expression-isolation-purification-soluble-insoluble-biotinylated

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

University of Akron

There are currently no acceptable cures for central nervous system injuries, and damage induced large gaps in the peripheral nervous system have been challenging to bridge to restore neural functionality. Innervation by neurons is made possible by the growth cone. This dynamic structure is unique to neurons, and can directly sense physical and chemical activity in its environment, utilizing these cues to propel axons to precisely reach their targets. Guidance can occur through chemoattractive factors such as neurotrophins and netrins, chemorepulsive agents like semaphorins and slits, or contact-mediated molecules such as ephrins and those located in the extracellular matrix. The understanding of biomolecular activity during nervous system development and injury has generated new techniques and tactics for improving and restoring function to the nervous system after injury. This review will focus on the major neuronal guidance molecules and their utility in current tissue engineering and neural regenerative strategies.

Neural regenerative strategies incorporating biomolecular axon guidance signals.

In this study we report the successful synthesis of N-(2-mercaptoethyl)-3-(3-methyl-3H-diazirine-3-yl) propanamide (N-MCEP-diazirine), with sulfhydryl and amine photoreactive ends to allow recombinant protein tethering to chitosan films. This regimen allows mimicry of the physiological endeavor of axon pathfinding in the nervous system where neurons rely on cues for guidance during development and regeneration. Our strategy incorporates strong covalent and noncovalent interactions, utilizing N-MCEP-diazirine, maleimide-streptavidin complex, and two custom biotinylated-fusion proteins, nerve growth factor (bNGF), and semaphorin3A (bSema3A). Synthetic yield of N-MCEP-diazirine was 87.3 ± 1.9%. Characteristic absorbance decrease at 348 nm after N-MCEP-diazirine exposure to UV validated the photochemical properties of the diazirine moiety, and the attachment of cross-linker to chitosan films was verified with Fourier transform infrared spectroscopy (FTIR). Fluorescence techniques showed no significant difference in the detection of immobilized proteins compared to absorbing the proteins to films (p < 0.05); however, in vitro outgrowth of dorsal root ganglia (DRG) was more responsive to immobilized bNGF and bSema3A compared to adsorbed bNGF and bSema3A over a 5 day period. Immobilized bNGF significantly increased DRG length over time (p < 0.0001), but adsorbed bNGF did not increase in axon extension from day 1 to day 5 (p = 0.4476). Immobilized bSema3A showed a significant decrease in neurite length (524.42 ± 57.31 μm) at day 5 compared to adsorbed bSema3A (969.13 ± 57.31 μm). These results demonstrate the superiority of our immobilization approach to protein adsorption because biotinylated-fusion proteins maintain their active confirmation and their tethering can be spatially controlled via a UV activated N-MCEP-diazirine cross-linker.

Specific immobilization of biotinylated fusion proteins NGF and Sema3A utilizing a photo-cross-linkable diazirine compound for controlling neurite extension.

Aleesha M. McCormick

Department of Chemical and Biomolecular Engineering

University of Akron

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Aleesha M. McCormick

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Biomolecular Engineering

University of Akron

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Department of Chemical and Biomolecular Engineering