University of Utah
Yang, Seung Ook.
In vivo construction of a nanoreactor using charge complementarity.
Degree: MS, Chemistry, 2012, University of Utah
Subcellular protein compartmentalization is one of the strategies that nature hasadapted for various functions, such as storage and protection of cargo molecules as wellas regulation of chemical reactions. There is a growing interest among nanotechnologiststo utilize protein capsids for encapsulating guest molecules in bioimaging, drug-delivery,nanoreactors, etc. To date, many viral and nonviral protein capsids have beeninvestigated for those applications.We used an engineered an Aquifex aeolicus lumazine synthase variant (AaLS-13)as a platform to encapsulate a guest enzyme by charge complementarity. The enzyme,esterase Est55 bearing a C-terminal deca-arginine (R10) tag, was placed in the lumen ofthe AaLS-13 during the self-assembly of the capsid when co-produced in E. coli. Thisencapsulated enzyme was characterized by colorimetric assay with the substrate, pnitrophenylacetate. Comparison of the specific esterase activity between the emptycapsid and the enzyme-loaded capsid showed 64-fold higher specific activity in theenzyme-loaded capsid compared to the empty capsid. Mass spectrometry and proteomicsanalysis verified that the presence of Est55-R10 in a sample of purified AaLS-13 that hadbeen co-produced with the esterase. Capsid loading was estimated by esterase activitymeasurements and SDS-PAGE analysis. Both methods gave a loading estimate of oneesterase molecule per eleven capsids.We plan to improve capsid loading using “supercharged” esterases. We also planto investigate the porosity of the capsid because size limit for small molecule diffusion inand out of capsid is not well defined. Esterase substrates of various sizes will be appliedto both esterase-loaded capsid and free esterase to determine the molecular weight cut-offof the AaLS-13 capsid.
Subjects/Keywords: Capsid; Charge Complementarity; Encapsulation; Esterase
to Zotero / EndNote / Reference
APA (6th Edition):
Yang, S. O. (2012). In vivo construction of a nanoreactor using charge complementarity. (Masters Thesis). University of Utah. Retrieved from http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/2072/rec/1338
Chicago Manual of Style (16th Edition):
Yang, Seung Ook. “In vivo construction of a nanoreactor using charge complementarity.” 2012. Masters Thesis, University of Utah. Accessed October 19, 2019.
MLA Handbook (7th Edition):
Yang, Seung Ook. “In vivo construction of a nanoreactor using charge complementarity.” 2012. Web. 19 Oct 2019.
Yang SO. In vivo construction of a nanoreactor using charge complementarity. [Internet] [Masters thesis]. University of Utah; 2012. [cited 2019 Oct 19].
Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/2072/rec/1338.
Council of Science Editors:
Yang SO. In vivo construction of a nanoreactor using charge complementarity. [Masters Thesis]. University of Utah; 2012. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/2072/rec/1338