Effect of Non-Thermal Atmospheric Pressure Plasma on the Expression of Green Fluorescent Protein in Pichia pastoris

Background and Aim: Non-Thermal Atmospheric Pressure Plasma (NTAPP) is an environmentally friendly technology that can be used for modifying protein activity and gene expression. Green fluorescent protein (GFP) is a reporter protein with an easily-detectable function. The aim of the present study was to study the effect of AC sinusoidal Atmospheric Pressure Plasma Jet on the expression and activity of GFP, as the model protein.Methods: Recombinant Pichia Pastoris expressing GFP was cultured in the YPG medium. After reaching OD۶۰۰ ~ ۲۰, the expression of GFP was induced by the addition of ۰.۵% methanol which was continued for more ۴ days. The culture media were treated with ۶۰ and ۲۴۰ sec of Helium plasma jet ignited with ۴kV, ۲۵ kHz source at the beginning of methanol feeding. The untreated culture and recombinant Pichia Pastoris expressing Endoglucanase were used as the positive and negative controls, respectively. The gap distance between the NTAPP nozzle and the culture surface was constant for all experiments (۱ cm). Optical emission spectroscopy was used for monitoring the free radicals including Reactive Oxygen/Nitrogen Species produced by NTAPP and plasma gas temperature measurement. Optical density was measured by spectrophotometer. The fluorescence intensity was quantified using the spectrofluorimeter with ۴۷۰ nm excitation and ۵۲۰ nm emission.Results: The obtained results indicated that the NTAPP treatment of recombinant Pichia Pastoris in the selected duration has no significant effects on the yeast cell mass, however, leads a significant reduction in fluorescent emission (~۵۰%).Conclusion: In conclusion, ۶۰ and ۲۶۰-sec treatment with NTAPP has no effect on the yeast cell viability but the expression of recombinant GFP reduced significantly. It is suggested that this NTAPP can be used for inactivation of some unwanted proteins in industrial application. Finding an optimum plasma treatment time can lead us to controllable gene expression.

کلیدواژه ها:

Non-Thermal Atmospheric Pressure Plasma ، Green Fluorescent Protein ، Gene expression