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Using Vitamin B1 as a Catalyst in Performing Multi-Component Reactions and Synthesis of Affinity Column ChromatograpHy for Purification of Recombinant Proteins

Lohi Khosroshahi, Ameneh | 2020

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 52761 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. Due to the importance of using pure proteins in the industry and basic research in order to study them in the structural contexts and interactions of proteins in recent years, purification methods have been extensively developed. The purification steps usually depend on the size of the protein, its physical and chemical properties, the binding affinity and the biological activity of the protein in question, In the purification process, the protein portion is separated from the non-protein portion. The biggest challenge is when we want to separate different proteins. Different chromatographic methods are used, depending on the properties of the impure proteins. One of these chromatographic methods, which are widely used for purification of proteins, is the affinity method. Proteins based on their specific amino acid groups, appropriate affinity chromatography columns have used for their purification. One of the preferred methods is immobilized metal affinity chromatography or imac, which is based on the specific amino acid interactions of proteins and metals (especially divalent metals such as nickel or cobalt). Since the recombinant protein often contains histidine groups, it is used to isolate and purify the protein from a nickel-affinity column. Imac methods have shown the highest selectivity over other purification methods. Our goal in this project has been to synthesize nanoparticles that could be used as an affinity column for purification of recombinant proteins. Therefore, these nanoparticles were used as central nuclei for the production of purified columns of histidine-binding recombinant proteins. TEM analysis revealed that the size of these nanoparticles was in the range of 12–6 nm and the IR method was used to study the functional groups of nanoparticles. The surface of the iron nanoparticles was enhanced by acrylamide and polymerized by ATRP. subsequently by binding to the EDTAD ligand, which retains nickel chelating, it was possible to stabilize the nickel on the surface of the nanoparticles. To determine whether the nanoparticles were stabilized at the surface, IR and CHN methods were used and EDX analysis was performed to ensure nickel deposition on the surface of the nanoparticles, which confirmed the presence of nickel as well as its exit during each use. In fact, after each particle application and EDX capture, the intensity of the nickel spectrum decreased. During the fabrication phase of the iron nanoparticles and after nickel fixation at the surface, the zeta potential of the particles was measured, with the numbers partially indicating good dispersion of the nanoparticles in aqueous media. The size of the nanoparticles was evaluated by DLS after the target groups and the increase in particle diameter in DLS analysis showed that the target groups were at the level of nanoparticles. The histidine-bearing protein SUT1 was induced and generated, and electrophoresis was used to study whether the SUT1 protein was purified by nanoparticles, and the protein was purified by particles at 30 kD containing histidine. The ability to recycle and reuse nanoparticles was also investigated and it was found that up to three times the usability of the nanoparticles was increased and the amount of purified protein increased as the nanoparticles weight increased. The concentration of the purified proteins was determined by spectrophotometry at 595 nm
  9. Keywords:
  10. Nanoparticles ; Protein Purification ; Histidine ; Recombinant Protein ; Immobilized Metal Affinity Chromatography (IMAC)

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