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AP plasmid and application thereof, PACE directed evolution system of PAP I enzyme and PAP I enzyme mutant
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A technology of directed evolution and mutants, applied in the fields of application, hydrolase, recombinant DNA technology, etc., can solve the problem that the wild-type PAPI is not ideal
Inactive Publication Date: 2022-08-05
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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Problems solved by technology
Considering that PAP I can be used for RNA modification in vitro, but wild-type PAP I is not ideal, this evolutionary system can be used to improve the enzymatic performance of PAPI
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Embodiment 1
[0025] Phage preparation:
[0026] 1. Press figure 1 The AP, DP, and SP plasmids required for the construction of the shown structure, in which DP contains the chloramphenicol resistance gene, and AP is the carbenicillin resistance gene; DP vector item number Addgene plasmid#140446, SP vector item number NEB N4040S;
[0027] 2. Take 50ng of DP and SP plasmids and add 100μL of Escherichia coli S1030 competent cells (Addgeneplasmid#105063) respectively, incubate in an ice-water bath for 30min, transfer to a 42°C water bath and incubate for 2min, add 900μL of pre-cooled LB liquid medium to the mixture , 37 ℃, 250rpm recovery 1h;
[0028] 3. Inoculate the recovered culture medium into 9 mL of LB medium, add anhydrotetracycline with a final concentration of 200 ng / mL to induce the expression of gIII protein, and chloramphenicol with a final concentration of 25 μg, and culture at 37 °C and 250 rpm overnight;
[0029] 4. Take 1 mL of culture solution, centrifuge at 8000×g at 4°C fo...
Embodiment 2
[0044] Example 2: Purification of Thermosensitive UDGase Mutants
[0045] 1. The obtained preferred PAP I amino acid sequence is optimized according to the codon preference of Escherichia coli, and the final DNA sequence is shown in SEQ NO.4;
[0046] 2. Synthesize the above DNA sequence and connect it to the pET21b(+) vector, take 10 ng of the ligated plasmid and add it to 100 μL of E. coli BL21(DE3) competent cells, incubate in an ice-water bath for 30 minutes, then transfer to a 42°C water bath and incubate for 2 minutes , add 900 μL of pre-cooled LB liquid medium to the mixture, and recover at 37 °C and 250 rpm for 1 h;
[0047] 3. Take 50 μL of the ligation solution and spread it on a carbenicillin-resistant plate, and invert overnight at 37°C;
[0048] 4. Pick a single clone and inoculate it in a liquid medium containing carbenicillin resistance, and cultivate overnight at 37°C and 250rpm;
[0049] 5. Inoculate the bacterial solution to 800mL 2×YT medium at a ratio of ...
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Abstract
The invention provides an AP plasmid which is characterized in that the plasmid expresses gIII-neg protein, and one or more RyhB binding sequences as shown in SEQ ID No.1 are inserted into the rear part of a ribosomebinding site of the plasmid. The invention further discloses application of the PACE gene in PACE directed evolution of PAP I enzyme and a PACE directed evolutionsystem of the PAP I enzyme. The invention also discloses a PAP I enzymemutant obtained by screening, a coding gene, a prokaryotic expression vector and a prokaryotic expressionsystem. According to the invention, escherichia coli non-coding RNA RyhB sRNA is introduced into a PACE directed evolution method, so that the translation of gIII-neg protein is inhibited, and the adverse effect of the gIII-neg protein on the packaging of offspring bacteriophages is relieved. The PAP I with stronger polyadenylation activity can prolong the half-life period of RyhB and promote the translation inhibition effect of the RyhB on gIII-neg protein. The bacteriophage forms population advantages after multiple passage and is separated and sequenced, and finally the PAP I with remarkably improved activity is obtained.
Description
technical field [0001] The patent of the present invention relates to an AP plasmid and its application, a PACE directed evolution system of PAP I enzyme, a PAP I enzyme mutant, an encoding gene, a prokaryotic expression vector and a prokaryotic expression system, which belong to the field of biotechnology. Background technique [0002] PAPI I is Escherichia coli Poly(A) polymerase I (Poly(A) polymerase I), which is responsible for the polyadenylation of the 3' end of RNA molecules, and can act on most intracellular mRNA transcripts to reduce the half-life of mRNA. PAPI has obvious cytotoxicity, so the intracellular level of PAP I is inversely proportional to the growth rate of cells, and excessive PAP I will lead to slow bacterial growth. This toxicity is due to the fact that PAP I polyadenylates the 3' end of mature tRNA, thereby preventing amidation by amido-tRNA synthetase, ultimately affecting protein synthesis and cell growth. For the above reasons, cells overexpressi...
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