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A protein coupling method based on catenation

A protein and coupling technology, applied in the field of preparation of biomacromolecular proteins

Active Publication Date: 2020-11-03
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this strategy has not yet been used in macromolecular systems

Method used

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  • A protein coupling method based on catenation
  • A protein coupling method based on catenation
  • A protein coupling method based on catenation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Expression and purification of reaction precursor protein

[0058] During the construction process, the reaction precursors BD-A, BD-ELP2-A and BD-DHFR-A were constructed in the pQE-80L vector, and c-S, c-S-E1, c-S-GFPrm were constructed in the pET-15b vector. The vector was transformed into Escherichia coli BL21(DE3) competent cells and cultured overnight. Then select the single clone grown on the plate, inoculate into 5 mL LB medium containing 100 μg / mL ampicillin sodium, and culture at 37° C. for 8-12 hours. Then transfer the obtained seed liquid into 1L of fresh LB medium at a ratio of 1:100 or 1:200, and culture it with shaking at 37°C until OD 600 Between 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and transfer to 16°C for expression for 20 hours. Bacteria were collected by centrifugation. Then the cells were dispersed in 30-35mL lysis buffer A (50mM disodium hydrogen phosphate, 300mM sodium chloride, 10m...

Embodiment 2

[0059] Embodiment 2: the characterization of reaction precursor

[0060] The purified reaction precursor was initially characterized by high performance liquid chromatography-electrospray mass spectrometry. figure 2 Precursors BD-A (a), c-S (b), BD-E2-A (c), c-S-E1 (d), BD-DHFR-A (e), and The mass spectrometry data of c-S-GFPrm(f), the experimental data and the theoretical calculation data are basically consistent.

Embodiment 3

[0061] Example 3: Preparation and Characterization of Protein Heterogeneous Catanenes

[0062] The purified reaction precursors BD-A / BD-E2-A / BD-DHFR-A were mixed with c-S / c-S-E1 / c-S-GFPrm at a concentration ratio of 1:1 at a reaction concentration of 50 μM. 10mM dithiothreitol (DTT) (5mM DTT was added to the BD-DHFR-A and c-S-GFPrm reaction system) was used to reduce and separate the intermolecular disulfide bonds. The mixed system was reacted at 4°C for 20h. After the reaction, 10 μL of the reaction precursor was mixed with the reaction mixture and 5× protein loading buffer, then heated at 98°C for 10 min, and the results were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. image 3 The reaction conditions of the three pairs of reaction precursors are shown, and new bands with molecular weights corresponding to the sum of the two reaction precursors appear in the reaction mixture, that is to say, a catenane structure is formed. Afterwards, the reactio...

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Abstract

The invention discloses a protein coupling method based on catenane. Each second-level component (SpyTag, BDTag and SpyStapler) in a SpyTag-SpyCatcher compound is connected renewedly, a chain entanglement structure designed by labor is introduced, a protein activity template method combining winding and catalyzing is developed, and mechanical key coupling of different target proteins connected in a catenane mode can be realized. The protein coupling method based on the catenane is used for performing heterogeneous catenane on disordered proteins or folding proteins, and the structure and performance of the proteins cannot be affected. The catenane process further can realize intracellular direct expression preparation through a coexpression system. Due to the fact that a catenane structure has a conformation limiting effect and space control on the target proteins, and the stability of the target proteins can be improved. According to the protein coupling method based on the catenane, concise synthesis of protein heterogeneous catenane is realized, and the protein coupling method based on the catenane is a very valuable and novel protein coupling strategy.

Description

technical field [0001] The invention relates to the preparation of biological macromolecular proteins, in particular to the coupling of proteins through heterogeneous catenate alkylation, and the application of catenate alkylation to improving the stability of target proteins. Background technique [0002] Traditional protein coupling mainly relies on covalent bond coupling to obtain fusion structures of linear or branched main chains. However, there are few examples of protein coupling using mechanical bonds. The spatial distribution of protein domains in conjugates The situation is also less considered. The ring structure can improve the stability of the protein due to the restrictive effect on the conformation of the main chain of the protein. Currently, work in the literature focuses on cyclization of individual proteins. Cyclization of multiple proteins will greatly weaken their confinement effect, so that the improvement of protein stability is also relatively limite...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/00C07K2319/21C07K2319/35C07K2319/50C12N15/70
Inventor 张文彬达晓娣
Owner PEKING UNIV
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