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Design of protein heterogeneous entanglement element and preparation method of complex isohydrocarbon structure

A protein and primitive technology, applied in the direction of animal/human protein, p53 protein, mammalian protein, etc., can solve the problems such as the efficiency to be improved, and achieve low folding structure and physiological activity, efficient and precise synthesis, and enhanced affinity. Effect

Active Publication Date: 2021-06-29
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above work is still limited to the preparation of relatively simple catenane structures, and the efficiency of catenanes needs to be improved.

Method used

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  • Design of protein heterogeneous entanglement element and preparation method of complex isohydrocarbon structure
  • Design of protein heterogeneous entanglement element and preparation method of complex isohydrocarbon structure
  • Design of protein heterogeneous entanglement element and preparation method of complex isohydrocarbon structure

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1: Design and synthesis of heterogeneous entanglement motif X + / X - representation of

[0069] The present invention is directed to p53 heterogeneous tangle motif X obtained by site-directed mutation + / X - Characterization was performed using polypeptides including unmutated homotangle dimer motif X and mutant X + / X - , prepared by solid-phase peptide synthesis technology (Beijing Deyi Biotechnology Co., Ltd.). First, through the circular dichroism spectroscopy (CD) X and mutant X + / X - Characterize the secondary structure of the entanglement motif, Figure 5 (a) X and mixed mutant X + / X - The system shows strong α-helical absorption signals, that is, negative absorption peaks at 208nm and 222nm, X + The system also has a certain α-helical absorption signal, but it is slightly weaker than the former two, while the X - There is no alpha helical absorption peak at all. The thermal stability of the aggregates formed by the above entanglement elemen...

Embodiment 2

[0071] Example 2: Expression and Purification of Protein Heterogeneous Catanenes and Higher Order Catanenes

[0072] The invention screens and optimizes the purification conditions of protein heterogeneous catenates and higher-order catenates, and determines the suitable conditions for corresponding systems.

[0073] Such as figure 2 As shown, the expression and purification of protein heterogeneous catenane are as follows: The precursor fusion protein 1 obtained by genetic engineering construction is NpuC1-X + -SUMO-NpuN1 is introduced into the MSCI of pACYCDuet-1 vector, fusion protein 2 is NpuC2-X - - sfGFP-NpuN2 was introduced into MSCII of the pACYCDuet-1 vector. Subsequently, the vector was transformed into Escherichia coli BL21 (DE3) competent cells, plated, and cultured at 37°C for 12h. A single clone was selected and inoculated into 10 mL of 2xYT liquid medium containing 33 μg / mL chloramphenicol, and cultured with shaking at 37° C. and 220 rpm overnight. Inoculat...

Embodiment 3

[0075] Example 3: Characterization and Topological Structure Proof of Protein Heterogeneous Catanes

[0076] After completing the purification of the protein heterogeneous cat-sfGFP-SUMO, the present invention utilizes sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), liquid phase mass spectrometry (LC- MS) to characterize it. Figure 7 (a) The results of gel electrophoresis showed that the prepared protein heterogeneous catenane had an obvious main band and its position was consistent with the expected molecular weight. The main band was quantified by gel densitometry and the yield was as high as 85%. Figure 7 The mass spectrometry data in (b) and (c) are also basically consistent with the theoretical calculation data, confirming the efficient and precise synthesis of heterogeneous catenates. During the construction process, the TEV enzyme cleavage site was inserted into the fusion protein 1, so the proof of the heterogeneous catenane topology was indir...

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Abstract

The invention discloses a design of a protein heterogeneous entanglement element and a preparation method of a complex isohydrocarbon structure. Site-directed mutagenesis is carried out on a tumor inhibition factor p53 dimerization entanglement element (X) to obtain a heterogeneous entanglement element X + / X-, and in combination with a protein coupling reaction pair, intracellular in-situ synthesis of protein heterogeneous Soxhdrocarbon and higher-order Soxhdrocarbon ([3] Soxhdrocarbon / [4] Soxhdrocarbon) is realized through an assembly coupling synergistic strategy. The method has the characteristics of modularization and gene coding, and can efficiently and accurately synthesize a protein topological structure. Through introduction of the target protein, soxazolylation construction of the target protein is realized, and soxazolylation does not obviously influence the structure and activity of the protein. An 'artificial antibody ' can be constructed by introducing an antibody or an antibody analogue into a higher-order soxarene skeleton, the affinity of the skeleton to receptor protein is remarkably improved due to the multivalence of the skeleton, and the skeleton has important significance in research and development of protein antibody drugs.

Description

technical field [0001] The invention relates to the design of heterogeneous entanglement motifs of biological macromolecular proteins, using the designed motifs to efficiently prepare protein heterogeneous catenane and higher-order catenane structures through one-step expression in cells, and to explore the effect of catenylation on target proteins The improvement of properties, especially the improvement of the affinity of antibody analogs by high-order cable alkylation, and its application to the design of "artificial antibodies". Background technique [0002] Most traditional proteins are formed by further folding of linear polypeptide chains. Although they have rich multi-level structures, their topology is relatively simple. In recent years, studies have found that proteins with nonlinear topological structures such as rings, knots, and catalanes can be obtained through complex post-translational modification processes in nature. These proteins show resistance to therma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K19/00C12N15/70
CPCC07K14/4746C12N15/62C12N15/70C07K2319/00C07K2319/92C07K2319/21C07K2319/95C07K2319/60C07K2319/735
Inventor 张文彬吴文豪
Owner PEKING UNIV