Method for conjugation of biomolecules and new use of gold donor for biomolecular complex formation

A technology of biomolecules and complexes, which is applied to the preparation methods of peptides, medical preparations of non-active ingredients, chemical instruments and methods, etc., and can solve problems such as toxicity of nanoparticles and obstacles to in vivo application

Pending Publication Date: 2021-03-26
JAGIELLONIAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of GNPs in the reaction is undesirable as 1.4nm nanoparticles are known to be toxic 12,13 , and can bind nonspecifically to the resulting structure, making purification of protein cage products from excess gold nanoparticles challenging and presenting an obstacle to potential future in vivo applications

Method used

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  • Method for conjugation of biomolecules and new use of gold donor for biomolecular complex formation
  • Method for conjugation of biomolecules and new use of gold donor for biomolecular complex formation
  • Method for conjugation of biomolecules and new use of gold donor for biomolecular complex formation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] TRAP complex Preparation - Reaction with Au-TPPMS

[0074] (See figure 1 )

[0075] Gold compound:

[0076] [Diphenyl (3-sulfonate] phosphine] Chloride (I), sodium salt hydrate (Au-TPPMS, MDL MFCD19443491) is purchased from STREM CHEMICALS UK, LIMITED, and achieved by dissolving in water The desired concentration (usually 5 mm). The gold nanoparticles (GNP) used are a diphenyl group (mexylphenyl) phosphine having 1-3 nm inner diameter from StREM CHEMICALS UK (MDL MFCD 17018839).

[0077] TRAP preparation:

[0078] The protein used to successfully use Au-TPPMS is a TRAP protein having introduced cysteine. Expression and purification of TRAP containing 35 residual lysine (K) to cysteine ​​and 64 residual argin (R) to serine (S) Additional mutation (called "TRAP-CS ") And the aforementioned TRAP-CS 11 (As described in detail above), significant changes are TCEP (three (2-carboxyethyl) phosphine) is not included in the cracking step. The final buffer is usually 20 mM Tris-HCl,...

Embodiment 2

[0083] Preparation of TRAP Composites - Reaction with Au (I) - Trobenzylphosphine

[0084] Trobhenylphosphine chloride (I) (Au-TPP, figure 1 B) A similar reaction instead of Au-TPPMS. The Au-TPP was dissolved in DMSO, reacted in 50 mM Tris, 150 mM NaCl, pH 7.9 and Au-TPP, and DMSO did not exceed 10% of the final volume, and the TRAP monomer was present at a concentration of about 1 mm. The ratio of Au-TPP and TRAP is 4: 2 to 4: 3. This also results in a TRAP cage formed from the same structure obtained in the Au-TPPMS reaction. The amount of the reagent is adjusted separately. The ratio and reaction conditions of the TRAP monomer / gold donor were the same as where Au-TPPMs was reacted as a gold donor.

[0085] Two embodiments having different halogen (triaryl phosphine) gold (I) gold donor reagents were carried out above. It shows a halogen (triaryl phosphine) gold (I) having a different aryl moiety (I) is adapted to form a complex according to the present invention.

Embodiment 3

[0087] Use Cryo-EM to confirm the TRAP complex structure

[0088] The initial (low resolution) Cryo-EM structure of the Trap cage was obtained using a Cryo-EM single particle recombination technique for using a TRAP cage formed using GNP. 10,11 And the structure data is used as an initial model of the high resolution Cryo-EM structure formed in the TRAP cage formed in the reaction in the reaction in the reaction in the reaction in accordance with the present invention.

[0089] The structure of the TRAP cage was resolved using Cryo-EM to 3.9 angstroms. This is sufficient to display the arrangement of 24 TRAP loops and demonstrate that there is a connection density (specified as AU) between the crystallysite chains of the relative ring (see figure 2 ).

[0090] figure 2 Examples of the gold sewing reaction and the formation of the complex are exemplified. figure 2 A shows the structure of a single TRAP ring (PDB4V4F) that is displayed in two mutually orthogonal views. The mutat...

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Abstract

The subject matter of the invention is a method for conjugation of free thiol group(s) containing biomolecules, leading to the biomolecular complex formation, comprising a reaction to connect biomolecules using a gold-donor agent in which a -S-Au-S- bond is formed, characterised in that a gold-donor agent is halogen(triarylphosphine)gold (I). The subject matter of the invention is also the use ofhalogen(triarylphosphine)gold (I) molecules as the gold-donor agent in the method of biomolecular complex formation.

Description

Technical field [0001] The invention belongs to the field of biochemistry. The present invention relates to a method for conjugating a free thiol group comprising a biomolecule, comprising reacting biomolecules with a gold donor reagent, wherein the -S-Au-S- bond is formed. Specifically, the method results in a complex as a protein cage. Background technique [0002] The protein complex in nature is manifested as an important and highly complex biological nano machine and nanostructure. The large protein complex in the nature is typically composed of many separate proteins that remain together by non-covalent interaction (i.e., hydrogen bonds, hydrophobic filled). This is particularly remarkable in the protein cage (e.g., the casing), wherein the same protein subunit of multiple copies is held together in this manner. In synthetic structural biology, the ability to design and construct artificial protein assembly may be useful, potentially allowing the introduction of the ability...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/13C07K17/14A61K47/52A61K47/62C07K14/245C07K14/32
CPCC07K1/1133C07K1/13C07K17/14C07K14/195
Inventor 乔纳森·赫德勒阿里·马来
Owner JAGIELLONIAN UNIVERSITY
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