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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 &#.......

Representative Results

BL21 (DE3) cell lysates expressing hFEN1 were passed through equilibrated Ni-NTA and TALON resins. The Ni-NTA resin is charged with Ni2+ ions and has a high binding capacity. The results show that the Ni-NTA resin yields a higher quantity of FEN1 compared to the TALON resin (Figure 7). The Ni-NTA resin is also known to non-specifically bind to other chromosomally expressed proteins. Cell lysate passed through the cobalt-based resin, was purified with high purity but a lower yield,.......

Discussion

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 .......

Acknowledgements

This work was funded by grants from the National Science Foundation (1929346) and the American Cancer Society (RSG-21-028-01). We would also like to thank members of the Balakrishnan laboratory for helpful discussions.

....

Materials

NameCompanyCatalog NumberComments
4x Laemmli sample bufferBioRad1610747
Acetic acidMerckUN2789
Beta-mercaptoethanol (BME)SigmaM-6250
Chromlab software version 6.1.27.0BioRadoperates the NGC system
Complete MINI protease inhibitor tabletRoche11836153001
Coomassie Brilliant Blue RSigmaB0149
Dithiothreitol (DTT)Dot ScientificDSD11000
Econo-Column glassBioRad7371512
Ethylene diamine tetraacetic acid (EDTA)Dot ScientificDSE57020
Flow adaptorBioRad7380014
GlycerolDot ScientificDSG22020
ImidazoleDot ScientificDSI52000
MethanolFisher ScientificA412
Mini PROTEAN TGX gelsBioRad4561084
NGC Chromatography SystemBioRadautomated liquid chromatography system 
Ni-NTA AgaroseQiagen1018244
Phenyl-methyl-sulfonyl fluorideDot ScientificDSP20270
PreScission Plus Protein Dual Color Standards BioRad1610374
Sodium chlorideDot ScientificDSS23020
TALON metal affinity resinTakara635502
Tris BaseDST60040

References

  1. Balakrishnan, L., Bambara, R. A. Flap endonuclease 1. Annu Rev Biochem. 82, 119-138 (2013).
  2. Asagoshi, K. FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells.

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6X His tagged ProteinAffinity PurificationFast Protein Liquid Chromatography FPLCFlap Endonuclease 1 FEN1Protein ExpressionProtein PurificationProtein StabilityProtein FunctionalitySample PreparationColumn PackingFlow RateElution ParametersChromatogram AnalysisRecombinant Protein Storage

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