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Intracellular synthesis of BLA traceless soxo hydrocarbon by using separated intein as connection means and preparation method of intracellular synthesis of BLA traceless soxo hydrocarbon

A connection method and intein technology, applied in the field of protein coupling, to achieve the effect of reducing fusion components and improving retention

Pending Publication Date: 2022-07-01
江苏贝奥泰克生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, during the refolding process of the catalane protein, the SpyCatcher / SpyTag complex may interact with the unfolding-folding process of the target protein, such as restricting the refolding process of the target protein to an intermediate state, thereby inhibiting the refolding process. sexual effect

Method used

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  • Intracellular synthesis of BLA traceless soxo hydrocarbon by using separated intein as connection means and preparation method of intracellular synthesis of BLA traceless soxo hydrocarbon
  • Intracellular synthesis of BLA traceless soxo hydrocarbon by using separated intein as connection means and preparation method of intracellular synthesis of BLA traceless soxo hydrocarbon
  • Intracellular synthesis of BLA traceless soxo hydrocarbon by using separated intein as connection means and preparation method of intracellular synthesis of BLA traceless soxo hydrocarbon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Plasmid construction

[0039] In the traceless catenation strategy, the cyclization process is accomplished by an isolated intein. Since the intein reaction pair that achieves trans-cleavage is still composed of two parts, the peptide segment and the protein chaperone. In the design of BLA catenane precursors, the possible influence between each fusion domain needs to be considered, and the importance of rational design of connecting peptide chains is greatly increased. The length and properties of the connecting peptide chain have a great impact on the expression, folding and functional retention of the fusion protein, especially for proteins with larger and more complex structures. During intein-mediated catenane process, I NTrans-shearing and removal occurs, and only p53dim and BLA remain in the final catenane structure, but I N The protein chaperone of the reaction still needs proper folding, so in the design of the catenane precursor, the amino acid sequence (GG...

Embodiment 2

[0072] Protein expression and purification

[0073] The corresponding plasmids were transformed into BL21(DE3) competent cells. Single clones were picked on an overnight cultured agar plate, inoculated into 5 mL of 2xYT liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking. The bacteria cultured overnight was diluted 1:50 into 200 mL of optimized LB liquid medium containing the same concentration of antibiotics (1% tryptone, 0.5% yeast extract, 25mM disodium hydrogen phosphate, 25mM dipotassium hydrogen phosphate, 50 mM potassium chloride, 5 mM sodium sulfate, 2 mM magnesium sulfate, 0.5% glycerol, 0.05% glucose, 0.3 μg / mL vitamin B1, 16 μM calcium chloride, 8 μM manganese chloride, 8 μM zinc sulfate, 1.6 μM cobalt chloride, 1.6 μM nickel chloride, 1.6 μM sodium molybdate, 40 μM ferric chloride), shaking at 37°C to OD 600 =0.6-1.5. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and transferred to 20°C...

Embodiment 3

[0076] Protein characterization

[0077] The concentration of BLA series samples was determined by the extinction coefficient (calculated by ProtParam): wt-BLA: 0.904L / (g·cm); SpyX-BLA-RL: 0.825L / (g·cm); intX-BLA-RL: 0.818 L / (g·cm); intX-BLA-FL: 0.818 L / (g·cm).

[0078] Product distribution was characterized by SDS-PAGE: protein samples were mixed with 5x loading buffer (250 mM Tris, 50% glycerol, 10% SDS, 250 mM mercaptoethanol, 0.05% bromophenol blue) and loaded after heating at 98oC for 10 minutes . The relative quantification of protein was carried out in two ways: Unicorn 6.3 software was used to analyze the size exclusion chromatographic curve; Image J was used to analyze the SDS-PAGE band. see the results figure 2 (a)

[0079] The molecular weights of the samples were characterized using ultra-high performance liquid chromatography-electrospray ionization mass spectrometry. The charge-to-mass ratio data were analyzed by MassLynx V 4.1 software, and the molecular w...

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Abstract

The invention relates to intracellular synthesis of BLA traceless soxo hydrocarbon by using separated intein as a connection means and a preparation method thereof, and belongs to the technical field of protein coupling. The preparation method disclosed by the invention comprises the following steps: (1) constructing a gene plasmid, namely respectively fusing an N end and a C end of a target protein with two parts IC and IN of the separated intein; the end-to-end connection of the target protein is realized through trans-shearing and IC and IN removal, and the unique reserved part is necessary amino acid residues for realizing trans-shearing, so that the intein gene cassette is obtained; (2) introducing the gridowta intein gene cassette constructed in the step (1) into a vector plasmid; (3) expressing the vector plasmid in a cell; and (4) carrying out protein expression and purification on the vector plasmid to finally obtain the BLA traceless soxarene. The traceless BLA isohydrocarbon intX-FL disclosed by the invention is relatively good in catalysis rate and catalysis capability, fusion components are reduced to the greatest extent, and an excellent platform is provided for realizing strict main chain cyclization.

Description

technical field [0001] The invention belongs to the technical field of protein coupling, and in particular relates to a method for intracellular synthesis of BLA trace-free catenane by using an isolated intein as a connecting means and a preparation method thereof. Background technique [0002] The choice of protein conjugation strategies has greatly expanded in recent years. Among them, the SpyCatcher / SpyTag complex will be introduced during the catenation process. For most proteins with low molecular weight (molecular weight between 20kDa and 30kDa), the presence of this complex reduces the proportion of target proteins after catenation. If catenylation is applied to a possible drug protein or polypeptide, the immunogenicity due to the redundant portion and the payload of the entire molecule must be considered. On the other hand, in the process of catenane protein denaturation, the unfolding-folding process of the SpyCatcher / SpyTag complex and the target protein may inter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
CPCC12N15/70C12N9/86C12Y305/02006C07K2319/21C07K2319/92
Inventor 张文彬王智辉
Owner 江苏贝奥泰克生物科技有限公司
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