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Development of Rapid and Scalable Method for Production of Recombinant Protein through a
Transient Expression System
Yves Saint D. Hall, Melissa R. Menie, Kevin Y. Wang
Northeastern State University of Broken Arrow, Oklahoma
Human therapeutic proteins have been produced in transgenic plants to fight various diseases.
Conventional method is time–intensive, and relatively low-yielding. In this study, a rapidly expressing, highyield transient expression system was developed, producing scalable recombinant protein within 2-4 days.
Agrobacterium-mediated vacuum-infiltration of lettuce was used to efficiently introduce gene-harboring
Agrobacterium into the majority of leaf tissue. Infiltration was demonstrated by visual observation of GFP
fluorescence in vivo under long-wavelength UV. Two protein extraction methods were tested: blenderextraction method and N2-extraction method. Both extracts were subjected to a stepwise ammonium sulfate
precipitation for purification.
The N2-extraction method is traditionally used when processing leaf tissue. In this study, N2-extractions
exhibited minimal fluorescence. In comparison, blender method extracts maintained fluorescence, gaining
intensity in the final steps, and was less time-consuming, requiring no use of specialized equipment.
(NH4)2SO4 provided enough purification of GFP for visibility under UV light. These steps allowed for timely,
and cost-efficient expression, purification, and identification of recombinant protein without denaturation.
Extracts were analyzed using native SDS-PAGE, allowing direct detection of fluorescence of GFP bands
in the gel. After detection, the gel was stained to further confirm the presence of GFP. Presence of target
protein was verified, confirming viability of these methods as an efficient production-purification paradigm.
This study is supported by NIH grants R03NS095246 and P20RR016478.