affinity column preparation and packing for fast protein liquid chromatography (fplc)

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

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 of a biological pathway necessitates interdisciplinary investigations.
With DNA being the genetic blueprint of life, understanding the mechanisms of DNA duplication and genome maintenance has been an area of active interest for over seven decades. Studies in the field of DNA replication have yielded copious data concerning the individual structures and functions of numerous replication proteins. These inquiries, which encompass mechanistic aspects and biochemical activity assays, have been made feasible through the purification of these proteins, enabling their meticulous examination in an in vitro milieu. Consequently, protein purification emerges as an indispensable and ubiquitous technique in the majority of research endeavors geared toward unraveling mechanistic insights into DNA replication.
This article presents a methodology for isolating a DNA replication protein tagged with 6X-histidine, which has been overexpressed in bacterial cells. The protein of interest is human flap endonuclease 1 (FEN1), a structure-specific nuclease that plays a pivotal role in lagging strand replication and is also a critical participant in DNA repair pathways like base excision repair (BER)1,2,3. FEN1&#39;s primary function is to cleave at the base of a 5&#39;-displaced flap structure, an intermediate that arises during DNA replication or BER. Initially, biochemical investigations assessing the enzymatic activity of FEN1 suggested a &#34;tracking&#34; mechanism, wherein the nuclease would recognize the free 5&#39;-phosphate end of a flap structure and then follow along the flap to its base before cleaving it4. Subsequent research revealed that FEN1 operates via a &#34;threading&#34; mechanism, wherein it first binds to the base of the flap and then threads the free 5&#39; end through its active site prior to cleavage (Figure 1)5. The ability to overexpress and isolate recombinant FEN1 has facilitated these breakthroughs, enabling researchers to employ it in biochemical and structural investigations.
Affinity chromatography is a commonly used separation method to purify DNA. This technique uses the reversible binding affinity of target proteins towards ligands immobilized on a resin to specifically trap the protein of interest. One of the most widely used bio-affinities is the robust interaction between the amino acid, histidine, and metal ions such as nickel and cobalt and hence, can be captured onto a resin charged with Ni2+ or Co2+.
The DNA sequence encoding a string of 6-9 histidine residues (His) is frequently incorporated into the plasmid construct which encodes the protein of interest (at either the N-terminus or C-terminus), tagging the protein with a 6X-His-tag or a poly-His tag. The His-tagged protein can then be easily purified by immobilized metal affinity chromatography (IMAC), a subtype of affinity chromatography whereby metal ions on the resin capture proteins with an affinity tag, which can later be eluted using appropriate elution agents. Transition metal ions such as Ni2+ and Co2+ can be immobilized onto agarose or silica gel matrices derived from N,N,N&#39;-tris-(carboxymethyl)-ethylenediamine or nitrilotriacetic acid (NTA) groups6.
Metal ligands are known to be robust against degradation by physical, chemical, and biological factors and hold this advantage over other types of ligands6,7. Additionally, the His-tag is a relatively small tag and does not significantly impact protein structure or function7. However, in a bacterial expression system, many chromosomally expressed proteins have an affinity towards metal ions and may co-purify with the target protein. Nickel and cobalt are the typical metal ions used in IMAC matrices. The Ni-NTA resin and the TALON cobalt-based resin are commonly used for the purification of His-tagged proteins.
Ni-NTA versus TALON 
The respective metal ions of both Ni-NTA and TALON are immobilized on the resin through NTA ligands. Ni-NTA is thought to have a higher binding capacity, binding up to 100 mg/mL of protein. This can result in a higher protein yield, with the caveat that contaminant proteins may be co-purified. In contrast, the resin has a higher binding specificity towards His-tagged proteins and may be able to produce fractions of higher purity. In this study, we aim to compare the purification efficiency of both resins using the referenced automated fast protein liquid chromatography system, the NGC system (see the Table of Materials).
Buffers and compatibility 
Buffers are required during protein purification for cell lysis, sample preparation, resin equilibration, and elution of the captured protein from the resin. Tris, MOPS, and HEPES buffers up to a concentration of 100 mM are the known compatible buffers for the Ni-NTA resin. Buffers often include reducing agents to prevent the oxidation of protein and protein aggregation. However, above a threshold limit, reducing agents could strip the resin of metal ions. The recommended concentration of reducing agents such as beta-mercaptoethanol (BME) or dithiothreitol (DTT) is below 1 mM for the above-mentioned nickel- and cobalt-based resins.
The buffers for the purification of hFEN1 are Tris buffers containing NaCl, BME, phenylmethylsulfonyl fluoride (PMSF), EDTA, and glycerol. NaCl maintains the protein in soluble form and disrupts molecular interactions such as DNA binding. BME reduces oxidized proteins and thereby prevents protein aggregation. PMSF) is a protease inhibitor that prevents protease-mediated degradation of target protein. EDTA eliminates divalent cations from the sample, preventing their access to nucleases and proteases. Glycerol enhances the stability of the protein in aqueous form. Additionally, the lysis buffer contains complete protease inhibitor tablets to ensure maximum protection of target protein from degrading proteases during cell lysis. The equilibration and elution buffers contain imidazole, with the elution buffer containing higher quantities for the imidazole to displace the bound protein from the resin during elution.
Next Generation Chromatography (NGC) system 
This automated, medium-pressure chromatography system designed for fast-flow protein liquid chromatography (FPLC) uses two pumps to simultaneously pump two different buffers and is capable of injecting a wide range of sample volumes from 250 &#181;L to 100 mL. The sample loop (known as the Dynaloop in this system), makes it possible to inject larger sample volumes. The system can be operated using the Chromlab software, which facilitates customized method creation, manipulation of purification runs, and analysis of UV peaks and protein fractions.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

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

In this study, we aim to purify flap endonuclease 1 or FEN1, a crucial DNA replication protein using affinity chromatography. This will allow us to investigate FEN1's function in DNA replication and repair in a controlled in vitro environment, advancing our understanding of its functions and biological roles. Various types of chromatography are used to purify DNA binding proteins, the most common being affinity chromatography.Within affinity chromatography, immobilized metal affinity chromatography is one of the strongest tools to purify His-tagged proteins. Since the interaction between the His-tagged and the metal ion, usually nickel or cobalt is very strong. Two crucial factors for successful protein purification process are protein yield and protein purity.Depending on the protein's intended use, either yield or purity might be preferred over the other. Our findings show that selecting the appropriate resin for the purification of His-tagged proteins might yield results desirable for the protein's downstream applications.

Affinity Column Preparation and Packing for Fast Protein Liquid Chromatography (FPLC)

To begin, fit the columns to a clamp stand as straight as possible. Plug the bottom of each column with a stopper. Add one to two milliliters of starting buffer to the bottom of each column to prevent air bubbles during column packing.Then open the top of the columns and pour in 10 milliliters of each slurry continuously against a metal spatula or glass rod held at the rim of the column to prevent air bubbles. Remove the stopper from the column and connect the column's bottom to one end of the tubing. Attach the other end of the tubing to the UV port of the FPLC system.Verify that the bed support housing is pulled into the flow adapter body and slide the flow adapter body onto the top of the column. With the cam latch in position one, lower the flow adapter. Then switch the cam latch to position two.Connect the tubing to the system and start the buffer flow for one to two minutes to eliminate air from the tubing and prevent air bubbles during packing. Afterward, switch the latch back to position one, lower the flow adapter smoothly into the buffer, avoiding air bubbles, and switch the latch back to position two. After switching the latch from position two to three, tighten both the cam base and the locking ring.Then, start the buffer flow at 10 milliliters per minute for three minutes or until the resin is well packed.

Research

JoVE Journal

Biochemistry

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...