3 research outputs found
Development of a Site-Selective Protein Immobilization Methodology Utilizing Unnatural Amino Acids
Proteins are nature’s catalysts and have evolved over millennia to be highly selective and efficient. As a result, many have sought to incorporate proteins into artificial systems to varying degrees of success. Immobilization of proteins onto solid supports can increase the stability of proteins in conditions that would normally induce denaturation but immobilization strategies can present their own challenges by using reactions that lack selectivity and can potentially disrupt protein function themselves. This work develops a new methodology for protein immobilization that uses an unnatural amino acid site-selectively incorporated into a protein as the functional handle for immobilization. As a model system, Green Fluorescent Protein was immobilized in this manner and found to retain its functional activity in conditions which would normally cause the protein to denature. Through the development of this methodology, it was found that immobilization efficiency is affected by the site where the functional handle is inserted, in addition to the accessibility and reactivity of the solid support. Finally, progress was made towards determining whether this methodology can be successfully incorporated into a system which utilizes microwave irradiation
Synthesis and Incorporation of Unnatural Amino Acids To Probe and Optimize Protein Bioconjugations
The utilization of unnatural amino acids (UAAs) in bioconjugations is ideal due to their ability to confer a degree of bioorthogonality and specificity. In order to elucidate optimal conditions for the preparation of bioconjugates with UAAs, we synthesized 9 UAAs with variable methylene tethers (2-4) and either an azide, alkyne, or halide functional group. All 9 UAAs were then incorporated into green fluorescent protein (GFP) using a promiscuous aminoacyl-tRNA synthetase. The different bioconjugations were then analyzed for optimal tether length via reaction with either a fluorophore or a derivatized resin. Interestingly, the optimal tether length was found to be dependent on the type of reaction. Overall, these findings provide a better understanding of various parameters that can be optimized for the efficient preparation of bioconjugates
Site-Specific Protein Immobilization Using Unnatural Amino Acids
Protein immobilization confers the
advantages of biological systems
to a more chemical setting and has applications in catalysis, sensors,
and materials development. While numerous immobilization techniques
exist, it is optimal to develop a well-defined and chemically stable
methodology to allow for full protein function. This paper describes
the utilization of unnatural amino acid technologies to introduce
bioorthogonal handles in a site-specific fashion for protein immobilization.
To develop this approach a range of solid-supports, organic linkers,
and protein immobilization sites have been investigated using a GFP
reporter system. Overall, a sepharose resin derivatized with propargyl
alcohol has afforded the highest yields of immobilized protein. Moreover,
an unnatural amino acid residue protein context has been demonstrated,
signifying a necessity to consider the protein site of immobilization.
Finally, a resin-conferred stabilization was demonstrated in several
organic solvents