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Protein compound based on Escherichia coli E family DNA enzyme and application of protein compound in artificial protein scaffold

A protein complex and colicin technology, applied in the application field of artificial protein scaffolds, can solve the problems of limited application and achieve the effect of efficient hydrolysis and unified structure

Active Publication Date: 2022-01-28
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, most protein interaction pairs need to interact under high salt ion conditions, such as Cohesin-Dockerin's action is regulated by calcium ions, which limits their application in vivo

Method used

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  • Protein compound based on Escherichia coli E family DNA enzyme and application of protein compound in artificial protein scaffold
  • Protein compound based on Escherichia coli E family DNA enzyme and application of protein compound in artificial protein scaffold
  • Protein compound based on Escherichia coli E family DNA enzyme and application of protein compound in artificial protein scaffold

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Expression and purification of CL protein and Im protein

[0044] 1. Through amino acid sequence comparison with CL7 protein, based on the high similarity of the four protein sequences, the carboxy-terminal DNase domains of CE family DNases CE2, CE7, CE8 and CE9 were protein engineered to obtain lost DNase activity, but still retain the mutants CL2, CL7, CL8 and CL9 with super high affinity to the corresponding immune protein Im protein, among which the four sets of CL-Im and four wild-type CE protein sequences are:

[0045] (1) CE2 protein: as shown in SEQ ID NO.1; CL2 protein: as shown in SEQ ID NO.2; Im2 protein: as shown in SEQ ID NO.3;

[0046] (2) CE7 protein: as shown in SEQ ID NO.4; CL7 protein: as shown in SEQ ID NO.5; Im7 protein: as shown in SEQ ID NO.6;

[0047] (3) CE8 protein: as shown in SEQ ID NO.7; CL8 protein: as shown in SEQ ID NO.8; Im8 protein: as shown in SEQ ID NO.9;

[0048] (4) CE9 protein: as shown in SEQ ID NO.10; CL9 protein: as s...

Embodiment 2

[0051] Example 2 Native-PAGE

[0052] 1. Native-PAGE non-denaturing acrylamide gel preparation

[0053] For the verification of the binding specificity of the CL-Im protein interaction pair, a 30% acrylamide (37.5:1), glycerol electrophoresis system was used, and the specific formula is shown in Table 1.

[0054] Table 1 Native-PAGE non-denaturing acrylamide gel recipe

[0055]

[0056] 2. Native-PAGE

[0057] In order to verify the specificity of the binding between CL protein and Im protein, in this example, 4 kinds of CL proteins and 4 kinds of Im proteins were mixed separately to obtain 16 combinations. After incubation at room temperature for 30 minutes, Native-PAGE was performed. The voltage of the upper gel was 120V, and the lower layer was The gel voltage was 180V, and the electrophoresis was ended when the bromophenol blue indicator band moved to the bottom of the gel, and then stained. The results of electrophoresis were as figure 2 As shown in -B, the electr...

Embodiment 3

[0059] Example 3 Construction and thermal stability verification of scaffold protein Scaf-CIQ

[0060] 1. In this example, based on the above-mentioned combination of CL and Im protein, an interaction pair can be formed, and the corresponding scaffold protein is constructed, as follows:

[0061] The scaffold protein Scaf-CIQ in the CIQ system is a cellulose-binding module CBM3a derived from Clostridium thermocellum (living environment 60-90°C), and is a fusion protein expressed in tandem with Im2, Im7, Im8, and Im9 through a linker, which is the scaffold Protein Scaf-CIQ. The presence of CBM3a endows the scaffold protein Scaf-CIQ with the ability to bind to cellulose matrices such as microcrystalline cellulose (Avicel), phosphoric acid swollen cellulose (PASC), and achieve the purpose of targeting cellulose matrices and immobilizing enzymes. The principle of the scaffold protein Scaf-CIQ is as follows image 3 As shown, each Im module on the scaffold protein Scaf-CIQ can onl...

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Abstract

The invention discloses a protein compound based on colicin E family DNA enzyme and application of the protein compound in an artificial protein scaffold. The protein complex comprises any one or more of an interaction pair formed by a CL2 protein and an Im2 protein, an interaction pair formed by a CL7 protein and an Im7 protein, an interaction pair formed by a CL8 protein and an Im8 protein, and an interaction pair formed by a CL9 protein and an Im9 protein. According to the invention, protein engineering transformation is carried out on carboxyl terminal DNase structural domains of DNA enzymes CE2, CE7, CE8 and CE9 of a CE family to obtain a mutant which loses DNA enzyme activity but still retains ultrahigh affinity with a corresponding immune protein Im, and CL2 / Im2, CL7 / Im7, CL8 / Im8 and CL9 / Im9 protein interaction pairs are constructed. Research finds that the protein interaction pairs have the properties of heat resistance, high affinity, high specificity, small molecular weight, high assembly speed and the like, an artificial protein scaffold is constructed based on the protein interaction pairs, and a novel platform is established for construction of an artificial multi-enzyme compound.

Description

technical field [0001] The invention belongs to the field of synthetic biology and nanobiology technology, and in particular relates to a protein complex based on colicin E family DNA enzymes and its application in artificial protein scaffolds. Background technique [0002] High-affinity protein interaction pairs are widely used in many fields, such as co-immunoprecipitation, protein pull-down, yeast two-hybrid and enzyme immobilization, etc., in which the multi-enzyme complex formed by self-assembly of protein interaction The system has attracted much attention due to its excellent synergistic catalytic ability. For the "one-pot" synthesis reaction, multiple orthogonal protein interaction pairs are used to assemble multiple enzyme molecules in a specific order, and cascade catalytic reactions are performed in the form of artificial multi-enzyme complexes. It is conducive to the formation of substrate channels, which can prevent the diffusion of intermediate products and pr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/22C12N15/70C12N9/96C12N9/42C12P19/02C12P19/14
CPCC12N9/22C07K14/245C07K14/33C12N15/70C12N9/96C12N9/2445C12N9/2437C12N15/52C12P19/02C12P19/14C12Y302/01021C12Y302/01004C12Y302/01091C07K2319/00C12N15/62C12P21/02C07K2319/20C12R2001/145C12R2001/01C12N9/2434C12Y301/21001C12Y302/01176C12P21/06
Inventor 马立新翟超杨军王飞
Owner HUBEI UNIV
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