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Isolation of polystyrene-protein corona complex from bovine serum using size exclusion chromatography and expression of Daphnia magna’s proteins using EDDIE fusion technology

Abbas, Ramy LU (2022) KEMR20 20221
Department of Chemistry
Computational Chemistry
Abstract
The comprehensive use of plastic globally has raised many questions about how it will affect living organisms once it is disposed of and reaches the ecosystem. Disposing of plastic in high amounts without recycling could lead to several toxic effects for aquatic organisms and eventually humans. Accumulation of plastic for a long time leads to its aggregation and/or degradation into small sizes in the micro and nano range due to several mechanical forces and ultraviolet (UV) radiation. It is of vital importance to understand how plastic nanoparticles will affect the environment and to evaluate their potential adverse effects on living organisms at various levels of organization. By using differential light scattering (DLS) and nano tracking... (More)
The comprehensive use of plastic globally has raised many questions about how it will affect living organisms once it is disposed of and reaches the ecosystem. Disposing of plastic in high amounts without recycling could lead to several toxic effects for aquatic organisms and eventually humans. Accumulation of plastic for a long time leads to its aggregation and/or degradation into small sizes in the micro and nano range due to several mechanical forces and ultraviolet (UV) radiation. It is of vital importance to understand how plastic nanoparticles will affect the environment and to evaluate their potential adverse effects on living organisms at various levels of organization. By using differential light scattering (DLS) and nano tracking analysis (NTA) for size measurements, we discovered that UV exposure causes aggregation and chemical surface changes in aminated polystyrene nanoparticles (PS-NPs) in less than 7 hours. In the biological fluid, proteins associate and get adsorbed on the NPs’ surface, leading to the formation of a complex known as protein corona (PC) and conferring NPs’ biocompatibility. Nanoparticles aggregation leads to different proteins composition on their surfaces depending on the aggregation level and aggregate size. Hence, new biological effects could take place in terms of protein interaction and cellular binding. Moreover, the distribution and accumulation of aggregated NPs in various body organs may result in unknown effects. In terms of PC composition analysis, isolating it from the bulk is the first step of the analysis, where centrifugation is the most commonly used technique. In our study, we used size exclusion chromatography (SEC) to successfully separate PC complex from bovine serum after pre-coating aminated PS-NPs with bovine serum albumin (BSA), which aids in preventing aggregation during separation. When compared to centrifugation, which can cause free protein sedimentation and impair the separation process, SEC is a superior method of separation as it helps in determining individual protein affinity for the NPs and estimation of protein exchange rate on the NPs' surface. Furthermore, we have attempted to express three Daphnia magna’s proteins using EDDIE fusion technology by utilizing BL21 STARTM cells as a bacterial host. The peptidases serine protease, chymotrypsin elastase, and carboxypeptidase B were selected to be expressed and intended to be used in pre-coating aminated UV-treated PS-NPs in order to determine whether pre-coating is instrumental in reducing NPs' toxicity upon being filtered with Daphnia.
Interestingly, serine protease and chymotrypsin elastase were not expressed using these cells, and the exponential cell growth was inhibited, suggesting cell toxicity. On the other hand, carboxypeptidase B was overexpressed, but EDDIE auto-cleavage activity was inhibited, resulting in not releasing the target protein. Other explanations could be that the auto-cleavage occurred successfully but the target protein was aggregated or rebounded with EDDIE, preventing its release. (Less)
Popular Abstract
Modern-day civilization is associated with the extensive use of plastic. Over the last decade, a staggering quantity and diversity of plastic materials have been produced. However, there is no proper balance between production and recycling of plastic. As a result, a substantial amount of plastic is accumulating in nature, posing a serious problem that must be addressed. Aside from the primary source of micro-plastics produced by factories during production, accumulated plastic can also be fragmented into extremely small sizes that are invisible to the naked eye. This can occur as a result of various environmental forces such as wind and waves, as well as ultraviolet radiation, resulting in what is called nanoplastics. Little is known... (More)
Modern-day civilization is associated with the extensive use of plastic. Over the last decade, a staggering quantity and diversity of plastic materials have been produced. However, there is no proper balance between production and recycling of plastic. As a result, a substantial amount of plastic is accumulating in nature, posing a serious problem that must be addressed. Aside from the primary source of micro-plastics produced by factories during production, accumulated plastic can also be fragmented into extremely small sizes that are invisible to the naked eye. This can occur as a result of various environmental forces such as wind and waves, as well as ultraviolet radiation, resulting in what is called nanoplastics. Little is known about how the living system will cope with and react to nanoparticles. Several studies have shown that nanoparticles can enter organisms via a variety of routes, causing accumulation and toxic effects. Polystyrene (PS) is one of the most commonly used material in the plastic industry. It is used in the production of several products, such as styrofoam, toys, CDs, and cup lids. In our study, we used amine-modified polystyrene nanoparticles as a raw model to see what could happen upon their exposure to ultraviolet (UV) radiation. We discovered that further reductions in particle size and/or aggregation occur in less than seven hours. One can imagine how long these particles have been exposed to UV radiation in nature. Once the nanoparticles reach any biological fluid such as blood, they will be decorated with various proteins attracted to their surfaces, forming a layer known as "protein corona" (PC), making them easily recognized by the cells. The deposition of these aggregated nanoparticles in the cells can lead to different undesirable effects that have to be taken into consideration. The first step in understanding nanoparticles' effects in the living system is to separate these PC complexes and identify the proteins surrounding them. In our study, we used a new practice for separation based on size. The principle of this technique depends solely on the fact that protein-nanoparticles complexes have a larger size than free proteins, so they will be separated first. However, to be able to use this method of separation, one has to stabilize the nanoparticles to prevent their aggregation, which can impair the separation process by getting stuck in the column used. After successful separation of the complex from the biological fluid, protein separation from the nanoparticles and identification is a routine procedure in order to understand the interaction between the proteins and the nanoparticles. This will help to evaluate the toxicity danger and find the suitable measures needed to cope with it. (Less)
Please use this url to cite or link to this publication:
author
Abbas, Ramy LU
supervisor
organization
course
KEMR20 20221
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Polystyrene nanoparticles (PS-NPs), Aggregation, Daphnia magnia, EDDIE, Protein corona (PC), Theoretical chemistry
language
English
id
9095046
date added to LUP
2022-07-01 14:10:12
date last changed
2022-07-01 14:10:12
@misc{9095046,
  abstract     = {{The comprehensive use of plastic globally has raised many questions about how it will affect living organisms once it is disposed of and reaches the ecosystem. Disposing of plastic in high amounts without recycling could lead to several toxic effects for aquatic organisms and eventually humans. Accumulation of plastic for a long time leads to its aggregation and/or degradation into small sizes in the micro and nano range due to several mechanical forces and ultraviolet (UV) radiation. It is of vital importance to understand how plastic nanoparticles will affect the environment and to evaluate their potential adverse effects on living organisms at various levels of organization. By using differential light scattering (DLS) and nano tracking analysis (NTA) for size measurements, we discovered that UV exposure causes aggregation and chemical surface changes in aminated polystyrene nanoparticles (PS-NPs) in less than 7 hours. In the biological fluid, proteins associate and get adsorbed on the NPs’ surface, leading to the formation of a complex known as protein corona (PC) and conferring NPs’ biocompatibility. Nanoparticles aggregation leads to different proteins composition on their surfaces depending on the aggregation level and aggregate size. Hence, new biological effects could take place in terms of protein interaction and cellular binding. Moreover, the distribution and accumulation of aggregated NPs in various body organs may result in unknown effects. In terms of PC composition analysis, isolating it from the bulk is the first step of the analysis, where centrifugation is the most commonly used technique. In our study, we used size exclusion chromatography (SEC) to successfully separate PC complex from bovine serum after pre-coating aminated PS-NPs with bovine serum albumin (BSA), which aids in preventing aggregation during separation. When compared to centrifugation, which can cause free protein sedimentation and impair the separation process, SEC is a superior method of separation as it helps in determining individual protein affinity for the NPs and estimation of protein exchange rate on the NPs' surface. Furthermore, we have attempted to express three Daphnia magna’s proteins using EDDIE fusion technology by utilizing BL21 STARTM cells as a bacterial host. The peptidases serine protease, chymotrypsin elastase, and carboxypeptidase B were selected to be expressed and intended to be used in pre-coating aminated UV-treated PS-NPs in order to determine whether pre-coating is instrumental in reducing NPs' toxicity upon being filtered with Daphnia.
Interestingly, serine protease and chymotrypsin elastase were not expressed using these cells, and the exponential cell growth was inhibited, suggesting cell toxicity. On the other hand, carboxypeptidase B was overexpressed, but EDDIE auto-cleavage activity was inhibited, resulting in not releasing the target protein. Other explanations could be that the auto-cleavage occurred successfully but the target protein was aggregated or rebounded with EDDIE, preventing its release.}},
  author       = {{Abbas, Ramy}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Isolation of polystyrene-protein corona complex from bovine serum using size exclusion chromatography and expression of Daphnia magna’s proteins using EDDIE fusion technology}},
  year         = {{2022}},
}