Journal of Biochemical and Biophysical Methods
Band 49, Weeks 1–3,
30. October 2001
, pages 607-624
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Abstract
This Review focuses on DNA affinity chromatography, the most powerful tool for purifying DNA-binding proteins. The purification of various DNA-binding proteins using non-specific, sequence-specific, and single-stranded DNA affinity columns is discussed. Purification strategies for transcription factors, restriction enzymes, telomerase, DNA and RNA polymerases, and DNA-binding antibodies are described. Various applications of DNA affinity chromatography are described.
introduce
Isolation of proteins from other cell components is important for cloning, characterization, and structural studies. Several chromatographic techniques have been developed for the separation of proteins, e.g. B. Size exclusion, ion exchange, reversed phase and affinity chromatography that separate proteins according to their size, charge, polarity and specificity. These techniques can be used in one or more combinations to produce a homogeneous protein.
Affinity chromatography is a type of liquid chromatography that uses specific chemical interactions between two substances to separate them. These interactions include the binding of an enzyme to an inhibitor or an antibody to an antigen. Base-pairing of nucleotide bases between two complementary polynucleotide strands also represents a very powerful tool for sequence-specific isolation of polynucleotides. The affinity of polynucleotide-binding proteins for DNA or RNA allows for separation based on their ability to bind polynucleotide sequences. Because affinity chromatography utilizes highly specific interactions between molecules, it is highly selective and provides high yield and purity.
This Review focuses on DNA affinity chromatography, which uses immobilized DNA to purify DNA-binding proteins. Aspects of the procedure, such as the nature of the DNA affinity chromatography column and its use in the purification of polymerases, telomerases, restriction enzymes, anti-DNA antibodies, and binding proteins from quadruplexes, Z-DNA, and aptamers, will be closely related to some discussion. Since the literature on DNA affinity chromatography for transcription factors has been adequately reviewed [1], [2], [3], [4], it will not be discussed in detail here. However, STAT proteins are an exception as they introduce a new form of DNA affinity chromatography.
partial extract
DNA affinity chromatography
Several cellular processes such as chromatin organization, gene regulation, cell cycle and differentiation are controlled by interactions between DNA and proteins such as histones, transcription factors and DNA polymerases. Hence, the purification and characterization of these proteins is of crucial importance. Because of its high selectivity, DNA affinity chromatography is often used as one, and sometimes as the only, step in the purification of DNA-binding proteins.
Purification of DNA-binding proteins
A large number of DNA-binding proteins such as helicases [16], gyrases [17] and histones [18] have also been purified using DNA affinity chromatography as one of the steps, but their DNA showed no significant differences in other uses. This section discusses the purification of DNA-binding protein moieties such as transcription factors, restriction enzymes, polymerases, and DNA antibodies.
Affinity columns made with alternative forms of nucleic acids
In addition to the three different forms of DNA affinity columns discussed in the previous sections, other forms of DNA such as Z-DNA, four-stranded DNA, etc. are sometimes used for protein purification. Sometimes RNA is also used as a ligand for protein purification. Ligands that mimic DNA, such as Cibacron and Procion dyes or heparin, have also been used to purify DNA-binding proteins. In this section, we briefly discuss these alternative affinity methods.
nucleic acid purification
In addition to purifying DNA-binding proteins, DNA affinity columns can be used to purify nucleic acids such as RNA and DNA. Poly-T columns are commonly used for purification of eukaryotic mRNA [3]. Most eukaryotic mRNAs have a 3' poly-A tail that easily attaches to poly-T on the column. After washing the column to remove unattached RNA, DNA and protein, the mRNA retained on the column can be eluted by increasing the temperature and reducing the salt
finally
DNA affinity chromatography is an efficient method for purifying DNA polymerases, pre-initiated RNA polymerase complexes, restriction enzymes, and many other DNA-binding proteins. Although it has been used for more than 30 years, new applications are developed every year and the field has produced some important innovations such as aptamers [62], catalytic chromatography [31] and HPLC adaptation [81]. This technique is the most powerful known chromatographic method for the separation of polynucleotides and their
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Design of artificial synthetic promoters for accurate, intelligent, and general gene expression in plants
2023, Factory Newsletter
With the development of high-throughput biotechnology and artificial intelligence, it is becoming increasingly possible to design and construct artificial biological components, modules, circuits and even entire systems. To overcome the limitations of natural promoters in controlling gene expression, artificial promoter design aims to synthesize short, inducible, and conditionally controlled promoters to coordinate the expression of multiple genes in different plant metabolic and signaling pathways. Synthetic promoters are versatile and can precisely control gene expression through intelligent responses. They show the potential to improve desirable traits in crops, thereby increasing crop yield, nutritional quality and food security. This Review first illustrates the importance of synthetic promoters, then presents promoter structures and comprehensively summarizes the advances in synthetic promoter design. The limitations of developing synthetic promoters and future applications of such promoters in synthetic plant biology and crop improvement are also discussed.
Chromatographic Techniques: Types, Principles, and Applications
2022, Analytical Technology for Life Sciences: From Basic Research to Application
Chromatography is considered to be one of the most important bioanalytical techniques used in various fields of life sciences and chemistry. It allows qualitative and quantitative separation, identification and purification of compounds of different origin, class and properties from complex mixtures. Isolation of compounds of desired shape, size, charge, and entity can be based on their ability to specifically bind. The nature of its ubiquitous use in separation science makes it one of the most versatile techniques, with many variants being used effectively for both laboratory and industrial scale separation purposes. In this chapter, different chromatographic techniques are briefly discussed in terms of bed shape, different phases, separation mechanism, principle, steps and applications. The focus is also on special techniques that add a new dimension to chromatography.
A novel porous polymer microsphere for the selective adsorption and separation of conalbumin
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Protein-based biotechnology has attracted great interest [1] and the use of efficient methods to isolate specific proteins from biological matrices is an important perspective for further research. Many methods are available to detect specific proteins from biological samples, such as B. liquid-liquid extraction [2,3], affinity chromatography [4,5], precipitation [6,7] and solid phase extraction (SPE) [8,9]. . Among these strategies, SPE has been extensively studied and widely used, as evidenced by a large number of custom materials for SPE processing during protein isolation and purification [10–12].
Prepared porous polymer microspheres, poly(styrene-divinylbenzene, PSDVB)-poly(ethylene glycol monoallyl ether, PEGMAE), called PSDVB-PEGMAE.happenInterfacial double emulsion polymerization strategy. PSDVB-PEGMAE microspheres have an average diameter of 2.98 μm and a heterogeneous porous structure with a pore volume of 0.354 cm3G−1The pore size is 34.3 nm. The PEGMAE portion resides on the outer surface of the microspheres, while the PSDVB and PEGMAE portions reside in the inner pores. PSDVB-PEGMAE microspheres show good selectivity for conalbumin (ConA) adsorption via hydrogen bonding and hydrophobic interactions.happenSurface adsorption and interpore adsorption. At pH 6 the adsorption capacity is 171.9 mg−1ConA was obtained. Captured ConA can be easily recovered by stripping with cetyltrimethylammonium bromide (CTAB) solution (0.1%, w/v). The microspheres were further used to isolate ConA from egg white, which resulted in highly pure ConA by SDS-PAGE assay.
Mass spectrometry-based identification of proteins interacting with nucleic acids
2013, Journal of Proteomics
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Multimerized oligonucleotides can be used to reduce the ends-to-binding site ratio and take advantage of short non-specific DNA competitors [34,35]. Although multimerization of DNA sequences has been widely used since the early days of DNA affinity purification [21], it has some putative disadvantages [33,36]. In fact, this can introduce new DNA binding sites, increasing the likelihood of false positive candidates during the detection step.
Identifying regulatory proteins that control DNA transcription and RNA stability and translation is a critical step in understanding the regulation of gene expression. These proteins can be purified by DNA or RNA affinity chromatography and then identified by mass spectrometry. While conceptually very simple, this poses a real technical challenge because regulatory proteins are less abundant than proteins that bind nucleic acids in large numbers in a non-sequence-specific manner. Here we review various strategies that have been developed to achieve this goal and discuss important parameters that should be considered to increase the chances of success. In general, two classes of biological problems can be distinguished: the identification of proteins that specifically interact with well-defined binding sites, which are mainly treated by quantitative mass spectrometry, and the identification of long regulatory regions composed of nucleic acids that are not comparatively good characterized. Finally, in addition to a large amount of research is devotedin vitro-Aggregated nucleic acid-protein complexes, few data on proteome analysisDirect- Describes complex complexes with particular emphasis on the challenges involved.
Proteomic analysis methods of transcription factors
2009, Acta Chromatography A
Studying the transcription factor (TF) proteome presents challenges, including the incorporation of a large number of low-abundance and post-translationally modified proteins. Specific purification and analysis methods have been developed over the last decades to facilitate the study of the TF proteome and are discussed here. In addition, generally applicable proteomic approaches that have already been used successfully are discussed. TFs are selectively purified by affinity techniques using DNA response elements (REs) as the basis for highly specific binding. Several reagents have been identified that can increase binding or decrease non-specific binding. Such an affinity method, called “capture”, allows the purification of TF bound at nM concentrations and the recovery of the TF complex in a highly purified state. The Electrophoretic Mobility Shift Assay (EMSA) is the most important TF assay as it provides a measure of the affinity and amount of TF present. Southwest (SW) blotting and DNA-protein crosslinking (DPC) make this possiblein vitroEstimate DNA-bound protein levels while chromatin immunoprecipitation (ChIP) confirms promoter bindingDirect.Two-dimensional gel electrophoresis (2-DE) and 3-DE methods combining EMSA with 2-DE can further resolve TF. The synergy between highly selective purification and analysis strategies has led to an explosion in knowledge about the proteome of TF and other DNA and RNA-binding proteins.
Trapping of transcription factors with symmetrical DNA using thiol–disulfide exchange chemistry
2008, Acta Chromatography A
Green fluorescent protein (GFP) fused to the C-terminal 100 amino acids of the CAAT enhancer binding protein (C/EBP), which also contains an N-terminal (His).6The tag (GFP-C/EBP) was used as a transcription factor model to test whether a thiol-disulfide exchange reaction can be used to purify transcription factors. Construction of symmetrical dithiol oligonucleotides with dual CAAT elements using 5' and 3' thiols. After reduction, circular dichroism confirms its spontaneous attachment to its internal complementary sequence to form a hairpin structure: in solution at nM concentrations, specific GFP-C/EBP protein-DNA complexes are formed, which are then recovered by thiols—disulfide (captured) Exchange with disulfidethiopropylagarose and elution with dithiothreitol. GFP-C/EBP was isolated from crude bacterial extracts treated with iodoacetamide; DNA binding of GFP-C/EBP was not altered by carboxamide methylation. The eluted GFP-C/EBP was of high purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After in-gel digestion with trypsin, the protein was also characterized by capillary reversed-phase liquid chromatography-nanoelectrospray ionization tandem mass spectrometry and the results analyzed using MASCOT software to search non-redundant protein databases. A score of 1874 and 51% sequence coverage (including terminal and internal sequences consistent with GFP-C/EBP) confirmed its identity and sequence. This method offers great potential for the identification and characterization of transcription factors and other DNA-binding proteins.
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Copyright © 2001 Elsevier Science B.V. All rights reserved.
FAQs
How do you purify DNA binding proteins? ›
Cleaning. Remove ionically bound proteins by washing with 0.5 CV of 2 M NaCl for 10 to 15 min. Remove precipitated or denatured proteins by washing with 4 CV of 100 mM NaOH for 1 to 2 h; or 2 CV of 6 M guanidine hydrochloride for 30 to 60 min; or 2 CV of 8 M urea for 30 to 60 min.
What is affinity purification of protein interaction? ›Affinity purification involves the separation of molecules in solution (mobile phase) based on differences in binding interaction with a ligand that is immobilized to a stationary material (solid phase). A support or matrix in affinity purification is any material to which a biospecific ligand is covalently attached.
What is the purpose of affinity purification? ›Contaminant removal. In some cases, the goal of affinity purification is to remove a particular class of undesirable sample components rather than to purify one target molecule.
Which method is used for DNA purification? ›DNA purification from detergents, proteins, salts, and reagents is used during the cell lysis step. The most commonly used procedures are: Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation.
What are the methods of DNA purification? ›DNA extraction techniques include organic extraction (phenol–chloroform method), nonorganic method (salting out and proteinase K treatment), and adsorption method (silica–gel membrane).
What is meant by affinity of protein? ›The binding of two proteins can be viewed as a reversible and rapid process in an equilibrium that is governed by the law of mass action. The binding affinity is the strength of the interaction between two (or more than two) molecules that bind reversibly (interact).
What happens in protein purification? ›The proteins are first separated by their size using SDS-PAGE (sodium dodecyl sulfate gel electrophoresis). They are then transferred to a porous membrane and blocked to prevent any non-specific binding of antibodies. After blocking, the membrane is incubated with a primary antibody specific to the protein of interest.
What is DNA affinity purification assay? ›We describe a DNA affinity purification method that allows for identification and analysis of protein complex components. For example, a DNA probe carrying a transcription factor binding site is used to purify proteins from a nuclear extract. The proteins binding to the probe are then identified by mass spectrometry.
What is affinity chromatography and how does it work? ›Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions.
What are the 4 steps of protein purification? ›There are four basic steps of protein purification: 1) cell lysis, 2) protein binding to a matrix, 3) washing and 4) elution.
What are the four methods of protein purification? ›
The four methods of protein purification are: (1) Extraction (2) Precipitation and Differential Solubilisation (3) Ultracentrifugation and (4)Chromatographic Methods. The methods used in protein purification, can roughly be divided into analytical and preparative methods.
What are the advantages of protein A affinity chromatography? ›Protein A/G purification has the advantage of simplicity compared to other affinity chromatography. A and G proteins are naturally occurring and therefore don't require engineering like the tagged version of affinity chromatography. They also have high purity and yield.
What does affinity chromatography remove? ›The process is often used to purify biomolecules such as enzymes, antibodies, and recombinant proteins. It can also help to remove harmful substances such as pathogens through the same principles.
What does affinity chromatography separate proteins based on? ›Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance.
How do you purify RNA binding proteins? ›In this approach, RNAs are immobilized on beads and incubated with protein lysates. After removing unbound fraction of proteins, the bound proteins are eluted by successive increasing of salt concentration. The lower the salt concentration, the lower is the binding affinity of a protein to the RNA.
How do you purify protein ion exchange? ›Ion-exchange chromatography is one of the most common procedures for protein purification. It relies on the charge-charge interactions between the proteins in the lysate and the charges immobilized on the ion-exchange resins. The resins are of two types: cation and anion-exchange.
How do you purify proteins? ›There are four basic steps of protein purification: 1) cell lysis, 2) protein binding to a matrix, 3) washing and 4) elution.
How do you purify protein from inclusion bodies? ›The conventional strategy to purify proteins from inclusion bodies consists of four major steps: isolation of purified inclusion bodies, solubilization of inclusion bodies, refolding of solubilized proteins and purification of refolded proteins by various chromatographic techniques [55].