New Strategies For Construction And Assembly Of Artificial Selenoenzymes Based On SP1 Protein

Selenium exists in selenoprotein in the form of selenocysteine and plays an important role in the catalytic process of antioxidant enzymes.In order to study the structure and functional characteristics of selenium in selenoprotein,scientists have put forward various strategies to design artificial selenoenzyme to solve the problems of instability and lack of natural sources of selenoenzyme.In all design strategies,the core point is to combine substrate recognition with catalytic selenium molecules.Through genetic engineering and site-specific introduction of catalytic selenium,a series of artificial selenoenzyme with high GPx activity have been developed.For example,from different catalytic angles and using different mechanisms,selenium-containing catalytic antibodies,semi-synthetic selenoenzyme and selenium-containing molecularly imprinted enzymes have been constructed.Taking advantage of host molecules such as cyclodextrins,dendrimers and hyperbranched polymers as main scaffolds,more selenoenzyme models were designed.Then,using electrostatic interaction,metal coordination and host-guest interaction,a variety of selenoprotein complexes and cascaded antioxidant nanoenzymes were constructed.Artificial simulation of selenoenzyme is not only helpful to understand the relationship between the structure and function of selenoenzyme,but also more importantly,the antioxidant ability of selenoenzyme can protect organism cells from oxidative stress,further avoid the accumulation of harmful substances inside and outside the cell membrane,and finally realize the dynamic balance of organism life,health and metabolic activities.Therefore,this thesis mainly focuses on the new strategy of design and assembly of artificial selenoenzyme and the functionalization of enzyme.The specific research contents of this thesis are as follows:1.Construction of artificial GPx based on SP1 proteinGPx is a magic enzyme,which has a very powerful ability to capture free radicals,thus realizing the stability and balance of free radicals in organisms.Because of its excellent antioxidant capacity,GPx has been highly concerned by scientists.However,the inherent shortcomings of natural GPx enzyme,such as the limited source and instability,will lead to its slow development and utilization.Therefore,scientists are committed to constructing natural GPx enzyme mimics through artificial methods,and studying their structure and catalytic properties.Considering the stability of the constructed artificial selenoenzyme,we selected SP1 protein as the scaffold for construction,which has extremely strong thermal stability.Firstly,we use software to simulate,so that we can intuitively select the appropriate groove on the surface of SP1 protein as the binding pocket of artificial selenoenzyme substrate.Then the plasmid of SP1 protein was further modified by genetic engineering to realize the targeted functionalization of the protein.In the process of modification of this site,the main modification sites and objectives are divided into two aspects.The first is to mutate the proline at the site 6 of SP1 protein into cysteine.After the mutation,the SP1 protein is named SP1-P6 C,because the SP1 protein sequence itself does not contain cysteine,which lays the foundation for the subsequent insertion of selenocysteine.The obtained plasmid is transformed into the expression system of E.coli for protein expression,and the mutant plasmid is transformed into the expression system of Cysteine-deficient E.coli,which can realize the transformation of cysteine to selenocysteine at position 6,thus endowing SP1 with extremely high selenoenzyme activity.The second aspect is to provide the possibility for protein assembly.We introduce histidine pairs at appropriate positions perpendicular to the surface of SP1 protein to facilitate the subsequent use of metal coordination drive to induce protein self-assembly to form protein nanotubes.So we mutated the glutamine and aspartic acid at the 78 and 82 positions of the SP1-P6 C plasmid into histidine,and named the constructed plasmid Se-SP1-P6C-Q78H-D82 H.2.Self-assembly of SP1 protein to construct intelligent GPx nanoenzymeIn the process of constructing artificial selenoenzyme using SP1,we found that its structure was stable and regular,so we tried to realize the series connection of regular single circular proteins,and could form highly ordered nanotubes.In the first part,when we modified the plasmid,we chose the position perpendicular to the SP1 protein,and mutated the amino acids at positions 78 and 82 into histidine(His).Through the metal coordination driving force of histidine and nickel ions,we realized the series connection of a single SP1 protein ring in the plane perpendicular to the protein,and further self-assembled to form protein nanotubes.It is noteworthy that we can realize the disassembly of the constructed selenoenzyme nanowires and the conversion between a single protein ring and a protein nanotube by introducing ethylenediamine tetraacetic acid(EDTA)chelating metal particles into the system.The catalytically active site of selenoenzyme is located in the center of the inner ring of the protein.Through assembly,the catalytically active site of selenoenzyme is shielded,resulting in the disappearance of selenoenzyme activity.However,adding nickel ions to the system again can dissociate the nanowires and restore them to a single SP1 protein ring,so that the active center of selenoenzyme is exposed again,realizing the reopening of selenoenzyme activity.In the whole system,we can control the activity of artificial selenoenzyme by adding nickel ion and EDTA,and realize the reversible conversion between "on" and "off" of selenoenzyme activity.This study provides a good idea for realizing the functionalization of artificial selenoenzyme.