AP plasmid and its application, PACE directed evolution system of PAP I enzyme and PAP I enzyme mutant

Abstract

The present invention provides an AP plasmid, characterized in that the plasmid expresses the gIII-neg protein and has one or more RyhB binding sequences shown in SEQ ID No. 1 inserted into the rear of its ribosome binding site. Also disclosed are its application in the PACE directed evolution of the PAP I enzyme and a PACE directed evolution system for the PAP I enzyme. Also disclosed are screened PAP I enzyme mutants, encoding genes, prokaryotic expression vectors, and prokaryotic expression systems. The present invention introduces Escherichia coli non-coding RNA RyhB sRNA into the PACE directed evolution method to inhibit the translation of the gIII-neg protein and eliminate the adverse effects of the gIII-neg protein on the packaging of progeny phages. The stronger the polyadenylation activity of the PAP I, the longer the half-life of RyhB can be extended, promoting the latter's translational inhibition of the gIII-neg protein. After multiple passages, the phage forms a population advantage and is isolated and sequenced, ultimately obtaining a PAP I with significantly improved activity.

Description

Technical Field

[0001] The patent of this invention relates to an AP plasmid and its application, a PACE directed evolution system of PAP I enzyme and PAP I enzyme mutants, encoding genes, prokaryotic expression vectors and prokaryotic expression systems, and belongs to the field of biotechnology. Background Art

[0002] PAP I, the enzyme responsible for polyadenylation at the 3' end of RNA molecules, acts on the vast majority of intracellular mRNA transcripts, thereby reducing their half-life. PAP I exhibits significant cytotoxicity, with intracellular PAP I levels inversely proportional to cell growth rate. Excessive PAP I levels can result in slow bacterial growth. This toxicity is due to PAP I polyadenylation of the 3' end of mature tRNAs, thereby hindering amidation by amido-tRNA synthetases, ultimately impairing protein synthesis and cell growth. For these reasons, cells overexpressing PAP I typically grow slowly and exhibit low PAP I expression levels. Because PAP I is a template-independent RNA polymerase, it can be used in in vitro RNA tailing experiments, stabilizing RNA and providing adapter binding regions for reverse transcription.

[0003] In addition to affecting mRNA and tRNA, PAP I has also been reported to act on the 3' end of sRNA, extending its half-life. sRNA is a type of non-coding RNA expressed by bacteria, typically ranging from 20 nt to 400 nt in length, and relies on local base pairing to regulate the translation of target mRNAs. In most cases, the action of sRNA requires the participation of the Hfq protein to enhance sRNA stability or the affinity of sRNA binding to mRNA. Due to the presence of specific secondary structures, there is a certain degree of conservation in the sequence of the binding regions of the same sRNA with different targets. According to Dhriti Sinha's report, PAP I plays an important role in the stability and function of Escherichia coli RyhB sRNA. Polyadenylation of RyhB can significantly enhance its stability, thereby promoting its interaction with the target, accelerating the degradation of some target mRNAs, and aborting translation.

[0004] PACE is a system for directed protein evolution that relies on rapid phage propagation. Higher expression levels of the phage packaging protein gIII, coupled to the desired enzymatic properties, lead to higher abundance of progeny phage. Through an in vivo mutagenesis system, the DNA sequences of the target mutants, i.e., progeny phage, are enriched as phage propagate through the phage. PACE's negative selection system relies on the protein concentration of the functionally defective gIII-neg protein; lower gIII-neg concentrations lead to greater propagation of progeny phage. This strategy is compatible with the negative regulation of translation by short RNAs (sRNAs), enabling the development of a unique protein evolution system. Given that PAP I can be used for in vitro RNA modification, but wild-type PAP I is suboptimal, this evolutionary system could be used to improve the enzymatic properties of PAP I. Summary of the Invention

[0005] The present invention provides an AP plasmid, characterized in that: the plasmid expresses gIII-neg protein, and one or more RyhB binding sequences shown in SEQ ID No. 1 are inserted at the rear of its ribosome binding site, and the sequence of RyhB-gIII-neg in the AP plasmid can be shown as SEQ ID No. 5.

[0006] The invention also discloses the application of the AP plasmid in the PACE directed evolution of the PAP I enzyme.

[0007] The present invention also discloses a PACE directed evolution system of PAP I enzyme, which is characterized by comprising: the above-mentioned AP plasmid, DP plasmid and SP plasmid, and host bacteria, wherein the DP plasmid carries the wild-type gIII gene and the wild-type PAP I expression gene, and the SP plasmid is an M13 phage genome, wherein the gIII gene is replaced by the wild-type PAP I expression gene.

[0008] Preferably, the AP plasmid and DP plasmid contain different resistance genes, wherein DP contains a chloramphenicol resistance gene and AP contains a carbenicillin resistance gene.

[0009] Preferably, the host bacteria is Escherichia coli, preferably S1030 competent cells.

[0010] The present invention also discloses a PAP I enzyme mutant obtained by the method, and its amino acid sequence is shown as SEQ ID No. 2.

[0011] The gene encoding the PAP I enzyme mutant is characterized by its nucleic acid sequence as shown in SEQ ID No.4.

[0012] The prokaryotic expression vector of the above-mentioned PAP I enzyme mutant.

[0013] The prokaryotic expression vector of the PAP I enzyme mutant is characterized in that the vector adopts the pET21b(+) plasmid.

[0014] The prokaryotic expression system of the PAP I enzyme mutant is characterized in that the prokaryotic expression vector is transformed into Escherichia coli BL21 (DE3) competent cells.

[0015] The present invention improves the negative-selection PACE directed evolution method and uses it to obtain a PAP I enzyme with enhanced in vitro polyadenylation activity. The PACE directed evolution method incorporates Escherichia coli non-coding RNA (RyhB sRNA) to inhibit the translation of the gIII-neg protein, thereby eliminating the adverse effects of gIII-neg on the packaging of progeny phage. PAP I with stronger polyadenylation activity can prolong the half-life of RyhB, promoting its translational inhibition of gIII-neg. The coding sequence of the highly active PAP I is then packaged into progeny phage. After multiple passages, the phages develop a population dominance and are isolated and sequenced, ultimately yielding PAP I with significantly enhanced activity.

[0016] Inhibition of gIII-neg protein translation is achieved by introducing a RyhB binding site into the 5'-UTR of the gIII-neg gene, preferably one or more RyhB binding sequences near the ribosome binding site. The RyhB binding sequence is shown in SEQ ID NO. 1.

[0017] The amino acid sequence of the PAP I enzyme mutant with significantly improved activity obtained in the present invention is shown in SEQ ID NO.2. Compared with the wild-type PAP I shown in SEQ ID NO.3, it has the following mutations: G67R, P198A, and T227S; the nucleotide sequence encoding the above amino acid sequence is shown in SEQ ID NO.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the PAP I enzyme evolution scheme. a) DP, AP, and SP plasmids used in the enzyme evolution PACE system. The DP plasmid provides the wild-type gIII protein and the mutagenic gene; the AP plasmid expresses the gIII-neg protein with a RyhB binding sequence inserted after its ribosome binding site; the SP plasmid is a modified M13 phage genome in which the gIII gene has been replaced by the PAP I expression gene pcnB. b) Schematic diagram of the interaction between the PAP I protein, RyhB sRNA, and gIII-neg mRNA.

[0019] Figure 2 Schematic diagram of the structure of AP plasmid.

[0020] Figure 3 Schematic diagram of the structure of SP plasmid.

[0021] Figure 4 The purification results of PAP I enzyme are shown in Figure 1. S represents the ultrasonic supernatant, Ft represents the flow-through, W represents the washing solution, and A1 represents the eluent.

[0022] Figure 5 The electrophoresis diagram shows the in vitro polymerization results of the variant PAP I enzyme and the competitor PAP I enzyme. The control is the competitor poly(A)polymerase I group, and PAP I is the purified variant protein group. DETAILED DESCRIPTION

[0023] Example 1:

[0024] Phage preparation:

[0025] 1. Press Figure 1 The AP, DP, and SP plasmids required for constructing the structure shown are shown. DP contains the chloramphenicol resistance gene, and AP contains the carbenicillin resistance gene. The DP vector is available as Addgene plasmid # 140446, and the SP vector is available as NEBN4040S.

[0026] 2. Take 50 ng of DP and SP plasmids respectively and add 100 μL of E. coli S1030 competent cells (Addgene plasmid # 105063). Incubate in an ice-water bath for 30 min, then transfer to a 42°C water bath and incubate for 2 min. Add 900 μL of pre-chilled LB liquid medium to the mixture and resuscitate at 37°C, 250 rpm for 1 h.

[0027] 3. Inoculate the revived culture medium into 9 mL of LB medium, add anhydrotetracycline at a final concentration of 200 ng / mL to induce gIII protein expression, and add chloramphenicol at a final concentration of 25 μg. Incubate at 37°C and 250 rpm overnight.

[0028] 4. Take 1 mL of culture medium, centrifuge at 8000 × g for 10 min at 4°C, separate the supernatant and filter with a 0.22 μm filter membrane;

[0029] 5. Take 100 μL of filtrate and dilute it to 10 with fresh LB gradient. -3 , 10 -5 The remaining filtrate was frozen at -20℃ for later use;

[0030] 6. Take 100 μL of 10 0 , 10 -3 , 10 -5The filtrate was mixed with 1OD of S1030 bacteria containing the DP plasmid, and then 10mL of semi-solid culture medium was added to the plate and cultured at 37℃ overnight.

[0031] 7. Calculate the number of plaques in the semi-solid plate and pick 1-2 plaques to inoculate until OD 600 The culture medium was DP / S1030 with a DP / S1030 value of 0.5, cultured at 37°C and 250 rpm for about 2 hours, and phage particles were isolated from the supernatant.

[0032] Host cell preparation:

[0033] 8. Co-transform the AP and DP plasmids into E. coli S1030 competent cells. After recovery, spread 100 μL of culture medium on chloramphenicol and carbenicillin double-resistance plates and culture at 37°C overnight.

[0034] 9. Take a single clone and inoculate it into 5 mL of chloramphenicol and carbenicillin double-resistance LB medium, and culture it at 37°C and 250 rpm overnight;

[0035] 10. Inoculate at a 1% ratio until AP&DP / S1030 grows to OD 600 When the value is about 0.5, take it out and set aside.

[0036] PAP I Evolution:

[0037] 11. Inoculate 200 μL of the supernatant from step 7 into the bacterial solution from step 10. In addition to adding chloramphenicol and carbenicillin to maintain plasmid stability, add L-arabinose at a final concentration of 0.02% and anhydrotetracycline at 200 ng / mL to induce the expression of the mutagenic gene and gIII protein, respectively. Cultivate at 37°C and 250 rpm for 8 h.

[0038] 12. Take 1 mL of culture medium, centrifuge at 8000 × g for 10 min at 4°C, separate the supernatant and filter with a 0.22 μm filter membrane;

[0039] 13. Repeat steps 11-12 about 10-20 times. The remaining supernatant after each round of inoculation can be frozen in a -20℃ refrigerator.

[0040] 14. Take 100 μL of filtrate and dilute it to 10 with fresh LB gradient. -3 , 10 -5 ,

[0041] 15. Take 100 μL of 10 0 , 10 -3 , 10 -5 The filtrate was mixed with 1OD of S1030 bacteria containing the DP plasmid, and then 10mL of semi-solid culture medium was added to the plate and cultured at 37℃ overnight.

[0042] 16. Take the plaques from the plate and sequence them to obtain the target sequence in the phage with high abundance. The amino acid sequence corresponding to the sequence is shown in SEQ ID NO. 2.

[0043] Example 2: Purification of PAP I enzyme mutants

[0044] 1. The obtained preferred PAP I amino acid sequence was optimized according to the codon preference of E. coli. The final DNA sequence is shown in SEQ NO. 4;

[0045] 2. Synthesize the above DNA sequence and ligate it to the pET21b(+) vector. Take 10 ng of the ligated plasmid and add it to 100 μL of Escherichia coli BL21(DE3) competent cells. Incubate in an ice-water bath for 30 min, then transfer to a 42°C water bath and incubate for 2 min. Add 900 μL of pre-chilled LB liquid medium to the mixture and resuscitate at 37°C, 250 rpm for 1 h.

[0046] 3. Take 50 μL of the ligation solution and spread it on a carbenicillin-resistant plate, and incubate it upside down at 37°C overnight;

[0047] 4. Pick a single clone and inoculate it into liquid culture medium containing carbenicillin resistance, and culture it at 37°C and 250 rpm overnight;

[0048] 5. Inoculate 1% of the bacterial solution into 800 mL of 2×YT medium and culture at 37°C and 250 rpm until the OD 600 When the value was about 0.8, IPTG with a final concentration of 1 mM was added to the shake flask to induce PAP I expression;

[0049] 6. After culturing at 37°C and 250 rpm for 4 h, collect the cells, wash twice with PBS, and resuspend in 100 mL of PBS;

[0050] 7. Ultrasonicate for 30 min at 60% power, 2s ON / 2s OFF in an ice water bath to break the cell wall. Centrifuge at 12,000 × g for 30 min at 4°C to separate the supernatant.

[0051] 8. The supernatant was filtered through a 0.22 μm pore size filter membrane and purified using a TOSOH 5 mL Heparin column. The results were as follows: Figure 4 As shown, the purity of PAP I is greater than 90%;

[0052] 9. Determine the concentrations of E. coli poly(A) polymerase (NEB M0276S) and purified PAP I protein. 0.1 μg, 0.2 μg, 0.4 μg, and 0.8 μg of each enzyme were added to the reaction. The reaction system is as follows:

[0053]

[0054] RNA template sequence: 5′-AUGACCAGGAUGCCAUUGCUGUGG-3′;

[0055] 10. The reaction system was placed at 37°C for 40 minutes, and then each reaction tube was heated to 90°C to terminate the reaction;

[0056] 11. Prepare 2% agarose gel, load the sample and run electrophoresis at 200V for 10 minutes. The results are as follows: Figure 5 As shown in the figure: at the same concentration, the product concentration of PAPI was significantly higher than that of the control group, and the product band length at each gradient was also longer than that of the control group, indicating that the PAPI obtained by the present invention has better enzymatic performance.

Claims

1. A PAP I enzyme mutant, characterized in that: The amino acid sequence of the PAP I enzyme mutant is shown in SEQ ID No.

2.

2. The gene encoding the PAP I enzyme mutant according to claim 1, characterized in that The nucleic acid sequence of the coding gene is shown in SEQ ID No.

4.

3. A prokaryotic expression vector containing the gene encoding the PAP I enzyme mutant according to claim 2.

4. The prokaryotic expression vector according to claim 3, wherein: The vector used was pET21b(+) plasmid.

5. A recombinant bacterium containing the prokaryotic expression vector of the PAP I enzyme mutant according to claim 3, characterized in that: The prokaryotic expression vector according to claim 3 is transformed into Escherichia coli BL21 (DE3) competent cells to obtain the expression vector.

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