Date of Graduation

5-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Suresh Kumar Thallapuranam

Committee Member

Navam Hettiarachchy

Second Committee Member

Paul Adams

Third Committee Member

Joshua Sakon

Keywords

binding affinity, Amlexanox, proteins, protein secretion, copper

Abstract

Recombinant DNA technology and the ability to produce recombinant proteins have significantly changed the world of pharmaceutical market. Recombinant DNA technology using E. coli cells has successfully produced many therapeutic proteins. In this study, the designed tag Ark-RUBY-tag facilitates rapid, and reproducible purification of recombinant proteins expressed as inclusion bodies in E. coli cells. Purification of Ark-RUBY-fused recombinant protein(s) can be obtained by using imidazole fractions. Target protein can be easily removed from the Ark-RUBY-tag by enzymatic cleavage. Ark-RUBY-fused recombinant proteins can be quantitatively detected using polyclonal antibodies. Ark-RUBY-tag can be used to purify small peptides. By using Ark-RUBY-tag, purified recombinant protein regains fully biologically active form. Therefore, bioactive protein has wide range of application in drug-delivery. Copper is crucial in many cellular functions. It is known that copper is necessary in non-classical secretion by inducing multiprotein realize complex, but the involvement of copper in complex formation is not clear. In this study, the role of copper in non-classical protein secretion of IL-1α was investigated. This framework demonstrates the role of copper to induce IL-1α dimerization under in vitro conditions. The dimerization mechanism is mediated by the single cysteine at position 143 that gets oxidized resulting in the formation of an inter-disulfide bond. The ratio of copper to induce IL-1α dimerization is one to two that means one copper can induce two molecules of IL1-α. The binding affinity of IL-1α to copper study showed that IL-1α binds to copper with Kd value 1.9 μM, which suggests IL-1α is copper binding protein. Also, some binding affinity experiments were conducted to elucidate whether copper is the only metal that induce dimerization. Some metals showed good binding affinity, but no one induced dimerization. Interestingly, corpus form showed high binding affinity, but it did not induce dimerization. Also, the monomer and the dimer forms of IL-1α have binding affinity to S100A13 with Kd values 1.30 μM and 1.34 μM respectively, which could be crucial in the non-classical secretion of signal-less IL-1α. The study also shows that Amlexanox (AMX), the anti-allergic drug, can bind to IL-1α and chelates copper with kd 1.9 μM and 1.6 μM respectively. It also inhibits the copper induced dimer formation of IL-1α. The mechanism that underlies dimer formation inhibition of AMX is either because it binds close to cysteine 143 of IL-1α where copper can bind and inhibits the induction of homodimer formation of IL-1α by masking the thiol group of Cys134 or by chelating copper and preventing it from binding to IL-1α. The results of AMX role in inhibiting dimer formation of IL-1α may help in designing a drug that inhibits non-classical secretion of IL-1α.

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