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A method for integrating unnatural amino acid pppa into Escherichia coli aqpz

A technology of unnatural amino acids and aquaporins, applied in the direction of microorganism-based methods, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of easy protein loss, affecting performance and lifespan, and improve soluble expression Efficiency, damage avoidance, and the effect of broadening one's thinking

Active Publication Date: 2019-11-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional biomimetic membranes embed AQPZ in amphiphilic diblock polymers or natural phospholipid bilayers in the flow mosaic mode, but this kind of aquaporin embedded with hydrophobic interaction is easy to lose, which affects the performance and performance of the entire membrane. life

Method used

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  • A method for integrating unnatural amino acid pppa into Escherichia coli aqpz
  • A method for integrating unnatural amino acid pppa into Escherichia coli aqpz
  • A method for integrating unnatural amino acid pppa into Escherichia coli aqpz

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1, the acquisition of MjpPpaRS gene, comprises the following steps:

[0027] 1. Using p15a-MjtyrRS as a template, using two pairs of primers RS F2 / RS R4 and RS F4 / RS R2 to amplify two DNA fragments by PCR, and homologously recombining these two DNA fragments to obtain three site mutations (Tyr32Ala / Glu107Pro / Leu162Ala) aminoacyl tRNA synthetase plasmid. Using the above-mentioned mutant plasmid as a template, two pairs of primers, RS F1 / RS R3 and RS F3 / RS R1, were used for the second round of mutation to obtain M. tyrosyl-tRNA synthetase (TyrRS), the mutated aminoacyl tRNA synthetase was named MjpPpaRS, and the p15a-MjpPpaRS plasmid was obtained. The gene sequences of the primers are shown in Table 1.

[0028] Table 1 Primers used for mutation

[0029]

[0030] 2. The PCR reaction conditions are: PCR conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 70°C for 5 minutes, ...

Embodiment 2

[0031] Embodiment 2, the preparation of Escherichia coli cell-free system extract, comprises the following steps:

[0032] 1. Simultaneously transform the recombinant vector p15a-MjpPpaRS and pUC-MjtRNA into Escherichia coli BL21 (DE3), and select positive transformants;

[0033] 2. Pick a single colony of recombinant Escherichia coli BL21(DE3) / p15a-MjpPpaRS / pUC-MjtRNA from the plate and inoculate it in 5 mL of LB medium, and add ampicillin (Amp) with a final concentration of 50 μg / mL. Shake at 200 rpm at 37°C overnight.

[0034] 3. Transfer all the seed solutions cultivated in step 2 to two 500 mL bottles of cell-free fermentation medium, and add Amp with a final concentration of 50 μg / mL.

[0035] 4.37℃, 200rpm culture to bacterial concentration OD 600 When it reaches 0.8-1, add IPTG with a final concentration of 0.5mM to induce until the bacterial concentration OD 600 When it reaches 3-4, the bacteria can be harvested.

[0036] 5. Pour the two bacterial solutions obtained...

Embodiment 3

[0047] Embodiment 3, the preparation of recombinant plasmid pIVEX2.4c-MjpPpaRS, comprises the following steps:

[0048] The cell-free expression vectors pIVEX2.4c and p15a-MjpPpaRS were digested with restriction endonucleases NcoI and BamHI at 37°C for 4 hours, respectively, and the digested products recovered from gel cutting were ligated overnight at 16°C and then transformed into Escherichia coli DH5α competent cells. Transformants were identified by NcoI and BamHI double enzyme digestion and sequencing, and the successfully constructed recombinant plasmid pIVEX2.4c-MjpPpaRS was obtained.

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Abstract

The invention provides a method for compounding non-natural amino acids pPpa in escherichia coli aquaporin AQPZ. The method comprises the following steps: carrying out site-directed mutation on tyrosine-tRNA synthetase of methanococcus jannaschii to obtain an MjpPpaRS gene; cloning the MjpPpaRS gene onto a pIVEX2.4c plasmid to obtain a recombinant plasmid pIVEX2.4c-MjpPpaRS; converting the recombinant plasmid pIVEX2.4c-MjpPpaRS and pUC-MjtRNA to escherichia coli BL21; selecting a positive transformant and fermenting to prepare escherichia coli cell-free extract; establishing a cell-free expression system; taking a pIVEX2.4c-AqpZ as a template and carrying out amber mutation on three sites including F10, F13 and F17 of the AQPZ; adding a decontaminating agent into the cell-free expression system to dissolve and express the AQPZ programmed into the pPpa; and carrying out affinity chromatography purification to obtain the AQPZ which is programmed into the pPpa at a fixed site. The AQPZ programmed into the pPpa is produced through utilizing the escherichia coli cell-free expression system, and the target protein is obtained through utilizing the affinity chromatography purification; and the non-natural amino acids are embedded, so that the affinity of the aquaporin and phospholipid is enhanced and the aquaporin is more stable after being embedded under the same condition.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a method for integrating unnatural amino acid pPpa in aquaporin AQPZ by using an Escherichia coli cell-free system. Background technique [0002] Organisms encode 20 natural amino acids and 3 termination signals by 64 codons. Due to the limited functional groups contained in the 20 natural amino acids, it cannot meet the needs of protein structure and function in biological sciences, chemical research and applications. More than 70 kinds of unnatural amino acids have been successfully encoded into proteins by gene-encoded unnatural amino acid technology. The addition of some unnatural amino acids can enhance protein performance, stabilize protein structure, and even generate new functions. These UAAs contain diverse functional groups such as amide, nitro, phosphate, sulfonic acid ketone, aldehyde, azide, alkynyl, and alkenyl groups, which can be used for various m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/245C12N15/31C12N15/70C12N1/21C12R1/19
CPCC07K14/245C12N15/70C12N2800/101
Inventor 徐志南庄冰佳唐云平蔡谨黄磊
Owner ZHEJIANG UNIV
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