Evaluation of GFP-based system for pre-crystallisation study of EgKUP membrane proteins

Mohd. Naqiuddin, . and Meilina Ong Abdullah, . Evaluation of GFP-based system for pre-crystallisation study of EgKUP membrane proteins. pp. 42-52. ISSN 1511-2780

Elucidation of atomic structures of membrane proteins (MP) is of great importance for comprehensive understanding of their functions and mechanism of actions. Determination of high resolution protein structures by X-ray crystallography requires crystallisation of a protein of interest. Obtaining wellordered protein crystals however is not an easy task and many factors can contribute to unsuccessful crystallisation. The situation will get much trickier when dealing with MP owing to the fact that they are not soluble in their innate state after extraction from the lipid bilayer environment. Unless appropriate detergents are used to stabilise proteins by mimicking their native environment MP tend to unravel thus becoming dysfunctional. This study evaluates the effectiveness of using Green Fluorescence Protein (GFP)-based system as C-terminal tag in Saccharomyces cerevisiae for pre-crystallisation study of Elaeis guineensis MP from the K Transporter/ K Uptake Permease/High Affinity K (KT/KUP/ HAK) superfamily. KT/KUP/HAK are one of the most important MP responsible for potassium uptake in plants. Development of recombinant expression system for high-level protein production is necessary as the vast majority of MP cannot be obtained in sufficient quantities from their native environment. The precrystallisation analyses were conducted on three EgKUP-GFP fusions namely EgKUP3-GFP EgKUP8- GFP and EgKUP11-GFP. The analyses involved whole-cell florescent count of each fusion protein using spectrofluorometer protein integrity assay by SDS-PAGE cellular localisation by confocal fluorescence microscopy and effects of various detergents towards EgKUPs stability and monodispersity using Fluorescence Size Exclusion Chromatography (FSEC). Results from the evaluation associated problems and proposed troubleshootings are discussed.

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Abstract

Elucidation of atomic structures of membrane proteins (MP) is of great importance for comprehensive understanding of their functions and mechanism of actions. Determination of high resolution protein structures by X-ray crystallography requires crystallisation of a protein of interest. Obtaining wellordered protein crystals however is not an easy task and many factors can contribute to unsuccessful crystallisation. The situation will get much trickier when dealing with MP owing to the fact that they are not soluble in their innate state after extraction from the lipid bilayer environment. Unless appropriate detergents are used to stabilise proteins by mimicking their native environment MP tend to unravel thus becoming dysfunctional. This study evaluates the effectiveness of using Green Fluorescence Protein (GFP)-based system as C-terminal tag in Saccharomyces cerevisiae for pre-crystallisation study of Elaeis guineensis MP from the K Transporter/ K Uptake Permease/High Affinity K (KT/KUP/ HAK) superfamily. KT/KUP/HAK are one of the most important MP responsible for potassium uptake in plants. Development of recombinant expression system for high-level protein production is necessary as the vast majority of MP cannot be obtained in sufficient quantities from their native environment. The precrystallisation analyses were conducted on three EgKUP-GFP fusions namely EgKUP3-GFP EgKUP8- GFP and EgKUP11-GFP. The analyses involved whole-cell florescent count of each fusion protein using spectrofluorometer protein integrity assay by SDS-PAGE cellular localisation by confocal fluorescence microscopy and effects of various detergents towards EgKUPs stability and monodispersity using Fluorescence Size Exclusion Chromatography (FSEC). Results from the evaluation associated problems and proposed troubleshootings are discussed.

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Item Type:	Article
AGROVOC Term:	Elaeis
AGROVOC Term:	Elaeis guineensis
AGROVOC Term:	Saccharomyces cerevisiae
AGROVOC Term:	Gene expression
AGROVOC Term:	Genes
AGROVOC Term:	Genetic vectors
AGROVOC Term:	Evaluation
AGROVOC Term:	Cloning
AGROVOC Term:	Polymerase chain reaction
AGROVOC Term:	Molecular weight
Depositing User:	Mr. AFANDI ABDUL MALEK
Last Modified:	24 Apr 2025 00:53
URI:	http://webagris.upm.edu.my/id/eprint/7798

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