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Accelerating membrane protein engineering and biocatalysis through phage display and vortex fluidics

  • Author(s): Meneghini, Luz Marina
  • Advisor(s): Weiss, Gregory
  • et al.
Abstract

Protein engineering has significantly contributed to the development of protein-based therapeutics, manufacturing of greener synthetic products, and has also expanded our knowledge of protein structure and function. Despite its achievements, designing proteins that exhibit a desired property or activity still remains a challenge. In particular, the inherent hydrophobicity of membrane proteins and their low expression yields makes site-directed mutagenesis for structure-function studies a difficult ordeal. Often expression tags are used to increase solubility, but liters of expression media are required to characterize a single variant. Another challenge in protein engineering is the enhancement of enzymatic activity of the native protein. This often requires directed evolution approach and a significant investment of both time and resources. The work here addresses to solve these bottlenecks by two innovative approaches: phage display of membrane proteins and a vortex fluidic device.

First, phage display and site-directed mutagenesis were used to expedite the protein charac- terization of the TonB dependent transporter (TBDT) ShuA from Shigella dysenteriae, and elucidate the mechanism of heme-uptake. Using site-directed mutagenesis, twenty-five ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable membrane protein variants for rapid characterization of binding to various ligands. The studies targeting each extracellular loop region of ShuA demonstrate that no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. This approach is generalizable to the dissection of other phage-displayed TBDTs and membrane proteins.

In another project, a vortex fluidic device was used to enhance enzymatic activity, for the first time without the need of directed evolution methods. Such device operates by spinning a 20 mm NMR tube at high rotational speeds to generates a thin film of solution. Within the thin film, the contents are subjected to high levels of shear stress, mass transfer, and vibrational energy input, which could accelerate a broad range of chemical reactions and protein folding. For our study, the technique was applied to four enzymes: deoxyribose-5-phosphate aldolase (DERA), alkaline phosphatase, esterase, and β-glucosidase. A systematic study of rotational speeds (i.e., frequency), processing time, and substrate concentration showed that rotational speeds are the most important control parameter for enzyme activity enhancement. Also, the activity enhancement presents narrow peaks at multiple frequencies. On average the enzymes displayed a seven-fold enzymatic activity enhancement, with DERA achieving a 15-fold acceleration. This study provides further evidence of the importance that mechanical forces can have on enzyme function, and sets the basis for investigating the possible use of vortex fluidic devices to accelerate protein–engineering studies.

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