A novel method for conjugation of biomolecules and new use of gold donor for biomolecular complex formation
Protein complexes in nature represent important and highly sophisticated biological nanomachines and nano-structures. Large protein complexes in nature are typically constructed of a number of individual proteins held together by non-covalent interactions (i.e. hydrogen bonds, hydrophobic packing). This is particularly noticeable in protein cages such as capsids where multiple copies of identical protein subunits are held together in this way. In synthetic structural biology the ability to design and construct artificial protein assemblies may be useful, potentially allowing the introduction of properties an capabilities not present in nature. To this end new ways of connecting individual proteins together in defined ways is desirable.
The reactions of gold compounds with the -SH groups are well-known and described in the literature. The use of gold compounds to incorporate gold particles into nanostructures or providing nanoparticles as nanoclusters, protein cages for multiple applications, among others as a targeting molecule in delivery systems, is also well described in the literature as well as in patent documents.
The novel approach for use of gold compounds in building biological molecules for different purposes is also shown in the publications, where gold nanoparticles (GNP) are used as a catalyst for linking together ring-shaped TRAP monomers presumably by the S-Au-S bond formation. The use of GNP in the reaction is not desirable as 1.4 nm nanoparticles are known to be toxic and may non-specifically bind to the resulting structures making purification of protein cage product from excess gold nanoparticles challenging and representing an obstacle to potential future in vivo applications.
The subject of the offer is a novel method for conjugation of free thiol group(s) containing biomolecules comprising a biomolecule reaction with a gold-donor agent in which a -S-Au-S- bond is formed. Specifically, the method leads to a complex formation that is a protein cage. The new method comprises following steps: (i) biomolecules preparation, (ii) conjugation of biomolecules by reaction of biomolecules with gold-donor and (iii) purification of conjugation product.
In the present invention a new approach is realised – instead of gold nanoparticles – (triarylphosphine)gold(I) halide is used as a catalyst for bond formation between protein units self-assembling into the protein complex, wherein SH groups are within the moiety, preferably cysteine moiety, naturally occurred or artificially incorporated in the protein structure. This innovative approach allows control of the assembly and disassembly of, in one embodiment, the capsid-like protein complex.
- innovative method for conjugation of biomolecules and new use of gold donor for biomolecular complex formation,
- an extremely attractive and desirable tool to produce new structures and ultimately new nanomachines / new smart drugs for medical application,
- switchability: assembly can be “switched on” by addition of gold,
- stability: the produced protein cage is stable to extremes of and over a wide pH range and a high concentration of agents usually disruptive to protein assemblies (e.g. urea),
- simplicity: no additional energy input is required.
The offered method for conjugation of biomolecules and its new use is patent pending. Further research and development of the invention are continued at the Malopolska Centre of Biotechnology, Jagiellonian University. Currently the Centre for Technology Transfer CITTRU is looking for entities interested in commercial application of the invention.