Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

Cold atmospheric plasma treatment enhances recombinant model protein production in yeast Pichia pastoris.

Cold atmospheric pressure plasma (CAP) has been described as a novel technology with expanding applications in biomedicine and biotechnology. In the present study, we provide a mildly stressful condition using non-lethal doses of CAP (120, 180, and 240 s) and evaluate its potential benefits on the recombinant production of a model protein (enhanced green fluorescent protein (eGFP)) in yeast Pichia pastoris. The measured eGFP fluorescence augmented proportional to CAP exposure time. After 240 s treatment with CAP, the measured fluorescent intensity of culture supernatant (after 72 h) and results of real-time PCR (after 24 h) indicated an 84% and 76% increase in activity and related RNA concentration, respectively. Real-time analysis of a list of genes involved in oxidative stress response revealed a significant and durable improvement in their expression at five h and 24 h following CAP exposure. The improvement of the recombinant model protein production may be partly explained by the impact of the RONS on cellular constituents and altering the expression of specific stress genes. In conclusion, using CAP strategy may be considered a valuable strategy to improve recombinant protein production, and deciphering the molecular background mechanism could be inspiring in the reverse metabolic engineering of host cells.

Functionalized Nanostructured Bioactive Carriers: Nanoliposomes, Quantum Dots, Tocosome, and Theranostic Approach.

BACKGROUND: Lipid nanocarriers have great potential for the encapsulation and delivery of numerous bioactive compounds. They have demonstrated significant benefits over traditional disease management and conventional therapy. The benefits associated with the particular properties of lipid nanocarriers include site-specific drug deposition, improved pharmacokinetics and pharmacodynamics, enhanced internalization and intracellular transport, biodegradability, and decreased biodistribution. These properties result in the alleviation of the harmful consequences of conventional treatment protocols. MATERIALS & METHODS: The administration of various bioactive molecules has been extensively investigated using nanostructured lipid carriers. In this article, theranostic applications of novel formulations of lipid nanocarriers combined or complexed with quantum dots, certain polymers, such as chitosan, and metallic nanoparticles (particularly gold) are reviewed. These formulations have demonstrated better controlled release features, improved drug loading capability, as well as a lower burst release rate. As a recent innovation in drug delivery, tocosomes and their unique advantages are also explained in the final section of this review. RESULTS AND CONCLUSION: Theranostic medicine requires nanocarriers with improved target-specific accumulation and bio-distribution. To this end, lipid-based nanocarrier systems and tocosomes combined with unique properties of quantum dots, biocompatible polymers, and metallic nanoparticles seem to be ideal candidates to be considered for safe and efficient drug delivery.