Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Podsumowanie

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Streszczenie

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Wprowadzenie

Numerous strategies are available for comprehending cellular biology. One approach involves a top-down strategy, wherein genetic mutations are introduced into a gene, followed by the evaluation of resulting phenotypic changes in a model organism. Conversely, a reductionist approach entails the initial elucidation of molecular mechanisms and enzymatic functions of a particular protein, accompanied by the characterization of its interactions with other cellular components. Subsequently, the impact of this protein on a biological pathway is assessed. Although each research approach possesses its inherent advantages and limitations, achieving a comprehensive understanding....

Protokół

1. Sample preparation

1. To purify recombinant FEN1, express the construct (pET-FCH-FEN1) in BL21(DE3) cells as previously described by Ononye et al.⁸.
   1. Inoculate LB media (Table 1) with 1% overnight culture and grow the cells at 37 °C until the OD reaches 0.6.
   2. Induce with 0.4 M isopropyl-beta-D-thiogalactoside (IPTG) and grow the culture for an additional 3 h.
   3. Harvest the cells by centrifuging the culture at 5,000 × g for 15 min at 4 °C. Store the pellet in a -80°C freezer.
   4. On the day of the purification, remove the cell pellet f....

Dyskusje

Affinity chromatography is a widely used technique to purify DNA-binding proteins. Immobilized metal affinity chromatography (IMAC) is a specific type of affinity chromatography that uses metal ions to capture the histidine residues of a peptide sequence. This is why the "6X-His tag" or "poly-His tag" is attached to the N-terminus or the C-terminus of proteins to be purified. Nickel and cobalt are the most commonly used metal ions and vary in their compatibility with reagents such as BME and DTT normally .......

Materiały

Name	Company	Catalog Number	Comments
4x Laemmli sample buffer	BioRad	1610747
Acetic acid	Merck	UN2789
Beta-mercaptoethanol (BME)	Sigma	M-6250
Chromlab software version 6.1.27.0	BioRad		operates the NGC system
Complete MINI protease inhibitor tablet	Roche	11836153001
Coomassie Brilliant Blue R	Sigma	B0149
Dithiothreitol (DTT)	Dot Scientific	DSD11000
Econo-Column glass	BioRad	7371512
Ethylene diamine tetraacetic acid (EDTA)	Dot Scientific	DSE57020
Flow adaptor	BioRad	7380014
Glycerol	Dot Scientific	DSG22020
Imidazole	Dot Scientific	DSI52000
Methanol	Fisher Scientific	A412
Mini PROTEAN TGX gels	BioRad	4561084
NGC Chromatography System	BioRad		automated liquid chromatography system
Ni-NTA Agarose	Qiagen	1018244
Phenyl-methyl-sulfonyl fluoride	Dot Scientific	DSP20270
PreScission Plus Protein Dual Color Standards	BioRad	1610374
Sodium chloride	Dot Scientific	DSS23020
TALON metal affinity resin	Takara	635502
Tris Base		DST60040