and a preparation method and application thereof. Background Art

[0002] Cell-free protein synthesis (CFPS) is a technology that uses enzymes, amino acid substrates and energy to synthesize proteins in vitro using exogenous mRNA or DNA as templates. Compared with traditional in vivo recombinant expression systems, in vitro cell-free synthesis systems have many advantages, such as the ability to directly use PCR products as templates to simultaneously synthesize multiple proteins in parallel, and to carry out high-throughput peptide or protein drug screening. In addition, in vitro cell-free synthesis systems also have unique advantages in the expression of peptides or proteins that are toxic to cells. Cell-free protein expression systems are an important supplement to cell-based protein expression systems.

[0003] Existing cell-free protein synthesis systems can be mainly divided into two categories: one is to combine and compound the ribosomes, enzymes, tRNA, etc. required for the synthesis of purified proteins in a certain proportion to obtain the ability to express proteins in vitro, that is, the purified cell-free protein expression system (PURE). This system has clear component information, and the formula can be easily and customized to adjust the formula to obtain different protein expression characteristics, but the disadvantage is that the preparation process is complicated and costly; the other 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 obtain 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. However, there is still huge room for optimization of the supporting technical processes and background strains of the system.

[0004] Protein synthesis is one of the most complex and energy-intensive cellular processes, requiring approximately 150 different proteins to facilitate the various processes involved in translation. Elongation factor 2 (eEF2) is one of the key players in the elongation cycle of protein synthesis. eEF2 is a 95 kDa GTP-binding protein that binds to the pre-translocation ribose. The role of this factor and its eubacterial homolog, elongation factor G (EFG), is to facilitate the translocation of the ribosome along the mRNA in the presence of simultaneous transfer of peptidyl-tRNA and deacylated tRNA to the ribosomal P- and E-sites. The yeast translation elongation factor eEF2 catalyzes ribosome translocation during protein synthesis and is a highly conserved essential gene, as shown in the attached Figure 1As shown, the sequence identity of eEF2 in Kluyveromyces lactis and Saccharomyces cerevisiae is very high. Summary of the invention

[0005] In order to solve the above technical problems, the present invention edits proteasome-related genes in the Kluyveromyces lactis strain through CRISPR-Cas9-mediated gene editing, and mutates threonine sites such as the phosphorylation modification sites T57 and T59 of the translation elongation factor eEF2, so as to effectively improve the in vitro protein expression activity of cell extracts.

[0006] To achieve the present invention, the first aspect of the present invention provides a protease translation elongation factor mutant protein, characterized in that it is obtained by mutating one or more threonine sites in the phosphorylation modification sites of the protease translation elongation factor, and the protein expression activity can be improved after mutation. Preferably, the translation elongation factor is eEF2.

[0007] Since the hydroxyl group on the amino acid is considered to be a reactive site for easy phosphorylation by the translation elongation factor, the threonine residues of the present invention are preferably mutated independently to amino acids not containing a hydroxyl group.

[0008] The threonine can be mutated to alanine (A), tyrosine (V), isoleucine (I), leucine (L), methionine (M), cysteine ​​(C), tryptophan (W), phenylalanine (F), asparagine (D) or a combination thereof.

[0009] The protease translation elongation factor mutant protein is derived from yeast, and the yeast is preferably Kluyveromyces, more preferably Kluyveromyces lactis.

[0010] The mutant protein is mutated in one or more threonine selected from the following group corresponding to SEQ ID NO.1 in the translation elongation factor (EEF2) derived from the wild type of Kluyveromyces lactis:

[0011] Threonine (T) at position 57;

[0012] Threonine (T) at position 59.

[0013] SEQ ID NO.1:

[0014] MVAFTVDQIRSLMDKVTNVRNMSVIAHVDHGKSTLTDSLVQRAGIISAAKAGEARFTDTRKDEQ

[0015] ERGITIKSTAISLFSEMSDDDDVKDIKQKTDGNAFLINLIDSPGHVDFSSEVTAALRVTDGALVVVDT

[0016] VEGVCVQTETVLRQSLAERIKPVVVINKVDRALLELQVSKEDLYQSFSRTVESVNVIISTYADEVLG

[0017] DVQVYPQRGTVAFGSGLHGWAFTVRQFANRYSKKKFGVDREKMMDRLWGDSYFNPKTKKWT

[0018] NKERDADGKPLERAFNMFVLDPIFRLFAAIMNFKKEEIPVLLEKLEINLKGDEKELEGKNLLKVVM

[0019] RKFLPAADALLEMIILHLPSPVTAQNYRAEQLYEGPSDDPACIAIKNCDPKSDLMLYVSKMVPTSD

[0020] KGRFYAFGRVFAGTVKSGQKVRIQGPNFIPGKKEDLFIKAIQRAVLMMGRFVEPIDDCPAGNIIGL

[0021] VGIDQFLLKTGTLTTFEGAHNMKVMKFSVSPVVQVAVEVKNANDLPKLVEGLKRLSKSDPCVLVS

[0022] MSESGEHIVAGTGELHLEICLQDLENDHAGIPLKISPPVVAYRETVEGESSQTALSKSPNKHNRIYL

[0023] KAQPIDEEVSLAIEGGKINPRDDFKARARIMADEFGWDVTDARKIWCFGPDGNGPNLVVDQTK

[0024] AVQYLNEIKDSVVAAFQWATKEGPIFGQMRSVRVNILDVTLHADAIHRGGGQIIPTMRRATYA

[0025] GFLLAEPKIQEPVFLVEIQCPEQAIGGIYSVLNKKRGQVVSEEQRPGTPLFTVKAYLPINESFGFTGE

[0026] LRQATGGQAFPQMVFDHWATLGTDPLDPSTKAGEIVLAARKRQGMKEEVPGWQEYYDKL

[0027] The mutant protein is mutated in one or more threonine selected from the following group corresponding to SEQ ID NO.2 in the wild-type translation elongation factor (EEF2) derived from Saccharomyces cerevisiae:

[0028] Threonine (T) at position 57;

[0029] Threonine (T) at position 59.

[0030] SEQ ID NO.2:

[0031] MVAFTVDQIRSLMDKVTNVRNMSVIAHVDHGKSTLTDSLVQRAGIISAAKAGEARFTDTRKDEQ

[0032] ERGITIKSTAISLFSEMSDDDVKDIKQKTDGNAFLINLIDSPGHVDFSSEVTAALRVTDGALVVVDT

[0033] VEGVCVQTETVLRQSLAERIKPVVVINKVDRALLELQVSKEDLYQSFSRTVESVNVIISTYADEVLG

[0034] DVQVYPQRGTVAFGSGLHGWAFTVRQFANRYSKKFGVDREKMMDRLWGDSYFNPKTKKWT

[0035] NKERDADGKPLERAFNMFVLDPIFRLFAAIMNFKKEEIPVLLEKLEINLKGDEKELEGKNLLKVVM

[0036] RKFLPAADALLEMIILHLPSPVTAQNYRAEQLYEGPSDDPACIAIKNCDPKSDLMLYVSKMVPTSD

[0037] KGRFYAFGRVFAGTVKSGQKVRIQGPNFIPGKKEDLFIKAIQRAVLMMGRFVEPIDDCPAGNIIGL

[0038] VGIDQFLLKTGTLTTFEGAAHNMKVMKFSVSPVVQVAVEVKNANDLPKLVEGLKRLSKSDPCVLVS

[0039] MSESGEHIVAGTGELHLEICLQDLENDHAGIPLKISPPVVAYRETVEGESSQTALSKSPNKHNRIYL

[0040] KAQPIDEEVSLAIEGGKINPRDDFKARARIMADEFGWDVTDARKIWCFGPDGNGPNLVVDQTK

[0041] AVQYLNEIKDSVVAAFQWATKEGPIFGEQMRSVRVNILDVTLHADAIHRGGGQIIPTMRRATYA

[0042] GFLLAEPKIQEPVFLVEIQCPEQAIGGIYSVLNKKRGQVVSEEQRPGTPLFTVKAYLPINESFGFTGE

[0043] LRQATGGQAFPQMVFDHWATLGTDPLDPSTKAGEIVLAARKRQGMKEEVPGWQEYYDKL

[0044] The mutant protease translation elongation factor mutant protein is selected from the following group:

[0045] The mutant protein is mutated in one or more threonines selected from the following group corresponding to SEQ ID NO.1 in the wild-type translation elongation factor (EEF2):

[0046] Threonine (T) at position 57;

[0047] Threonine (T) at position 59.

[0048] The mutant protein is selected from the group consisting of:

[0049] In another preferred embodiment, the 57th threonine (T) is mutated to one or more amino acids selected from the following group: alanine (A), tyrosine (V), isoleucine (I), leucine (L), methionine (M), cysteine ​​(C), tryptophan (W), phenylalanine (F), asparagine (D) or a combination thereof.

[0050] In another preferred embodiment, the 59th threonine (T) is mutated to one or more amino acids selected from the following group: alanine (A), tyrosine (V), isoleucine (I), leucine (L), methionine (M), cysteine ​​(C), tryptophan (W), phenylalanine (F), asparagine (D) or a combination thereof.

[0051] In another preferred embodiment, the mutation is selected from the following group: T57A, T57C, T57F, T57V, T57M, T57I, T57L, T57M, T57D, T57W, T59A, T59C, T59F, T59V, T59M, T59V, T59L, T59M, T59D, and T59W.

[0052] In another preferred embodiment, the mutation is selected from the following group: T57A and T59A, T57A and T59C, T57A and T59W, T57A and T59V, T57A and T59M, T57A and T59I, T57A and T59L, T57A and T59M, T57A and T59D, T57A and T59W.

[0053] In another preferred embodiment, the mutation is selected from the following group: T57C and T59A, T57C and T59C, T57C and T59W, T57C and T59V, T57C and T59M, T57C and T59I, T57C and T59L, T57C and T59M, T57C and T59D, T57C and T59W.

[0054] In another preferred embodiment, the mutation is selected from the following group: T57F and T59A, T57F and T59C, T57F and T59W, T57F and T59V, T57F and T59M, T57F and T59I, T57F and T59L, T57F and T59M, T57F and T59D, T57F and T59W.

[0055] In another preferred embodiment, the mutation is selected from the following group: T57V and T59A, T57V and T59C, T57V and T59W, T57V and T59V, T57V and T59M, T57V and T59I, T57V and T59L, T57V and T59M, T57V and T59D, T57V and T59W.

[0056] In another preferred embodiment, the mutation is selected from the following group: T57M and T59A, T57M and T59C, T57M and T59W, T57M and T59V, T57M and T59M, T57M and T59I, T57M and T59L, T57M and T59M, T57M and T59D, T57M and T59W.

[0057] In another preferred embodiment, the mutation is selected from the following group: T57I and T59A, T57I and T59C, T57I and T59W, T57I and T59V, T57I and T59M, T57I and T59I, T57I and T59L, T57I and T59M, T57I and T59D, T57I and T59W.

[0058] In another preferred embodiment, the mutation is selected from the following group: T57L and T59A, T57L and T59C, T57L and T59W, T57L and T59V, T57L and T59M, T57L and T59I, T57L and T59L, T57L and T59M, T57L and T59D, T57L and T59W.

[0059] In another preferred embodiment, the mutation is selected from the following group: T57M and T59A, T57M and T59C, T57M and T59W, T57M and T59V, T57M and T59M, T57M and T59I, T57M and T59L, T57M and T59M, T57M and T59D, T57M and T59W.

[0060] In another preferred embodiment, the mutation is selected from the following group: T57D and T59A, T57D and T59C, T57D and T59W, T57D and T59V, T57D and T59M, T57D and T59I, T57D and T59L, T57D and T59M, T57D and T59D, T57D and T59W.

[0061] In another preferred embodiment, the mutation is selected from the following group: T57W and T59V, T57W and T59M, T57W and T59I, T57W and T59L, T57W and T59M, T57W and T59D, T57W and T59W.

[0062] In another preferred embodiment, the mutation is selected from the following group: T57W and T59A, T57W and T59C, T57W and T59W, T57W and T59V, T57W and T59M, T57W and T59I, T57W and T59L, T57W and T59M, T57W and T59D, T57W and T59W.

[0063] The second aspect of the present invention provides a polynucleotide, characterized in that the polynucleotide encodes the mutant protease translation elongation factor mutant protein described in the first aspect.

[0064] In a preferred example, the polynucleotide further contains auxiliary elements selected from the following group on the flank of the ORF of the mutant protease translation elongation factor mutant protein: a signal peptide, a secretory peptide, a tag sequence or a combination thereof.

[0065] In another preferred embodiment, the polynucleotide is selected from the following group: a DNA sequence, an RNA sequence or a combination thereof.

[0066] The third aspect of the present invention provides a vector, characterized in that the vector contains the polynucleotide described in the second aspect of the present invention.

[0067] In another preferred embodiment, the vector comprises one or more promoters, which are operably connected to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selected marker, nucleic acid restriction site and / or homologous recombination site.

[0068] In preferred examples in the field, the vector includes a plasmid and a viral vector.

[0069] In another preferred embodiment, the viral vector is selected from phage vectors, baculovirus vectors, animal virus vectors, and plant virus vectors. Further preferably, the soldier vector is selected from papovavirus vectors, adenovirus vectors, herpes virus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors and Epstein-Barr virus vectors, retrovirus vectors, and adeno-associated virus (AAV) vectors.

[0070] The fourth aspect of the present invention provides a strain, characterized in that the host cell contains the vector of the third aspect of the present invention, or the polynucleotide of the second aspect is integrated into its genome, or it expresses the mutant protein described in the first aspect.

[0071] In a preferred example, the strain is selected from prokaryotic cells and eukaryotic cells.

[0072] In another preferred embodiment, the strain is a prokaryotic cell, such as Escherichia coli.

[0073] In another preferred embodiment, the strain is a eukaryotic cell, such as yeast, source cells, Chinese hamster, ovarian cells, insect cells, wheat germ cells, and rabbit reticulocytes.

[0074] Preferably, the yeast is selected from yeast of one or more sources of the following group: a combination of one or more of Saccharomyces cerevisiae and Kluyveromyces yeast. In another preferred embodiment, the Kluyveromyces yeast is selected from a combination of one or more of Kluyveromyces lactis, Kluyveromyces marxianus and Kluyveromyces dobsonii.

[0075] The lactic acid Kluyveromyces strain is a genetically engineered strain in which the expression or activity of the KlEXN53 gene (nuclease gene) is reduced (the specific preparation method is prepared by the method disclosed in CN108949801A), a genetically engineered strain in which the genome is integrated with a first exogenous gene expression cassette of a first nucleic acid construct expressing a fusion protein (such as a fusion protein formed by KlPAB1 and KleIF4G) (the specific preparation method is prepared by the method disclosed in CN111778169A and CN110938649A), and a genetically engineered strain in which the genome is integrated with a first exogenous gene expression cassette of a first nucleic acid construct expressing a fusion protein (such as a fusion protein formed by KlPAB1 and KleIF4G) and the expression or activity of a nuclease (such as KlEXN53) is reduced (the specific preparation method is prepared according to the method disclosed in CN110093284A or CN111778169A). Unless they conflict with the purpose of the present invention, patents CN108949801A, CN111778169A, CN110938649A and CN110093284A and their references are cited in their entirety and for all purposes.

[0076] The fifth aspect of the present invention provides a cell lysate or cell extract, which is obtained by lysing the host cells described in the fifth aspect of the present invention.

[0077] The sixth aspect of the present invention provides a cell-free in vitro protein synthesis system, characterized in that it comprises the cell lysate or cell extract described in the fifth aspect of the present invention.

[0078] The seventh aspect of the present invention provides a use of the above-mentioned mutant protease translation elongation factor mutant protein, characterized in that the mutant protease translation elongation factor mutant protein is used to improve the expression purity, efficiency and / or acquisition rate of exogenous proteins in an in vitro cell-free synthesis system.

[0079] In an eighth aspect, the present invention provides a method for preparing the host cell for in vitro cell-free protein synthesis according to the first aspect of the present invention, comprising the steps of:

[0080] Cultivating the host cell according to claim 9 under conditions suitable for expression, thereby expressing the protease translation elongation factor mutant protein; and / or isolating the protease translation elongation factor mutant protein.

[0081] In a preferred embodiment, the steps include:

[0082] (1)1. Design of gRNA targeting sequence

[0083] Select the ORF region of the targeted protease gene and use CRISPOR (tefor.net)

[0084] gRNA was designed on the website (http: / / crispor.tefor.net / ), and gRNA with higher scores was selected as the candidate sequence. The corresponding primer pairs were synthesized according to the candidate gRNA targeting sequence;

[0085] (2) Construction of gRNA_Cas9 expression vector

[0086] The primer pairs obtained in step (1) were diluted separately and added to PCR tubes for mixing, and then annealing was completed on a PCR instrument. The pCasMFR vector treated with BsaI and purified was mixed in a T4 ligase system, and the annealed primers were added for ligation reaction; all the ligation products were transformed into Escherichia coli DH5α competent cells, and screened on an LB plate containing kanamycin, and the clones with correct sequencing were selected to extract the gRNA_Cas9 co-expression plasmid and determine the concentration before use in subsequent electroporation experiments;

[0087] (3) Construction of double-stranded DNA donor

[0088] The DNA donor sequence was obtained by Overlap-PCR method. Four primers P1, P2, P3 and P4 were selected from the ORF of the selected protease gene, p1+p2 and p3+p4 were used as forward and reverse primer pairs, respectively, and the genomic DNA of K.lactis Y1140 was used as a template. Phanta Super Fidelity DNA polymerase was used to perform PCR amplification to obtain PCR products H1 and H2; and p1+p4 was used as a template for the second round of PCR, and the double-stranded DNA donor product was obtained. The product was dissolved in D2O and frozen below -20°C for storage for later use after precipitation with 70% ethanol;

[0089] (4) Transformation and screening of yeast strains

[0090] The yeast strain was transformed by electroporation. Competent cells were prepared according to the electroporation method. Before electroporation, 40 μL of the competent cell mixture was fully mixed with the gRNA_Cas9 expression plasmid prepared in step (2) and the DNA Donor prepared in step (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.

[0091] Pick a single colony for PCR verification, select paired primers for verification, and sequence the positive PCR product; streak the single colony with correct sequencing on a plate, and pick a single colony for a second round of PCR verification. Select the strains that have been verified correctly in two rounds to prepare glycerol cryopreservation tubes and store them in a -80℃ refrigerator.

[0092] Furthermore, in step (1), the length of the ORF region is preferably 100 bp-2000 bp;

[0093] Preferably, the method for synthesizing the corresponding primer pair is: for each gRNA, a 4-base sequence of AATC and AAAC is added to the 5'-end of the forward sequence and the reverse complementary sequence thereof, respectively, to obtain a 24-base primer pair.

[0094] Further, in step (2), preferably, the primer pairs are diluted to a concentration of 1-10 μM;

[0095] Preferably, the annealing process is completed on the PCR instrument according to the following program: 95°C, 3 min; 72°C, 2 min; 65°C, 2 min; 60°C, 2 min; 55°C, 2 min; 50°C, 2 min; 16°C, 2 min.

[0096] Preferably, the temperature of the ligation reaction is 16°C and the time is 1h;

[0097] Preferably, in the ligation reaction, the ratio of the pCasMFR vector treated with BsaI and purified and the annealed primer added is 1-100 ng: 0.1-1 μL.

[0098] Further, in step (4), the yeast strain is derived from yeast from one or more sources selected from the group consisting of: Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces; preferably Kluyveromyces, more preferably Kluyveromyces lactis (Kluyveromy ceslactis, K. lactis);

[0099] In some feasible preferred embodiments, the lactic acid Kluyveromyces strain is a genetically engineered strain in which the expression or activity of the KlEXN53 gene (nuclease gene) is reduced (the specific preparation method is prepared by the method disclosed in CN108949801A), a genetically engineered strain in which the genome is integrated with a first exogenous gene expression cassette of a first nucleic acid construct expressing a fusion protein (such as a fusion protein formed by KlPAB1 and KleIF4G) (the specific preparation method is prepared by the method disclosed in CN111778169A and CN110938649A), and a genetically engineered strain in which the genome is integrated with a first exogenous gene expression cassette of a first nucleic acid construct expressing a fusion protein (such as a fusion protein formed by KlPAB1 and KleIF4G) and the expression or activity of a nuclease (such as KlEXN53) is reduced (the specific preparation method is prepared according to the method disclosed in CN110093284A or CN111778169A). Unless they conflict with the purpose of the present invention, patents CN108949801A, CN111778169A, CN110938649A and CN110093284A and their references are cited in their entirety and for all purposes.

[0100] Preferably, the mixing ratio of the competent cell mixture, gRNA_Cas9 expression plasmid and DNA Donor is 10-100 μL: 300-600 ng: 1-2 μg;

[0101] Preferably, the operation flow of the electroconversion process is:

[0102] Prepare transformation buffer: PEG 3350 (50% (w / v)) 260 μL, LiAc (1.0 M) 36 μL, carrier DNA (5.0 mg / mL) 20 μL, gRNA_Cas9 expression plasmid 5 μL, DNA Donor 10 μL, add sterile water to a final volume of 360 μL. After heat shock, centrifuge at 13000 g for 30 seconds to remove the supernatant. Add 1 mL of YPD liquid medium, culture for 2-3 hours, draw 200 μL and apply it to solid YPD (200 μg / mL G418) medium, and culture for 2-3 days until single colonies appear.

[0103] The beneficial effects of the present invention are:

[0104] 1) The present invention systematically analyzes and selectively modifies genes in the genome of Kluyveromyces lactis strains by means of CRISPR-Cas9-mediated gene editing, thereby providing a general method for regulating in vitro biosynthetic activity.

[0105] 2) Editing proteasome-related genes in the Kluyveromyces lactis strain and mutating the phosphorylation modification sites T57 and T59 of the translation elongation factor eEF2 can effectively improve the in vitro protein expression activity of cell extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The amino acid sequence alignment of eEF2 from Kluyveromyces lactis (Q6CPQ9) and from Saccharomyces cerevisiae shows that the protein sequences of eEF2 in these two yeasts are highly identical.

[0107] Figure 2 Design diagram for CRISPR-Cas9-mediated gene mutation method. The targeting sequence of gRNA just covers the coding sequence of T57 and T59, so the primer sequences of p2 and p3 can be designed to introduce phosphate site mutations and other synonymous mutations. These mutations include T57M / T59W

[0108] Figure 3 Design scheme for gRNA position and primer-mediated nucleotide mutation. The figure shows the sequence information, relative position and matching details of primers p2 and p3 relative to the original sequence.

[0109] Figure 4 The results of the activity of the mutant strain expressing mEGFP are shown in Figure 2. It can be seen that T57L can significantly increase the IVTT activity of the strain lysate, and the IVTT activity at 3h and 20h can be increased by 57.0% and 19.4%, respectively. However, the T57M mutation has no significant effect on the IVTT activity of the strain lysate; on the contrary, the T57M / T59W (TTm) mutation slightly reduces the IVTT activity of the lysate.

[0110] Figure 5 This is the growth of mutant strains at 30°C. It can be seen that there is no significant difference in the growth of all strains at 30°C.

[0111] Figure 6 Figure 2 is the growth of mutant strains at 37°C. It can be seen that the growth of T57I and T57V mutants at 37°C is slightly better than that of the control strain, while the growth of T57C, T57M, T59M, T59V and T57M / T59W mutants at 37°C is slightly weaker than that of the control strain. DETAILED DESCRIPTION

[0112] The advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0113] the term

[0114] Engineered strains

[0115] The "engineered strain" and "genetically engineered strain" described in the present invention are used interchangeably and both refer to the engineered strain for improving the efficiency of in vitro protein synthesis of the present invention, i.e., the strain described in the first aspect of the present invention. The genetically engineered strain is a yeast derived from one or more sources selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces; preferably Kluyveromyces, and more preferably Kluyveromyces lactis.

[0116] elongation factor eEF2

[0117] The "translation elongation factor eEF2", "elongation factor eEF2" and "transcription factor eEF2" mentioned in the present invention can be used interchangeably. There is a unique transcription factor eEF2 encoding gene in the K. lactis Y1140 genome, and its gene name is KLLA0_E02993g, while there are two corresponding homologous genes in the model fungus Saccharomyces cerevisiae, which are named EFT1 (YOR133W) and EFT2 (YDR385W) respectively.

[0118] The yeast translation elongation factor eEF2 catalyzes ribosome transfer during protein synthesis and is a highly conserved essential gene, such as Figure 1 As shown, the sequence identity of eEF2 in Kluyveromyces lactis and Saccharomyces cerevisiae is very high.

[0119] D2P

[0120] In the present invention, the "D2P" system includes but is not limited to IVTT reaction (in vitro transcription and translation reaction). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as D-to-P system, D_to_P system, DNA-to-Protein system; the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method, which has the same meaning as "in vitro cell-free protein synthesis system", "in vitro expression system", "in vitro protein synthesis system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system" and other expressions. Protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system) and other descriptions. Including in vitro translation system, in vitro transcription and translation system (IVTT system), etc. We also call the in vitro protein synthesis system a "protein synthesis factory". In vitro protein synthesis reaction refers to the reaction of synthesizing proteins in an in vitro cell-free synthesis system, which at least includes the translation process.

[0121] Foreign DNA

[0122] The exogenous DNA comes from prokaryotes or eukaryotes; in another preferred embodiment, the exogenous DNA comes from animals, plants, or pathogens; in another preferred embodiment, the exogenous DNA comes from mammals, preferably primates, rodents, including humans, mice, and rats.

[0123] Foreign protein

[0124] The exogenous protein is selected from any one or more combinations of the following groups: luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of antibody, luciferase mutant, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, xylanase, toxin, hemoglobin, sweet protein. However, the above is only an exemplary introduction to exogenous proteins, not only including the above exogenous proteins; it can be any protein that can be expressed exogenously.

[0125] In vitro protein synthesis system

[0126] The in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, various detection methods and other technical elements of the present invention can also be independently selected from the following documents to select appropriate implementation methods or implementation methods, including but not limited to CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN10942 3497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845722A, CN110938649A, CN110964736A, etc. Unless otherwise in conflict with the purpose of the present invention, these documents and their citations are cited in their entirety and for all purposes.

[0127] One of the specific embodiments of the in vitro protein synthesis system also includes, but is not limited to, for example, the cell-free protein synthesis system based on Escherichia coli described in WO2016005982A1. Other references of the present invention, including but not limited to in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus described in the direct and indirect references, are also included in the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineerin, 2019, Chapter 2, 23-45" includes but is not limited to the in vitro cell-free protein synthesis system described in the referenced documents on pages 27-28 of the "2.1 Systems and Advantages" section, which can be used as an in vitro protein synthesis system for implementing the present invention.For example (unless they conflict with the present invention, the following documents and their references are cited in their entirety and for all purposes), documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN10942 3509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN11040 8635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN11084 5722, CN110938649A, CN110964736A, CN111378706A, CN111378707A, CN111378708A, CN111718 419A, CN111748579A, CN2019107298813, CN2019112066163, CN2018112862093, CN2019114181 The in vitro cell-free protein synthesis systems and the methods for constructing and amplifying DNA templates described in 518, CN2020100693833, CN2020101796894, CN202010269333X, CN2020102693382, CN2020113574616 and the references thereto can all be used as the in vitro protein synthesis systems and the methods for constructing and amplifying DNA templates of the present invention.

[0128] In the present invention, the standard preparation method of the yeast cell extract is not limited. A preferred preparation method comprises the following steps:

[0129] (i) providing a yeast cell with a protease gene knocked out provided by the first aspect of the present invention;

[0130] (ii) washing the yeast cells to obtain washed yeast cells;

[0131] (iii) disrupting the washed yeast cells to obtain a crude yeast extract;

[0132] (iv) subjecting the crude yeast extract to solid-liquid separation to obtain a liquid portion, namely, a yeast cell extract.

[0133] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0134] In a preferred embodiment, the centrifugation is performed in a liquid state.

[0135] In the present invention, the centrifugation conditions are not particularly limited. A preferred centrifugation condition is 5000g-100000g, preferably 8000g-30000g.

[0136] In the present invention, the centrifugation time is not particularly limited. A preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

[0137] In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

[0138] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate or a combination thereof.

[0139] In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen cryogenic) disruption.

[0140] The nucleoside triphosphate mixture in the in vitro protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of various mononucleotides is not particularly limited, and usually the concentration of each mononucleotide is 0.5-5mM, preferably 1.0-2.0mM.

[0141] The amino acid mixture in the in vitro 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 0.01-0.5mM, preferably 0.02-0.2mM, such as 0.05, 0.06, 0.07, 0.08mM.

[0142] In a preferred embodiment, the in vitro protein synthesis system further contains polyethylene glycol or its analogs. The concentration of polyethylene glycol or its analogs is not particularly limited. Generally, the concentration (w / v) of polyethylene glycol or its analogs is 0.1%-8%, preferably 0.5%-4%, more preferably 1%-2%, based on the total weight of the protein synthesis system. Representative examples of PEG include (but are not limited to): PEG3000, PEG8000, PEG6000 and PEG3350. It should be understood that the system of the present invention may also include polyethylene glycols of various other molecular weights (such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).

[0143] In a preferred embodiment, the in vitro protein synthesis system further contains sucrose. The concentration of sucrose is not particularly limited, and generally, the concentration of sucrose is 0.03-40wt%, preferably 0.08-10wt%, and more preferably 0.1-5wt%, based on the total weight of the protein synthesis system.

[0144] A particularly preferred in vitro protein synthesis system contains the following components in addition to yeast extract: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphate kinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, 8-20 ng / μl firefly luciferase DNA, and 0.027-0.054 mg / mL T7 RNA polymerase.

[0145] To verify the present invention, more specific embodiments are described below.

[0146] The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions, such as the experimental conditions described in documents such as "Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free Protein Synthesis Experiment Manual" "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008", or according to the conditions recommended by the manufacturer, or according to the conditions indicated by the specific embodiments described above. Unless otherwise specified, the percentages and parts mentioned in the present invention are weight percentages and weight parts.

[0147] Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

[0148] The present invention uses Kluyveromyces lactis (K. lactis) as an example, but the same design, analysis and experimental methods are also applicable to other yeasts and other lower eukaryotic cells and higher animal cells. The gene modification method in the present invention is CRISPR-Cas9 technology, but it is not limited thereto, and can be any known and existing gene modification method.

[0149] Example 1: Design of gRNA targeting sequence

[0150] Select the ORF region (100-2000 bp) of the target gene and complete the gRNA design on the CRISPOR (tefor.net) (http: / / crispor.tefor.net / ) website, such as Figure 2 The gRNAs shown are all designed inside the ORF, while the H1 and H2 homology arms with a length of 500-1000 bp are designed outside the ORF, and H1 and H2 are assembled by Overlap-PCR. To achieve the successful assembly of H1 and H2, the annealing temperature of primers P1-P4 is 6061°C, and 8-15nt sequences that can be reverse complementary to each other's 5'-ends are added to the 5'-ends of P2 and P3, respectively.

[0151] The gRNAs with higher scores were selected as candidate sequences. For each selected gene, two sequences with the highest comprehensive scores were selected as the final gRNA sequences. The specific information is shown in Table 1. According to the designed gRNA targeting sequence, the corresponding 24-base primer pair was synthesized, that is, for each gRNA, the 4-base sequences of AATC and AAAC were added to the 5'-end of its forward sequence and reverse complementary sequence, respectively, and the final pair of 24-base primers constituted a 24-base primer pair.

[0152]

[0153]

[0154] Example 2. Construction of gRNA_Cas9 expression vector

[0155] The paired 24-base primers obtained in Example 1 were diluted to 10 μM, and 10 μL of each was added to the PCR tube and mixed, and the annealing process was completed on the PCR instrument according to the following procedures: 95°C, 3min; 72°C, 2min; 65°C, 2min; 60°C, 2min; 55°C, 2min; 50°C, 2min; 16°C, 2min. 0.5 μL of the annealed primer pair and 20 ng of the pCasMFR vector treated with BsaI and purified were mixed in 10 μL of the T4 ligase system and ligated at 16°C for 1h. All the ligation products were transformed into Escherichia coli DH5α competent cells and screened on LB plates containing 50 μg / mL kanamycin. Two single bacteria were randomly selected for sequencing verification, and the clones with correct sequencing were selected to extract the gRNA_Cas9 co-expression plasmid and determine the concentration for subsequent electroporation experiments.

[0156] Example 3. Construction of double-stranded DNA donor

[0157] like Figure 3 Shown is the gRNA position and primer-mediated nucleotide mutation design scheme. The DNA donor sequence used in the present invention is obtained by Overlap-PCR method. First, the genomic DNA of K.lactis Y1140 is used as a template, and xxxN-p1+xxxN-p2 and xxxN-p3+xxxN-p4 are used as forward and reverse primer pairs (xxxN is the corresponding gene name), respectively. The primer list is shown in Table 1, and Phanta Super Fidelity DNA polymerase is used for PCR amplification to obtain PCR products H1 and H2; and xxxN-p1+xxxN-p4 is used as a template for a second round of PCR, using the products H1 and H2 of the previous step of PCR as templates. The products of the second round of PCR were dissolved in 50 μL ddH2O after 70% ethanol precipitation and frozen at -20°C for use.

[0158] To achieve successful assembly of H1 and H2, the annealing temperatures of primers P1-P4 were all 60-61°C, and 8-15 nt sequences were added to the 5'-ends of P2 and P3, respectively, which could be reverse complementary to each other's 5'-ends.

[0159] Example 4. Transformation and screening of yeast strains

[0160] The transformation of Kluyveromyces lactis strains was completed by electroporation. Competent cells were prepared according to the requirements of the electroporation method. Before electroporation, 40 μL of the competent cell mixture was fully mixed with the corresponding 500 ng gRNA_Cas9 expression plasmid and 1-2 μg DNA Donor. The electroporation process was completed according to the standard operation procedure. The electroporated yeast cells were screened on a YPD plate containing 250 μg / mL G418. Single colonies were picked for PCR verification. The verification primers are shown in Table 3. Paired primers were selected for verification. 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 into glycerol cryovials and stored in a -80°C refrigerator.

[0161] Example 5. Lysate preparation and IVTT activity detection

[0162] After streaking the YPD plate, pick a single colony from the strain to be tested stored in a -80°C refrigerator 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 was carried out at 200rpm in a 30°C shaker until the harvest period, and the cells were collected by high-speed centrifugation. The growth was compared at 30°C and 37°C, and the cell lysate was prepared under liquid nitrogen protection for subsequent IVTT activity test.

[0163] 1. Lysis solution test:

[0164] Take a certain amount of lysate prepared according to the standard process, and complete the detection and analysis of D2P expression activity of the lysate using mEGFP as the reporter protein according to the operating procedures, and judge the IVTT activity of the corresponding lysate based on the detected reporter fluorescence intensity (RFU value). Figure 4 As shown: The activity results of mutant strains expressing mEGFP, K. lactis is a control example of Kluyveromyces lactis strain. It can be seen that T57L, T57S, T57M, T57I, T57V, T59M, T59V, T57M / T59W (TTm) can significantly improve the IVTT activity of the strain lysate, and the IVTT activity at 3h and 20h can be increased by 60.0% and 28%, respectively. However, the T57M mutation has no obvious effect on the IVTT activity of the strain lysate; on the contrary, the T57M / T59W (TTm) mutation slightly reduces the IVTT activity of the lysate.

[0165] 2. Cell Growth Test

[0166] The mutant bacterial cells were cultured and grown under the same conditions of 30℃ and 37℃. The results are as follows Figure 5 and Figure 6 As shown in the figure, K. lactis is the control Kluyveromyces lactis strain: it can be seen that there is no significant difference in the growth of all strains at 30°C, but the growth of T57I and T57V mutants at 37°C is slightly better than that of the control strain, while the growth of T57C, T57M, T59M, T59V and T57M / T59W mutants at 37°C is slightly weaker than that of the control strain.

[0167] The preferred embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in this field do not need to invent

[0168] Therefore, any technical solution that can be obtained by a technician in the technical field through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the existing technology should be within the scope of protection determined by the claims.

Claims

1. A protease translation elongation factor mutant protein, characterized in that It is obtained by mutating one or more threonine sites in the phosphorylation modification site of the protease translation elongation factor, and the protein expression activity can be improved after mutation. Preferably, the translation elongation factor is eEF2.

2. The protease translation elongation factor mutant protein according to claim 1, characterized in that The threonine residues are each independently mutated to an amino acid not containing a hydroxyl group.

3. The protease translation elongation factor mutant protein according to claim 2, characterized in that The threonine can be mutated to alanine (A), tyrosine (V), isoleucine (I), leucine (L), methionine (M), cysteine ​​(C), tryptophan (W), phenylalanine (F), asparagine (D) or a combination thereof.

4. The protease translation elongation factor mutant protein according to claim 3, characterized in that The protease translation elongation factor mutant protein is derived from yeast, and the yeast is preferably Kluyveromyces, and more preferably Kluyveromyces lactis.

5. The protease translation elongation factor mutant protein according to claim 1, characterized in that The mutation occurs in one or more threonine selected from the group consisting of: Threonine (T) at position 57; Threonine (T) at position 59.

6. The protease translation elongation factor mutant protein according to claim 5, characterized in that The 57th threonine (T) is mutated to one or more amino acids selected from the following group: alanine (A), tyrosine (V), isoleucine (I), leucine (L), methionine (M), cysteine ​​(C), tryptophan (W), phenylalanine (F), asparagine (D) or a combination thereof; and / or, The 59th threonine (T) is mutated to one or more amino acids selected from the following group: alanine (A), thymidine (V), isoleucine (I), leucine (L), methionine (M), cysteine ​​(C), tryptophan (W), phenylalanine (F), asparagine (D) or a combination thereof.

7. The protease translation elongation factor mutant protein according to claim 5, characterized in that The mutations include mutations selected from the group consisting of T57A, T57C, T57F, T57V, T57M, T57I, T57L, T57M, T57D, T57W, T59A, T59C, T59F, T59V, T59M, T59V, T59L, T59M, T59D and / or T59W.

8. A polynucleotide, characterized in that The polynucleotide encodes the mutant protease translation elongation factor mutant protein according to any one of claims 1-7.

9. A carrier, characterized in that The vector contains the polynucleotide according to claim 8.

10. A strain characterized by: Containing the vector of claim 9, or the polynucleotide of claim 8 integrated into its genome; preferably, the strain is selected from prokaryotic cells or eukaryotic cells; preferably, the strain is a prokaryotic cell, such as Escherichia coli; more preferably, the strain is a eukaryotic cell, such as yeast, source cells, Chinese hamster, ovary cells, insect cells, wheat germ cells, rabbit reticulocytes.

11. A method for producing a mutant protein of a protease translation elongation factor according to any one of claims 1 to 7, characterized in that: Includes steps: Cultivating the strain according to claim 10 under conditions suitable for expression, thereby expressing the protease translation elongation factor mutant protein; and / or, The protease translation elongation factor mutant protein is isolated. A cell lysate derived from the strain according to claim 10.

13. A cell-free in vitro protein synthesis system, characterized in that: Containing the cell lysate according to claim 12.

14. A biological product, characterized in that: The enzyme preparation comprises the protease translation elongation factor mutant protein according to any one of claims 1 to 7.

15. Use of the mutant protease translation elongation factor mutant protein according to any one of claims 1 to 7, or the polynucleotide according to claim 8, or the vector according to claim 9, or the strain according to claim 10 in improving the expression purity, efficiency and / or yield of foreign proteins, preferably for cell-free in vitro protein synthesis or for preparing a cell-free in vitro protein synthesis system.

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