Laboratory Report: Expression and Purification of His6-tagged Recombinant GFP from E. coli using Ni2+ Affinity Chromatography

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Green Fluorescent Protein (GFP) is a powerful tool in molecular biology, allowing the visualization of gene expression and protein localization in living organisms. This experiment aimed to express and purify a His6-tagged recombinant form of GFP (rGFP) from E. coli using Ni2+ -agarose affinity chromatography. The GFP used in this study was UV-optimized and expressed in the E. coli strain BL21 (DE3)<pLysS><pRSETA-GFPuv>. The His6 tag facilitated purification through Ni2+ affinity chromatography.

GFP's unique fluorescence originates from the chromophore, consisting of modified amino acid residues protected within an 11-stranded Beta-barrel.

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This experiment focused on expressing rGFP with an additional N-terminal His6/Xpress epitope tag, allowing for efficient purification.

Growth and Expression of rGFP in E. coli:

The BL21(DE3)<pLysS><pRSETA-GFPuv> strain was induced with IPTG, and samples (G0, G3, G3-15ml) were collected at different time points. The GFPuv plasmid contained an Ampicillin-resistant gene, ensuring plasmid stability and preventing contamination.

Cell lysis was achieved by slow freezing and quick thawing of the bacterial pellet (G3-15ml). The resulting crude extract (GCE) was obtained by centrifugation.

Ni+2 Agarose Column Purification:

A Ni+2 agarose column was used for purification. Wash fractions (W1-W10) and elution fractions (E1-E10) were collected. The qualitative fluorescence of fractions was observed using a hand-held UV light.

Determining Protein Amount with Bradford Assay:

The Bradford Assay was employed to determine protein concentrations in W1-W6 and E1-E6. A standard curve with known BSA concentrations was used for quantification.

SDS-PAGE with Coomassie blue staining estimated purity and molecular weight. Gel electrophoresis was performed with samples (G0, G3, GCE, E2, E3, W3, W4) and a molecular weight ladder.

Western blot analysis confirmed the presence of rGFP in G3, GCE, E2, E3, W3, and W4 fractions using anti-Xpress epitope antibodies.

GFP Expression and Ni2+ Agarose Chromatography:

G3 sample post-IPTG induction showed successful GFP expression. Ni2+ agarose chromatography resulted in elution fractions (E2 and E3) with the highest fluorescence and protein content.

SDS-PAGE revealed a distinct band for rGFP at approximately 37 kDa, consistent with the estimated molecular weight. Purity estimation for E3 was around 55%, and the yield of rGFP was calculated as 47.2 μg.

Western blot analysis confirmed the presence of rGFP in fractions G3, GCE, E2, E3, W3, and W4, reinforcing the successful expression and purification.

The experiment successfully expressed and purified His6-tagged rGFP using Ni2+ affinity chromatography. The estimated molecular weight closely matched the SDS-PAGE results. Although the purity in E3 was slightly lower than expected, the western blot confirmed the presence of rGFP in the desired fractions.

rGFP can be employed in various biological studies, such as gene expression monitoring and protein localization. Further experiments could focus on modifying the chromophore to enable multicolor fluorescence, expanding the utility of GFP in simultaneous tagging of different cellular processes.

GFP's ability to fluoresce without exogenous substrates makes it a valuable tool for monitoring gene expression and protein localization in living organisms. The potential applications of GFP in cancer cell tracking and other biological studies highlight its significant impact on scientific research.

This laboratory experiment provides valuable insights into the expression and purification of rGFP, contributing to the broader understanding and application of GFP in molecular biology.

Formula 1: Molecular Weight Estimation
MWestimated=33.5kDa
MWSDS−PAGE=37kDa

Growth and Expression of rGFP in E. coli:

Formula 2: Cell Growth
OD600≈0.5

Preparing GCE:

Cell lysis was achieved by slow freezing and quick thawing of the bacterial pellet (G3-15ml). The resulting crude extract (GCE) was obtained by centrifugation.

Ni+2 Agarose Column Purification:

Formula 3: Specific Activity

SpecificActivity=mgRFU
GFP Expression and Ni2+ Agarose Chromatography:

G3 sample post-IPTG induction showed successful GFP expression. Ni2+ agarose chromatography resulted in elution fractions (E2 and E3) with the highest fluorescence and protein content.