Antibodies are proteins secreted by a plasma cell in the human body. At the production stage, antibodies are usually present in complex cells, and most of their applications require purification. The isolation and purification of recombinant antibodies and their fragments are relatively simple compared to other recombinant proteins. The ideal purification method can obtain antibody products of sufficient purity in one step from the crude extract of recombinant E. coli protein. Therefore, the rational selection of purification methods is particularly important. A series of processes for protein isolation and purification are designed to isolate a single type of protein from a complex mixture. Protein Separation offers a wide range of purification systems for simple protein separation and purification.
Antibodies can identify some foreign substances in the human immune system, such as viruses, bacteria, etc. All antibodies belong to immunoglobulins, so antibodies can be said to belong to proteins, but not all proteins are antibodies. Antibodies are constantly being used in a variety of scientific research, including but not limited to basic research, chromatographic analysis, targeted analysis, imaging, diagnosis and treatment. However, most of these applications require high purity homogeneous antibodies. Thus, for the purification of antibodies from complex mixtures such as plasma, serum, ascites, cell culture media, egg yolks, plant extracts and bacterial or yeast cultures, there is an increasing demand for simple, efficient and economical purification methods. Protein separation companies often use affinity chromatography to separate and purify proteins. Medicilon is a preclinical CRO company with extensive experience in recombinant protein expression and purification services, with a variety of protein expression systems, including prokaryotic protein expression systems, yeast protein expression systems, and insect cell protein expression systems (baculovirus). The mammalian cell protein expression system, with a variety of fusion technologies, provides customers with a wide range of options for protein expression and purification.
Purification of recombinant antibody and fragments thereof can generally be accomplished in several ways:
1. Bacterial affinity chromatography.
Affinity chromatography is a common method for purifying recombinant proteins. It utilizes the specificity of the protein structure, can bind to the corresponding specific molecule, and can be dissociated under specific conditions, and can obtain high-purity protein in one step. For example, the specific enzymes, receptors, antibodies, and the like can be purified by utilizing specific binding of an enzyme to a substrate, binding of a receptor to a ligand, and binding of an antibody to an antigen. It is also possible to add a tag such as His, Flag, GST, HA, c-Myc, GFP, SUMO, MBP, Avi, etc., when the recombinant protein is expressed.
Some bacteria can synthesize proteins that specifically recognize and bind to higher mammalian immunoglobulins, such as proteins A, B, G, and L. The binding sites are mainly located in the constant region of the antibody molecule, and only a few are present in the variable region. Therefore, this method is more suitable for isolating recombinant antibody intact molecules as well as monovalent and bivalent FAB fragments, but is ineffective for the isolation of FV or scFV fragments.
2. Antibody affinity chromatography
Antibody fragments expressed as fusion proteins can often be separated by selection of a suitable antibody affinity chromatography column depending on the characteristics of the target protein. Currently used target proteins such as alkaline phosphomonoesterase, peroxidase and some toxin proteins have corresponding commercial antibody affinity maturation services media, but if E. coli itself can also synthesize this target protein or target protein The homologous protein, the specificity of this method will be affected. To overcome this difficulty, the Flag and Myc TAG tag sequences in the pIG vector can also serve as target sequences for anti-Flag antibodies and anti-Myc TAG antibodies, and fusion proteins containing these sequences can be separated by corresponding antibody affinity chromatography columns. However, the above antibodies are expensive and generally only used on a laboratory scale.
3. Antigen affinity chromatography
If a specific antigen corresponding to a recombinant antibody or antibody fragment is readily available, separation of the expression product using this antigen affinity chromatography column is the best choice because it is not only highly selective but also can be obtained from any non- Rapid separation of the target antibody or antibody fragment in a properly folded protein mixture. During hapten affinity chromatography, the isolated product typically requires elution with a soluble hapten under very mild conditions. However, for some harmful antigens (such as tumor antigens, etc.), it is generally inappropriate to use this method to separate antibody fragments for use in vivo.
4, ligand affinity layer column method
Phosphocholine affinity chromatography columns, which were used to isolate intact antibody molecules, can also be used directly to purify recombinant FAB, FV, scFV fragments and various bivalent minibodies from crude E. coli protein extracts, all with the proviso that all The recombinant antibody fragment must have a good folded structure. In addition, the His tag tag sequence mounted on some pIG vectors was designed specifically for the ligand affinity chromatography purification process for expression products. The histidine residue in the polypeptide chain can be combined with a plurality of divalent heavy metal ions, and the protein in which the histidine residue is concentrated in the primary sequence can theoretically be separated by a divalent heavy metal ion affinity chromatography column. The good separation effect depends to a large extent on the combination of metal ions and eluent. For example, the Zn2+ column is usually eluted with a diacetic acid imide solution, while the Ni2+ column is eluted with an imidazole or triacetonitrile solution. Because of the low cost of heavy metal ion affinity chromatography media, this method is more suitable for large-scale production of recombinant antibody fragments.
Affinity chromatography as a method for purification of recombinant protein, according to its uniqueness, easy operation, relatively high yield and throughput, make it the most efficient and widely used chromatographic technology. A general technique for antibody purification.
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