HriGFP fluorescentni protein: Izraz in aplikacije

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Edited according to the article: Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen et Sezerman, U: HriGFP Novel Flourescent Protein: Expression and Applications. Molecular Biotechnology. 2020, 62: 280-288

Author: Almina Tahirović

Contents

Introduction

Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greatest threats are in fact, industrial wastes and pollutants which are constantly released into the enviroment, including the pesticides that consist of organo-phosphorous compounds or either their derivatives [1]. Those pesticided are often used to increase the food production, even though the high amounts of it can have lethal consequences when it comes to human health or food safety. Since the proper remoting of agricultural areas have been problem for decades, because the usual methods like Chromatography Mass Spectroscopy or Liquid/Gas Chromatography are costly, non-portable and require specialized laboratories, scientist have come up with the idea of using cost-effective portable biosensors which could effectively detect the methyl parathion (one of the organo-phosphorous compounds) on spot [2].

Green fluorescent protein

The green fluorescent protein (GFP) is a protein that sucessfully exhibits green fluorescence when exposed to light in the blue to ultraviolet range and GFP usually refers to the protein first isolated from the jellyfish Aequorea victoria. Also, GFP-like coral proteins exhibit a broad spectral diversity ranging from blue to red FPs, so both the stability and expression of it could be optimized by amino acids substitutions possessing different kinds of biophysical properties, therefore such modifications could ensure better rate of maturation and stability of mentioned FPs. Such modificated and more stable monomeric FPs could be used as biosensors [3].

Matherials and methods

Materials used in this study were of analytical grade - stock and arsenium solution, both obtained from Sigma. HriGFP gene was cloned into pET28a+vector (containing the T7 lac promoter, induced using isopropyl β-d-1-thiogalactopyranoside) and sucessfully transformed into E. coli BL21 (DE3) cells. In order to sucessfully transform it in Bacillus megaterium, the gene had to be sub-cloned into the shuttle vector pHIS1522. Kanamycin was added as well, with the role of the resistant marker and the plates were incubated at 37 °C overnight. Structure prediction of the HriGFP was done by homology modeling and entire statistical analysis was performed with Graph-pad Prism. Lastly, all the biosensing experiments were done in total of three biological replicates [1].

Results

Research team sucessfully transformed and expressed the HriGFP in E. coli and Bacillus megaterium which was presented by fluorescent microscopy. The entire HriGFP structure was modeled with the help of MODELLER - it contained 7 beta sheets, resembling to beta barrel which is specific for GFP molecules. Research team also analyzed the HriGFP interaction with the metal ions where strong interaction was observed with divalent iones, such as Mg and some slightly less strong interactions with iones like Zn and Mn. It was also observed that exposure of HriGFP to the monovalent iones did not show any change in fuorescence intensity and at final, it was concluded that strongest fuorescence quenching was observed in the case of copper as opposed to mercury and arsenic. Alltoghether, the biosensing ability of the HriGFP producing cells was tested and signifcant increase in the fuorescence intensity was observed with the corresponding decrease in the concentration of methyl parathion, which is considered to be detected from 500 mg to 0.05 mg/L (1.89 mM- 189 μM) concentration [1].

Conclusion

HriGFP could be used efciently as biosensor for the detection of heavy metals and harmful organo-phosphorous compounds like methyl-parahtion.

Literature

[1] Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen, Z., Sezerman, U. HriGFP Novel Flourescent Protein: Expression and Applications. Molecular Biotechnology. 2020;62: 280-288.

[2] Gong, J., Wang, L., Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase immobilized onto Au–polypyrrole interlaced network-like nano-composite. Biosensors and Bioelectronics. 2009;24: 2285–2288.

[3] Mérola, F., Erard, M., Fredj, A., Pasquier, H. Engineering fuorescent proteins towards ultimate performances: lessons from the newly developed cyan variants. Methods and Applications in Fluorescence. 2016;4: 1200.

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