Dariana Chavez
IUUR STEM Summer Research Program
Major: Molecular Life Sciences
Mentor: Cristina Landeta
Disulfide bonds are formed from covalently bonded sulfhydryl side chains of two cysteine residues and serve to activate and stabilize proteins in prokaryotes and eukaryotes. Most
disulfide bond formation pathways in bacteria utilize a DsbAB system where DsbA, a thiol disulfide oxidoreductase (TDOR), oxidizes nascent proteins and is reduced. DsbB, a disulfide
bond formation protein, restores DsbA to its oxidized state and regenerates the pathway. Escherichia coli carries a DsbAB system to fold proteins in the periplasm while eukaryotes,
actinobacteria, cyanobacteria, and some deltaproteobacteria have disulfide bond formation pathways that use the enzyme, vitamin K epoxide reductase (VKOR) instead of DsbB to oxidize DsbA. VKOR and DsbB are non-homologous, yet they both perform the similar function of oxidizing DsbA. Furthermore, recent research has found inverted VKOR homologues present in viruses. Here, we sought to complement DsbB using human VKOR (HsVKOR) and two different viral VKORs (HvVKOR and BsVKOR) in the disulfide bond formation pathway of E. coli and test the functionality of the pathway using motility and alkaline phosphatase assays. Synthesized segments of HsVKOR, BsVKOR, and HvVKOR were used as inserts in two
separate vectors, and the plasmid was then used to transform competent E. coli cells. HsVKOR was cloned successfully in E. coli under an IPTG-inducible promoter, and the pathway was
tested through a motility assay. At first, the concentration of IPTG used was too toxic for the cells, so human VKOR was inserted in a vector carrying a weaker promoter and the assay was
repeated. Additionally, the viral VKORs were cloned and transformed successfully in E. coli cells and will be used to test the disulfide bond formation pathway functionality using an alkaline phosphatase assay.
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