Method for rapidly screening escherichia coli chassis cell mutant expression high protein

Abstract

The invention discloses a method for rapidly screening escherichia coli chassis cell mutant expression high protein. The method disclosed by the invention comprises the following steps: effectively fusing a target gene and a lipase gene together through Linker to obtain a fusion gene of which the respective functions are not influenced, constructing recombinant plasmids by introduced plasmid multiple cloning sites, and then transferring the recombinant plasmids of the fusion gene into a mutated mixed escherichia coli strain, a flat plate containing glyceride is coated with the strain for culture, a transparent circle appears on the periphery of monoclone after glyceride is hydrolyzed by lipase in the fusion gene, and escherichia coli chassis cell mutants capable of highly expressing target protein are rapidly screened by observing the size of the transparent circle. The method disclosed by the invention does not need to use expensive equipment, is low in cost and high in efficiency, and has better application potential and practical application value.

Description

Technical field:

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to a method for rapidly screening Escherichia coli chassis cell mutants expressing high protein. Background technology:

[0002] With the development of science and technology, the development of biological enzymes and their application in the synthesis of chemical drugs have been popularized and applied to a certain extent around the world. The efficient expression of enzyme proteins in chassis cells has become a hot topic in research, especially in Escherichia coli chassis cells. Highly expressed enzyme proteins can not only reduce production costs and improve conversion efficiency, but also facilitate downstream separation.

[0003] At present, conventional operation methods mainly focus on optimizing the parameters of direct protein expression, but ignore the relationship between exogenous proteins and host E. coli cells. The expression of exogenous proteins in the host E. coli requires the expression of T7 RNA polymerase on the E. coli genome itself; after T7 RNA polymerase binds to the T7 promoter on the exogenous plasmid, the subsequent exogenous gene expression is initiated. Therefore, effective control of the reasonable expression of T7 RNA polymerase can regulate the expression of exogenous genes on the plasmid. This relationship closely binds the level of exogenous gene expression to different chassis cells (see Appendix). Figure 1 ), the expression of exogenous genes can be increased by mutating the sequences on the chassis cell genome.

[0004] At present, in all enzyme protein qualitative evolution, efficient screening methods are the key to obtaining mutants. Correlating the mutation of the chassis cell genome with the expression of exogenous proteins is a reverse screening method. There are two main methods for screening mutants that have been reported. One is to use enzymes to react with substrates, and screen efficient mutants through TLC spot plates or microplate reader detection; the other is to use flow cytometry to perform microdroplet mutant reactions, or combine fluorescent signals to screen mutants. However, both methods require the use of expensive instruments, and the screening test cycle is long. For example, cells need to be cultured in 96-well plates first, and then catalyzed with substrates. The operation time is long, which seriously affects the efficiency and cost of screening.

[0005] Therefore, we need to develop a mutant screening method based on existing technologies that is simple to operate, highly efficient, sensitive to detection, and low in cost. Summary of the invention:

[0006] In view of the shortcomings of the prior art and in combination with the principle of expressing foreign proteins in host Escherichia coli, the present invention provides a method for rapidly screening Escherichia coli chassis cell mutants expressing high protein.

[0007] The technical scheme adopted by the present invention is as follows: the target gene and the lipase gene are effectively fused together through a linker to obtain a fusion gene whose respective functions are not affected, the introduced plasmid multiple cloning site is constructed into a recombinant plasmid, and then the fusion gene recombinant plasmid is transferred into a mutated mixed Escherichia coli strain, and the strain is spread on a plate containing glyceride for culture, and the lipase in the fusion gene hydrolyzes the glyceride to present a transparent circle around the single clone, and the Escherichia coli chassis cell mutant that can highly express the target protein is quickly screened by observing the size of the transparent circle.

[0008] Furthermore, the linker fuses the target gene and the lipase gene together, which means that the target gene and the lipase gene are transcribed and translated together under the connection of the linker to obtain a fusion protein, such as the attached Figure 2 shown.

[0009] Furthermore, Linker is selected from a short peptide chain of ((GGGGGS)*3), and then the Linker is fused to the N-terminus of the lipase gene under overlap PCR amplification technology to obtain a short peptide sequence with (GGGGGS)*3 at the N-terminus. The sequence is tightly and seamlessly connected to the ATG start codon of lipase to obtain the Linker-lipase gene.

[0010] Furthermore, the Linker-lipase gene is fused to the C-terminus of the target gene, wherein the termination codon TAA is removed from the C-terminus of the target gene to ensure that the Linker and lipase are translated together during protein translation to obtain a target gene-Linker-lipase protein with a large molecular weight.

[0011] Furthermore, the target gene is selected from ketoreductase, transaminase, aldolase, nitroreductase, alcohol oxidase, monoamine oxidase, nitrile reductase, amine dehydrogenase, preferably ketoreductase and transaminase.

[0012] Further, the lipase source is selected from Thermomyces lanuginosus, Mucor miehei, Pseudomonas fluorescens, Aspergillus niger, Rhizomucor miehei, Candidalipolytica, Rhizopus oryzae, Candida antarctica, Burkholderia sp., Candida rugosa and Bacillus Subtilis, and a mixture of one or more genetically modified strains thereof, preferably Bacillus Subtilis.

[0013] Furthermore, Crispr-Cas9 gene editing technology was used to construct DNA random mutations in the pribnow box upstream of the T7 RNA polymerase promoter of Escherichia coli BL21 (DE3), thereby generating genes with different pribnow boxes and forming a BL21 (DE3) mixed strain containing different pribnow boxes.

[0014] Furthermore, the ester is selected from glyceryl tributyrate, glyceryl triacetate, glyceryl diacetate, preferably glyceryl tributyrate.

[0015] The beneficial effect of the present invention is that the method disclosed by the present invention is simple and easy to operate, and the size of the transparent zone obtained by lipase hydrolysis of ester can quickly screen mutants expressing high protein. Compared with the traditional method, the method of the present invention can complete the screening of mutants in a short time, and does not require the use of expensive equipment, only needs to be cultured on a plate, and has low cost, good application potential and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Expression of foreign proteins in host E. coli

[0017] Figure 2 Fusion of target gene and lipase gene

[0018] Figure 3 Example 1 Linker-lipase fusion gene amplification results

[0019] Figure 4 Example 2 KRED-linker-lipase fusion gene amplification results

[0020] Figure 5 Example 3 Protein electrophoresis of KRED fusion gene

[0021] Figure 6 Example 4 Protein clearing zone and electrophoresis diagram of KRED fusion gene

[0022] Figure 7 Example 6 Protein clearing zone and electrophoresis diagram of ATA fusion gene DETAILED DESCRIPTION

[0023] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0024] Example 1 Construction of linker-lipase gene fragment

[0025] The lipase gene (KAF4430836) from Bacillus Subtilis was selected from the NCBI website. The amino acid sequence is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2.

[0026] SEQ ID NO.1

[0027] AEHNPVVMVHGIGGASFNFAGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSRFVQKVLDETGAKKVDIVAHSMGGANTLYYIKNLDGGNKVANVVTLGGANRLTTGKALPGTDPNQKILYTSIYSSADMIVMNYLSRLDGARNVQIHGVGHIGLLYSSQVNSLIKEGLNGGGQNTN

[0028] SEQ ID NO.2

[0029] GCAGAACATAATCCGGTTGTGATGGTTCATGGCATTGCGGGTGCAAGTTTTAATTTTGCCGGTATTAAGAGCTATCTGGTGAGCCAGGGTTGGAGCCGTGATAAACTGTATGCCGTGGATTTTTGGGATAAAACCG GTACAAATTATAACAATGGTCCGGTGCTGAGCCGTTTTGTGCAGAAAGTGCTGGATGAAACCGGTGCCAAAAAAGTGGATATTGTTGCCCATAGCATGGGCGGCGCCAATACCCTGTATTATATTAAGAATCTGGAC GGCGGTAATAAGGTTGCCAATGTGGTGACCCTGGGTGGCGCCAATCGCCTGACCACCGGCAAAGCCCTGCCGGGTACAGATCCGAATCAGAAAATTCTGTATACCAGTATCTATAGCAGTCCCGATATGATTGTTA TGAATTATCTGAGTCGTCTGGATGGCGCACGTAATGTTCAGATTCATGGTGTTGGTCATATTGGTCTGCTGTATAGTAGTCAGGTTAATAGTCTGATTAAGGAAGGTCTGAATGGTGGCGGTCAGAATACCAATTAA

[0030] The amino acid sequence of Linker is: GGGGGSGGGGGSGGGGGS.

[0031] The nucleic acid sequence of Linker is: GGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC.

[0032] Primer PF: GCGGCGGCGGCAGCGCAGAACATAATCC.

[0033] PR:TTAATTGGTATTCTGACCGCCACCAT.

[0034] The linker-lipase gene fragment was amplified using the above primers. The amplification conditions are shown in Table 1. The amplified product was subjected to 1% agarose gel electrophoresis analysis. The results are shown in Table 1. Figure 3 , there are obvious gene bands, indicating that the linker-lipase fusion gene was amplified successfully.

[0035] Table 1 Primer amplification conditions

[0036]

[0037] Example 2 Fusion of target gene and linker-lipase

[0038] The KRED sequence information was found from the NCBI database (NCBI number is XP_022456152.1), the amino acid sequence is shown in SEQ ID NO. 3, and the gene sequence is shown in SEQ ID NO. 4. The linker-lipase gene obtained in Example 1 was fused with the KRED gene using the over-lap PCR method.

[0039] SEQ ID NO.3

[0040] MSALSKTQAGYIFKKGAGHIVKAEVPIPKPTGAQSLLRVKAAGMCHSDLHVIGETLEVPTDGYVLGHEIAGELVEIGDSVNPEVFKVGGRYAVHGLNSCGSCEMCRTGHDNDCTGNESKWYGLGISGGYQQYLLVPNSHHLLPIPDNVSYEVAAATSDAVLTPYHAIKNS GVTPSSKVLMFGLGLGSNALQILKAFGAYVVAVDVKPASKAIADEFKADEFYTDISQSSWKPASFDYCFDFVSLQVTFDICQKYIKSHGTIFPVGLGSSKLTFDLGNLALREVKIVGNFWGTSQEQIEAMELVSSGRVKPQVHTTELENLPESLEKLEEGKINGRLVMLP

[0041] SEQ ID NO.4

[0042]

[0043] According to the nucleic acid sequences of the target genes KRED and linker-lipase, the following two pairs of primer sequences were designed. The specific design information is shown in Table 2.

[0044] Table 2 Primers for overlap PCR

[0045] Primer name Sequence length KRED-F <![CDATA[CGTA CCATGG ATGTCTGCTCTCTC]]> KRED-R GCCGCCGCCGCCGCCGGAAGATTGGTTATGCTTCCA LL-F TGGAAGCATAACCAATCTTCCGGCGGCGGCGGCGGC LL-R <![CDATA[ACTG GAATTC TTAATTGGTATTCTGACCGCCAC]]>

[0046] Under the action of primers KRED-F and KRED-R, a KRED sequence with an NcoI restriction site at the N-terminus and a partial linker sequence at the C-terminus was amplified. Under the action of primers LL-F and LL-R, a linker-lipase sequence with a partial KRED sequence at the N-terminus and an EcoRI sequence at the C-terminus was amplified. Finally, the entire KRED-linker-lipase sequence was amplified using the two primers KRED-F and LL-R. The electrophoresis results are shown in Figure 4 , there are obvious gene bands, indicating that the KRED-linker-lipase fragment was successfully amplified.

[0047] Example 3: Transformation of recombinant plasmid into BL21(DE3) competent cells

[0048] The fusion gene KRED-linker-lipase obtained sticky ends under the action of NcoI and EcoRI endonucleases, and then was connected in vitro with the plasmid pET28a containing the same sticky ends to obtain the fusion gene recombinant plasmid. The recombinant plasmid was transferred into the competent Escherichia coli BL21 (DE3) and in the presence of Kan + The single clone was selected from the resistance plate and transferred to a test tube containing LB for culture at 37°C. After 16 hours of culture, 2 mL of the seed solution was transferred to 400 mL of 2YT medium and cultured at 37°C for 3 hours. 600 When the value reaches 0.6-0.8, 0.1mM IPTG inducer is added to induce expression at 25℃ for 12h. Then the bacteria are ultrasonically broken, and the solution after ultrasonication is divided into supernatant and precipitate for electrophoresis analysis. The results are as follows Figure 5 As shown, it can be found that the protein bands in the supernatant are obvious, but the expression level is not high.

[0049] Example 4: Transformation of recombinant plasmid into saturated mutagenesis BL21 (DE3) competent cells

[0050] The CRISPR-Cas9 technology was used to saturate the gene of the pribnow box upstream of lacUV5 on the genome of Escherichia coli BL21 (DE3) to form a BL21 (DE3) mixed strain containing different pribnow boxes. The mixed strain was prepared into competent cells, and the pET28a-KRED-linker-lipase recombinant plasmid was transferred into the above mixed strain, and the transformed strain was spread on a plate containing Kan + The cells were cultured on LB, lactose and tributyrin plates with resistance and incubated at 37°C for 12 h. Transparent circles of varying sizes were observed on the plates (see Figure 6 ), this is because the expressed lipase has a hydrolysis effect on tributyrin. The larger the transparent circle, the more lipase is expressed, and the higher the corresponding KRED expression level is. Figure 6 The protein electrophoresis results were the same as Figure 5 In comparison, the protein band in the supernatant became obviously thicker, indicating that the expression of the fusion protein increased.

[0051] Example 5: Transformation of recombinant plasmid into saturated mutagenesis BL21 (DE3) competent cells

[0052] The CRISPR-Cas9 technology was used to saturate the gene of the pribnow box upstream of lacUV5 on the genome of Escherichia coli BL21 (DE3) to form a BL21 (DE3) mixed strain containing different pribnow boxes. The mixed strain was prepared into competent cells, and the pET28a-KRED-linker-lipase recombinant plasmid was transferred into the above mixed strain, and the transformed strain was spread on a plate containing Kan + The cells were plated on LB, lactose and triacetin plates and cultured at 37°C for 12 h. Transparent circles of varying sizes could be seen on the plates.

[0053] Example 6 Screening of strains with high expression of lipase fusion and transaminase

[0054] The lipase gene (KAF4430836) from Bacillus subtilis and the linker short peptide GGGGGSGGGGGSGGGGGS were selected, and the linker was fused to the N-terminal ATG of lipase, and then fused with the transaminase ATA to obtain the ATA-Linker-Lipase gene. After digestion with restriction enzymes NcoI and EcoRI, the ATA-Linker-Lipase gene was inserted into the multiple cloning site of the vector pET28a to obtain the pET28a-ATA-Linker-Lipase recombinant plasmid. The recombinant plasmid was transferred into the BL21 (DE3) mutant obtained by Crispr-Cas9 technology, and then the transformed strain was spread on a plate containing Kan + The cells were cultured on LB, lactose and tributyrin plates with resistance and incubated at 37°C for 12 h. Transparent circles of varying sizes were observed on the plates (see Figure 7 ), and screened out highly expressed ATA enzyme mutant strains according to the size of the transparent zone, and significantly increased the soluble expression level of the protein compared with the wild type.

Claims

1. A method for rapidly screening Escherichia coli chassis cell mutants expressing high protein, characterized in that: The method effectively fuses the target gene and the lipase gene together through a linker to obtain a fusion gene whose respective functions are not affected, constructs a recombinant plasmid through an introduced plasmid multi-cloning site, and then transfers the fusion gene recombinant plasmid into a mutated mixed Escherichia coli strain, and the strain is spread on a plate containing glyceride for culture, and a transparent circle is presented around the single clone after the lipase in the fusion gene hydrolyzes the glyceride, and the Escherichia coli chassis cell mutant that can highly express the target protein is quickly screened by observing the size of the transparent circle.

2. The method according to claim 1, characterized in that The Linker is selected from (GGGGGS)*3 short peptide chain.

3. The method according to claim 1, characterized in that The target gene is selected from ketoreductase or transaminase.

4. The method according to claim 1, characterized in that The lipase gene is derived from Bacillus Subtilis.

5. The method according to claim 1, characterized in that The mixed Escherichia coli strain mutant is obtained by using Crispr-Cas9 gene editing technology.

6. The method according to claim 1, characterized in that The ester is selected from tributyrin, triacetin, and diacetin.

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