Keywords: affinity chromatography; Basic principles; Type; App application
China Library Classification Number: Q8 19
Document ID: a
Article number:1007-7847 (2006) 01-0012-06.
Affinity chromatography is a kind of liquid chromatography which uses the affinity adsorption medium of coupled affinity ligands as the stationary phase to adsorb the target product, thus separating and purifying the target product. In recent decades, affinity chromatography technology has developed very rapidly and made remarkable achievements in the separation and purification of biotechnology products, biomolecules and tissues. Now it has been widely used to separate and purify egg white, peptides, enzymes and their substrates and inhibitors, antibodies and antigens, nucleic acids and their specific effectors, hormones and receptors, cells and cell surfaces. Based on this, this paper introduces the basic types of affinity chromatography, the selection of ligands and the application of affinity chromatography in biology, especially protein omics.
1 Types of affinity chromatography
Immobilized ligand in affinity chromatography column is the key factor to determine the success of affinity chromatography. According to the different interaction systems between ligands and biomacromolecules, affinity chromatography can be divided into the following four types.
1. 1 biological affinity chromatography (BAFC)
Bio-affinity chromatography is an affinity chromatography, which uses pairs of bio-specific interacting substances existing in nature. Usually with high selectivity. Typical substance pairs include enzyme-substrate, enzyme-inhibitor, hormone-receptor and so on.
1.2 immunoaffinity chromatography (1AFC)
Immunoaffinity chromatography is a separation system, which uses one of antigen and antibody as ligand and affinity adsorbs the other. Immunoaffinity chromatography is widely used. Many typical affinity chromatography methods use monoclonal antibodies as affinity ligands. At present, monoclonal antibodies against each target protein can be obtained by using antibody-antigen model, and then the target protein can be separated and purified by affinity chromatography with monoclonal antibodies as ligands. This method has a high recovery rate of purification multiple activities. As antibody binding proteins, protein A and protein C have been applied to immunoaffinity chromatography, and they are good ligands for the analysis of human immunoglobulin, especially IgG antibodies. Various types of immunodetection of immunochromatographic column are called immunodetection method, which is especially suitable for detecting samples with low content. Immunoassay uses labeled antibodies or simulated analytes for indirect analysis. Labels such as enzyme labels, fluorescent labels and chemiluminescent labels are commonly used.
1.3 metal ion affinity chromatography (IMAC)
Affinity chromatography of metal ions is a separation system that utilizes the complex of metal ions or the ability to form an integrated body to adsorb protein. The amino acid residues exposed on the surface of protein, such as imidazole group of histidine, sulfhydryl group of cysteine and indole group of tryptophan, are very beneficial to the combination of protein and immobilized metal ions, and are the basis for the separation and purification of protein by IMAC. It has been found that metal ions, such as zinc and copper, can bind imidazole groups of histidine and sulfhydryl groups of cysteine well. Protein containing different amounts of these groups can be separated by metal ion affinity chromatography.
1.4 bionic affinity chromatography (bionic)
Biomimetic affinity chromatography uses the interaction between some molecules to simulate the structure of biomolecules or specific parts. Affinity chromatography using synthetic ligands as stationary phase to adsorb protein, such as dye affinity chromatography (DAFC) and amino acid affinity chromatography (including polypeptide affinity chromatography (AALA)). Dye ligands, such as triazine or triphenylmethane compounds, can be firmly bound to the matrix and carrier through valence bonds. Dye ligands interact with many active sites of protein and enzymes to mimic the substrates, cofactors or binders of these biomolecules.
Design of Affinity Chromatographic Ligands and Screening of Ligands by Combination Method
Affinity chromatography is a very effective separation technology for large-scale purification of biotechnology products, which has been widely used in protein omics and other fields. Although it has great potential, the application of affinity chromatography is limited by whether suitable ligands can be obtained for each target. In recent years, the combination of molecular model design and combinatorial chemistry has become an innovative method to produce new and specific ligands needed by biotechnology.
2. Design of1Affinity Chromatographic Ligands
Design ligands according to protein structure: The strategy of designing ligands reasonably includes obtaining structural information about the target protein from a suitable database and determining possible binding sites on protein. The target site can be an active site, an area exposed to a solvent or a site involved in the binding of the same neutral complementary ligand. Two important factors need to be considered when designing ligands according to protein structure: one is the ability to correctly predict the designed ligand image; Second, the ability to correctly predict the affinity of ligands. Some successful methods for designing ligands using proteins have been reported. For example, anthraquinones designed for L- lactate dehydrogenase mimic biological dye ligands.
Design ligands according to protein function: When the three-dimensional structure of target protein is unknown, ligands can be designed according to the function of protein, and some structural characteristics of ligands are considered. Summarized as follows:
1) meets certain requirements. For example, linear negatively charged molecular heparin is a conventional method to purify DNA-binding enzymes. In addition, the ligand triazine dye CibacronBlue 3GAca used to purify nucleotide binding enzymes has several structures, ions and hydrophobic groups needed to form appropriate interactions with nucleotide binding sites of some enzymes.
2) Ligands have specific functional groups. For example, benzamidine is used as the ligand of trypsin, and hydroxyl amino acid is used as the ligand of metal binding protein.
3) Structural patterns derived from the combination of two or more known structural motifs (such as substrates, inhibitors, effectors or cofactors). For example, a ketone-carboxyl biomimetic chimeric dye-ligand is designed as a ligand for purifying an enzyme that recognizes (ketone) carboxyl groups.
2.2 Combination method for screening ligands
Using combinatorial chemistry to establish a polypeptide library, and then using the polypeptide library to screen polypeptide ligands that specifically bind to the target protein has greatly promoted the development of affinity chromatography screening and ligand synthesis technology. In the past decade, there have been methods to generate as many as 10 different protein, peptide chains or nucleic acid libraries. Such a large library makes it possible to select molecules with high affinity and specificity. When looking for ligands, two strategies can be applied. Firstly, the targets that can bind to biomolecules are screened from various types of large libraries, and then a design method is introduced to reduce the size of the targeted library. This method mainly depends on what information we have at the beginning. If there is information about the structure of the analyte, it is possible to design ligands. However, without this information, combinatorial method may be the only way to screen ligands. Binding molecules obtained from the first generation library can be used as matrix scaffolds to generate other new libraries, from which binding ligands with higher activity can be screened. The application of combinatorial methods for screening affinity ligands from libraries of (poly) peptides, polynucleotides and substituted triazine compounds has been reported.
2.2, 1 synthetic peptide library
A synthetic peptide library is a collection of random peptide molecules containing all possible sequences of a given peptide chain length. Chemical synthesis of peptide molecules by solid-phase peptide synthesis. The synthesized peptide can be broken from the soluble peptide library or remain on the resin of the peptide library with one structure in the bead. A basic method for screening combinatorial chemical libraries in vitro is to let the mixture in the library flow through a channel on which the protein to be detected has been immobilized. Ligands that have affinity for protein molecules immobilized on the surface are peptides that are resisted in their channels, and they are usually good candidates for affinity chromatography ligands. In this way, peptide ligands with F-L-L-V-P-L sequence have been synthesized and used to purify human fibrinogen.
2.2.2 Phage display
Phage display technology has matured rapidly and developed into a tool for affinity chromatography to find high affinity ligands. This technique is based on the rapid identification of highly selective ligands in a screening process called biological elutriation using phage display libraries. Peptides displayed by phage can be selected by affinity method in two ways. The library can be directly cultured with the immobilized target, or pre-cultured with the target before being immobilized on a solid support, just like affinity chromatography, and the interacting peptides or protein are eluted specifically or non-specifically. Bacterial infection can amplify these interacting phages and increase their copy number. This screening/amplification process can be repeated many times to obtain phage display peptides with high affinity. By sequencing the isolated phage DNA sequence, the desired sequence can be obtained. Phage display library has been successfully applied to epitope localization, vaccine development, identification of protein kinase substrates, peptide simulation of bioactive peptides and non-peptide ligands, and is very suitable as a source of affinity ligands for chromatographic analysis. The immunoglobulin binding domain of protein A has been displayed on the surface of phage, which makes it possible to screen the affinity chromatography conditions of protein A variants with improved specificity or mild elution conditions. Gaskin et al. described the use of a 7- amino acid peptide library displayed on the surface of filamentous phage M 13 as a possible source of affinity ligands for Rhizomucormiehei lipase.
2.2.3 Ribosome display and affinity selection by SELEX
Ribosome display is especially suitable for scanning and screening folded egg whites. The principle is summarized as follows: First, a DNA library is transcribed into mRNA and translated in vitro. Due to the lack of terminator, mRNA and peptide are prevented from being released from ribosomes, and stable ternary complexes are formed under appropriate conditions. The complex is then exposed to the target molecules immobilized on the surface. If the peptide binds to the target with sufficient affinity, the complex remains, the peptide with low affinity is washed away, the attached ribosome ternary complex can be dissociated by EDTA, and the mRNA can be reverse transcribed into cDNA and amplified by PCR. SELEX is widely used to screen nucleic acid ligands, and in vitro screening has been used to determine the target nucleic acid ligands. These targets cover a wide range of volumes, including simple ions, peptide chains and so on.
In the past decades, the role of synthetic chemical ligands in protein chromatography is very real. Ion-exchange, hydrophobic and metal chelating adsorbents together constitute an ultra-stable and non-toxic synthetic functional body fixed on the matrix skeleton, which has affinity with charged, non-polar or metal binding groups on the surface of protein. For example, combinatorial chemistry technology has begun to have a great influence on the discovery and design of new fusion peptide metal ligands.
2.3 ab initio design of ligands
Because the knowledge of X-ray crystallography, NMR or homologous structure can be combined with limited or combinatorial chemical synthesis and advanced computer technology, it is more feasible, effective, reasonable and fast to design affinity ligands reasonably. This method relies on the structural information of the target protein, guides the synthesis of ligands purposefully, and limits the synthesis efforts to the direction that has been evaluated by experiments and can be combined with the detected target or expected to be combined with the target. The method includes the following steps: selecting an appropriate site on the target protein, designing a ligand compatible with the three-dimensional structure of the site, combining it into a solid-phase binding ligand library related to the structure, and scanning the target protein in the library.
According to the X-ray crystal structure of the complex composed of B domain of protein A and Fc fragment of lgG, the non-peptide bionic ligand of IgG was established. Using computer-aided molecular model, a series of bionic molecules were designed around protein A dipeptide Phel32-Tyrl33. One such ligand can bind to human IgG, which has 3- aminophenol and 4- amino-1- naphthol moieties on the triazine ring. When this biomimetic ligand is immobilized on agarose, it has been successfully used to purify human plasma IgG. A similar method for designing bionic ligands for recombinant insulin precursor M 13 1 and recombinant human coagulation factor vlar 35 1 was also established.
Application of affinity chromatography
3. Application of1affinity chromatography in protein omics.
Two-dimensional electrophoresis and mass spectrometry are the most commonly used experimental techniques in protein omics. However, affinity chromatography is also an important separation method. Affinity chromatography was mainly used for selective preconcentration and pretreatment of samples before protein was separated by 2-DE. It should include removing one or a class of protein that will interfere with 2-DE resolution. A typical example is the removal of albumin and immunoglobulin IgG from serum and cerebrospinal fluid samples by affinity resin adsorption, which can significantly improve the resolution of protein spots on 2-DE gel. In addition, affinity chromatography can concentrate protein with low abundance so that they can be seen on two-dimensional gel. All protein can also be divided into two or more categories for further analysis.
In many cases, those samples that have not been separated by 2-DE are determined by mass spectrometry, and affinity chromatography can be used to separate them before determination to reduce the complexity of the samples. For non-2-DE-based research, affinity chromatography can be used to separate protein and peptide chains before or after protein hydrolysis and digestion. For example, lectin affinity chromatography has been used to separate and extract a special kind of glycoprotein after protein hydrolysis and digestion, and to concentrate glycopeptides. This method is particularly useful for studying post-transcriptional modified protein. Affinity chromatography is also used to study the site of post-transcriptional modification. Affinity chromatography is used to analyze glycoproteins, which can be recognized by lectins even after protein hydrolysis. Affinity chromatography using antibodies and some IMAC can be used to separate and concentrate glycopeptides.
Understanding how cells function as a system depends on determining the complex network of cell protein interactions. Protein spectrum identification of protein complex after affinity purification has become a powerful tool to generate protein-protein interaction map and complex localization map in fine cells. All affinity purification methods for identifying protein complexes are carried out by labeling protein at the gene level or at the level of extracting protein from cells. Protein affinity chromatography may be superior to yeast two-hybrid method, because it produces fewer false positive results, and it is more suitable for Qualcomm quantity (a large number of samples to be detected) operation. Affinity chromatography can purify protein, which forms a complex in cells, and can purify target protein together with those related protein from, for example, cell lysate. Recombinant DNA technology uses peptide fragment markers such as GST, epitope or TAP to mark "bait" protein. Large-scale methods can label all bait proteins with these commercial peptides. The protein complex containing TAP-labeled protein was purified by two successive affinity steps: lgG beads and calmodulin beads.
Although the parallel method of two-dimensional electrophoresis and tandem mass spectrometry has been able to relatively quantify protein in two-dimensional electrophoresis gel, the quantitative analysis of protein omics can be achieved by using stable isotope labeling. A class of small molecule reagents, isotope-coded affinity labels, have been used for quantitative analysis of protein omics. One method is to analyze the complex protein mixture with the help of MALDI-TOF mass spectrometer and ICAT labeling reagent.
3.2 Separation, purification and quantitative determination of target protein
In recent years, with the rapid development of biopharmaceutical industry, affinity chromatography has been widely used as an important tool for clinical drug separation, purification and quantitative determination. Affinity chromatography is most commonly used in the separation and purification of target proteins, especially vaccines, especially fusion proteins, because fusion proteins have specific binding ability. Affinity chromatography is widely used in the separation and purification of genetically engineered subunit vaccines, among which Ni-NAT and GST affinity chromatography are widely used in fusion proteins with (His)6 and GST affinity tags. Metal chelating chromatography and immunoaffinity chromatography have also been applied to some extent.
In recent years, the principle of affinity chromatography has been applied to quantitative analysis, mainly combined with immune technology to analyze antigens and antibodies, and some technologies such as radioimmunoassay, enzyme-labeled immunity and fluorescent antibody have been developed. In addition, affinity chromatography technology is also widely used to determine the binding constant with biological macromolecules and their specific substrates and quantitative analysis of complexes. Affinity chromatography has been widely used in clinic, such as quantitative analysis of glycosylated proteins. In addition, the immobilized immunoaffinity chromatography column against human transferrin was applied to the purification and identification of transferrin isomers in diluted serum.
3.3 Identify the interaction between biomolecules
Affinity chromatography is also used to study the interaction between biomolecules in biological systems. This field has been used to test the interactions between various biological systems, including lectin//sugar, enzyme/inhibitor, egg white/protein and DNA// protein. Clinically, this technique is mainly used to study the combination between drugs or hormones and serum proteins. There are two chromatographic methods that can be used to study the interaction between organisms. One is the study of regional elution, and the other is the study of frontier front analysis.
3.4 Affinity chromatography combined with other analytical techniques
In recent years, another development trend is to seek more optimized system design and arrangement in order to obtain fast, excellent selectivity and a large number of test samples. Affinity chromatography can be combined with other analytical techniques to achieve this goal. At present, there have been many successful applications, such as affinity chromatography-mass spectrometry (AFC-MS), AFC -HPLC, AFC-HPLC-MS, AFC-CE (capillary electrophoresis) and so on. This combination will continue to develop because of its irreplaceable advantages.
4 conclusion
The challenge of finding and producing protein for treating diseases makes it necessary to reconsider the design and purification process. This choice is mainly determined by the introduction speed, effectiveness, stability and economic affordability of this method. The high selectivity and maturity strategy based on affinity chromatography is replacing the traditional purification procedure. This technology provides a reasonable design for purification, and stimulates and explores the natural biological process of molecular recognition to selectively purify the target protein. Affinity chromatography may be the only separation technique that focuses on the key issues of Qualcomm quantity and protein omics. The main problem is to design new techniques to determine the goal of high selectivity affinity ligand binding hypothesis. Designing ligands with high selectivity and stability by rational and combinatorial methods will have a great impact on the future application of affinity chromatography. Acknowledgement: Special thanks to Professor Liang Songping, Professor Zhang Jian and Dr. Zeng for their valuable comments on this article! This article is the full text of the original. Users who don't have a PDF browser should download and install the original text first.