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Linear polyfunctional multimer biomolecule coupled to polyubiquitin linker and use thereof

A biomolecular, multifunctional technology for fusion with degradation motifs, peptides containing affinity tags, biochemical equipment and methods, etc., to solve problems such as separation or analytical efficiency limitations

Pending Publication Date: 2021-01-08
ONEGENE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods only describe the use of ubiquitin in the process of expressing proteins, and do not describe or teach the preparation of proteins in the form of multimers, so that the proteins to be separated and purified are freely combined with ubiquitin, so they still have separation or analysis efficiency. upper limit

Method used

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  • Linear polyfunctional multimer biomolecule coupled to polyubiquitin linker and use thereof
  • Linear polyfunctional multimer biomolecule coupled to polyubiquitin linker and use thereof
  • Linear polyfunctional multimer biomolecule coupled to polyubiquitin linker and use thereof

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preparation example Construction

[0069] In one embodiment of the present invention, a method for preparing a linear multifunctional multimeric biomolecule is provided, which includes: (i) recombinantly expressing a biomolecule from a host cell including a prokaryotic cell or a eukaryotic cell, the biomolecule Fusion to or binding to a ubiquitin C-terminal tag via an adapter and (ii) addition of E1, E2 and E3 enzymes or E1 and E2 enzymes for ubiquitination to the lysates of the above host cells and performing a reaction comprising two or more binding moieties each having specificity for mutually different binding sites as a biomolecule, the above-mentioned biomolecule being bound to a polyubiquitin backbone formed by covalent bonding of two or more ubiquitins . Thus, in the present invention, the initiator that initiates the formation of a linear multifunctional multimeric biomolecular polymer or complex can be E3, E2, El, free ubiquitin, or a target substrate of E3. Wherein, the above-mentioned E2 enzyme can...

preparation example 1

[0074] Preparation Example 1: Cloning, expression and purification of C-terminal fusion protein

[0075] Genscript Inc. was commissioned to prepare the gene encoding the protein fusion of Ubiquitin C-terminal Tag (Sequence: 1) used in the examples of the present invention.

[0076] In order to prepare Ub out gene constructs that do not contain ubiquitin tags at the C-terminus, a fast cloning system (fast cloning system) was used (Li C, Wen A, Shen B, Lu J, Huang Y, Chang Y (2011). Fastcloning : a highly simplified, purification-free, sequence-and ligation-independent PCR cloning method. BMC Biotechnol 11, 92.). This method is the following technology: under the condition that there is no restriction enzyme and binding enzyme, if only Dpn1 is directly processed on the polymerase chain reaction (PCR) product, then Dpn1 and the polymerase are restricted by a mechanism that has not been identified so far. Enzymes and binding enzymes act to link genes (insertion, deletion or sub...

preparation example 2

[0080] Preparation Example 2: Preparation of linear multifunctional polymer fusion protein linear structure

[0081] In the present invention, the reactions for preparing fusion proteins in the form of linear multimers are respectively named linear multimer fusion protein reactions. In linear multimer fusion protein buffer (25mM HEPES (Sigma-aldrich), pH 7.5, 50mM NaCl, 4mM MgCl 2) was performed in a linear multifunctional multimer fusion protein reaction (total volume of 50 μL), the linear multifunctional multimer fusion protein reaction for linear multifunctional multimer The reaction was initiated with a sex polymer fusion protein mixture (0.5 μM E1, 5 μM E2, 1 μM E3, 4 mM ATP). The ratio of the protein used for the reaction is as follows, that is, 10uM to 20uM of the ubiquitin C-terminal tag protein fusion (1:10 to 1:20 ratio) is used per 1uM of E3 enzyme (enzyme), this is for, by The linear multifunctional multimer fusion protein reacts within 1 hour to make at least ...

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Abstract

The present invention provides a linear multimer biomolecule polymer wherein a biomolecule is bonded to a polyubiquitin scaffold formed of two or more covalently bonded ubiquitins, by obtaining, froma host cell, a biomolecule bonded with a ubiquitin C-terminal tag through recombinant expression, and polyubiquitinating the biomolecule in vitro in the presence of proteins involved in ubiquitination, E1 (activation enzyme), E2 (conjugation enzyme), and E3 (ligase), and a substrate. The polymer according to the present invention may be used in the separation and purification of a biomolecule, theseparation of a target material that binds to the biomolecule, etc.

Description

technical field [0001] The present invention relates to methods for preparing biomolecules comprising proteins as polymers in the form of multimers. In particular, the present invention relates to methods for preparing recombinantly expressed biomolecules from host cells into linear multifunctional multimeric biomolecular polymers using the ubiquitination system. Background technique [0002] Biomolecules and / or smallmolecule chemical compounds including proteins, peptides, polypeptides, antibodies, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) in a multimeric form has several advantages. For example, two or more homogeneous or heterogeneous proteins are fused (fusion) or a cross-linker (cross linker or cross-linking agent) is used to link two or more homogeneous or heterogeneous proteins, thereby improving protein Physical and chemical properties such as solubility, gelation, thermal stability and pH stability. For example, when starch oxidation is carried out...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/14G01N33/535C12N9/10
CPCC07K14/47G01N33/535C12N9/0006C12N9/0008C12N9/16C12N9/88C12N9/90C07K1/14C12N9/104C12Y101/01009C12Y101/01307C12Y102/03003C12Y203/02C12Y401/01003C07K2319/95C07K14/001C07K14/4702C07K2319/21C07K2319/23C12N9/93C12Y602/01C12Y603/02019C12Y301/02015C12Y603/02021
Inventor 朴成真任大成崔载永
Owner ONEGENE BIOTECH INC
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