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dc.contributor.advisorHecht, Michael
dc.contributor.authorJiang, Alex
dc.description.abstractRecombinant protein expression in microbial hosts is needed to produce high yields of proteins that are expressed in endogenous tissues. Heterologous expression also has an increasing influence in the development of biopharmaceutical products. To assist with the demands for high and stable expression of recombinant proteins, expression-augmenting tags that confer stability and solubility are often fused to difficult-to-express proteins. To date, all current fusion tags are natural proteins with well-folded structures. Here, I describe the design of DEEP1 (De novo Expression Enhancer Protein 1), the first highly stable de novo protein that allows for increased expression and greater purification yields for three peptide targets—Trp cage, Aβ42, and LS3—which respectively represent a soluble, aggregating, and hydrophobic sequence. Using a Ni(II)-immobilized metal affinity chromatography (Ni[II]-IMAC) and high-performance liquid chromatography, we purified and quantified DEEP1 constructs and compared the yields to constructs fused to a standard expression tag, SUMO. We found that DEEP1 outperforms SUMO in all three constructs, leading us to hypothesize that DEEP1 is a versatile tag for the expression of proteins varying in biochemical profiles. DEEP1 was next engineered for enhanced Ni(II)-IMAC purification by replacing four consecutive residues with histidine residues. This new construct, DEEP2, shows similar stability and expression but stronger binding to the Ni(II) column than DEEP1. Taken together, these findings support the hypothesis that DEEPs can serve as adaptable expression tags for sequences that are difficult to purify and/or express. In future studies, we aim to test this pioneering technology with more target proteins and compare yields to those when tagged with SUMO. Additionally, we plan to elucidate the mechanism for DEEP1’s expression-enhancement properties.
dc.titleA “DEEP” alternative to heterologous protein expression
dc.typePrinceton University Senior Theses
pu.departmentMolecular Biology
pu.certificateGlobal Health and Health Policy Program
Appears in Collections:Molecular Biology, 1954-2022
Global Health and Health Policy Program, 2017-2022

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