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The Introduction of Fab Fragment Antibody

What is Fab fragment antibody?

With the rapid development of DNA recombination technology and the further understanding of antibodies, the third generation of antibodies, recombinant antibodies, following polyclonal antibodies and monoclonal antibodies, has emerged. Small molecule antibodies in recombinant antibodies have become the main members and research hotspots of the recombinant family because of their characteristics.The main small molecular antibodies are Fab fragment antibody, scFv fragment antibody, single domain antibody, bispecific antibody, double chain antibody, tribody and minibody.



Figure1.Fab fragment

Fab fragments, also known as antigen-binding fragments, are regions in the structure of antibodies that can bind to antigens. Fab fragment consists of a complete VH and CH1 domains of light and heavy chains. Both light chain and heavy chain have a constant region and a variable region, and there are disulfide bond links between light chain and heavy chain. Like monoclonal antibodies, the development of Fab antibodies has gone through four stages: murine, chimeric, humanized and fully human monoclonal antibody.

Structure of Fab

Natural polyclonal antibodies and monoclonal antibodies are immunoglobulin molecules. IgG molecule consists of two identical 5×104 Dolton heavy chains and two identical 2.5×104 Dolton light chains. Fab molecule is composed of light chain and heavy chain, about 5×104 Dolton. The interaction between the two chains and the disulfide bond formed play a stable role in the double-chain structure of Fab molecule.The antigen binding sites of Fab and IgG are composed of six complementarity determination regions (CDR), which are located on light chain and heavy chain respectively, and form the core of antigen binding sites. Among the three-dimensional structure of the variable region, two opposite parallel beta-folded peptide chains form the framework region. The outer side is connected by highly variable CDR rings, forming a sandwich structure. The inner side is connected by conservative disulfide bonds, which makes the CDR stand out.

Fab fragments are obtained mainly by chemical, enzymatic and genetic engineering methods. Chemical method is to use chemical reagents to act on the N-terminal of disulfide bond between heavy chains, open the disulfide bond, and obtain two identical antigen binding fragments. It is also a common method to digest full-length IgG antibodies with papain, pepsin and fig protease.Fab fragment antibodies of functional antibodies obtained by genetic engineering are expressed mainly through E. coli system, Pichia pastoris system and insect system, the most common of which is E. coli system. However, the low expression of Fab fragment antibody in E. coli system is the main factor restricting its large-scale production, so we will learn about the optimization measures of E. coli production today.

1. Optimization of host bacteria

Because the structural stability of Fab fragments is determined by the disulfide bond between the heavy chain Fd segment and the complete light chain, and the formation of disulfide bond requires oxidative environment, it is difficult to obtain functional antibody fragments in the cytoplasm of reductive environment. It is necessary to guide light chain and heavy chain Fd segment to the periplasmic cavity by signal peptide for correct folding. Recently, the cavity environment is particularly important.In addition, modification of strains, co-expression of catalysts and formation of oxidative environment in cytoplasm are also alternative measures. The difference of expression hosts has a significant effect on the total expression of Fab, mainly due to some proteases in host bacteria, especially periplasmase or outer membrane protein-related genes. The protease of host itself can degrade foreign proteins, and the deletion of outer membrane protein-related genes can release more Fab fragments into the culture medium, which is more conducive to purification and has practical application value in production.

2. Selection of expression vectors

When selecting expression vectors, we should start from promoter, replicator, selective label and fusion protein expression sequence, and select the appropriate combination of expression elements to express Fab fragment.

Selection of replicators

The choice of replicators depends on the number of copies. In general, it can be considered that plasmids with high copy number mean higher protein expression. However, a large number of plasmids exert more metabolic pressure on E. coli, which in turn reduces the growth and reproduction rate of E. coli, and consequently reduces the expression of the target protein. Therefore, high copy number does not represent high protein expression.

Promoter selection

In protein expression, a strong transcription promoter is needed to control the high level of expression, while avoiding the basic expression as far as possible.

3. Vector design (target gene sequence)

The target gene sequence is the key factor affecting the expression level. The position order of light chain and heavy chain is different, and the expression level varies greatly. The classical vector design method is based on the same promoter and the ribosome binding sites of the light and heavy chains. The N-terminal of the heavy and light chains are added with their respective signal peptide sequences to guide the heavy and light chains into the periplasmic cavity for correct folding.

4. Choice of molecular chaperones

Because of the role of strong promoter, the cell resources of host bacteria are used to express the target protein. The level of target protein expression is often high. As the molecular chaperone of host bacteria cannot meet the correct folding of the peptide chain, inclusion bodies will be formed. Introducing exogenous molecular chaperones to assist with the correct folding of target proteins is a way to increase the production of active proteins and increase the production of active proteins.

5. Optimization of culture conditions

The secretion position of Fab antibody produced by Escherichia coli is closely related to the culture conditions. Specific culture conditions can make Fab molecules more secreted and expressed in the supernatant, possibly because the accumulation of Fab molecules in the periplasmic cavity causes the breakdown of the bacteria, or because the culture medium or culture temperature causes the permeability of the outer membrane of E. coli to increase.

6. Selection of culture medium

In the production of Fab fragment antibody, the composition of culture medium has an important influence on soluble expression. Buffer solution rich in metal ions and salt ions is more conducive to Fab expression, especially soluble expression. Metal ions can help fold enzymes form disulfide bonds and increase the expression of soluble proteins.

7. Purification of Fab antibody

In addition to constructing better vectors to increase the expression of Fab antibody, improving the purification process to enhance the purification efficiency can also improve the production of Fab.

Compared with full-length antibody, Fab antibody has attracted more and more attention. It has been widely used in the treatment of tumors, immune diseases and ophthalmic diseases. As a tracer diagnostic agent, it also has a good effect.
publié le samedi 17 août à 13:26

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