A Brief Discussion on Prokaryotic Expression of Recombinant Antibodies-Special topic on antibody modification-Tekbiotech-Yeast Display Service,Phage display technology

A Brief Discussion on Prokaryotic Expression of Recombinant Antibodies

Through recombinant DNA technology and antibody engineering techniques, antibody genes can now be successfully cloned and expressed as fragments in bacteria, mammalian cells and yeast, plants, and insect cells. One advantage of this new technology is that they can retain the intact antigen binding site (paratope) while reducing the size of the antibody molecule. Compared with the parent antibody, these minimized antibodies have several advantages in clinical practice, including better tumor penetration, faster blood clearance, shorter non-target tissue retention time, and reduced immunogenicity. This may also lead to the expression of functional antibodies and their fusion in bacteria, and also allow them to be displayed on filamentous phages. In addition, combining small antibody molecules with efficient microbial production systems can ultimately produce sufficient amounts of homogeneous proteins for diagnosis and treatment as well as structural studies.

In microorganisms such as Escherichia coli, Fab has been successfully produced in the exoplasm. However, due to the limited space in the bacterial periplasm, the polypeptide folds and aggregates improperly, and the product titer is usually <1g/L. To overcome these problems, Skerra introduced a one-step advancement technology that uses only part of the antibody molecule (Fab or Fv fragment) for expression.

I. Single-chain Antibody Structure

The Fv fragment is the smallest unit of an immunoglobulin molecule with antigen-binding activity. Antibodies in the form of scFv (single-chain fragment variable) consist of the variable regions of the heavy chain (VH) and the light chain (VL), which are linked together by a flexible peptidase that can be easily expressed in a functional form in E. coli, allowing protein engineering to improve the properties of scFv, such as increased affinity and altered specificity.

II. Obtaining Single-chain Antibodies

First, mRNA is isolated from hybridomas (or spleen, lymphocytes, and bone marrow) and then reverse transcribed into cDNA as a template for antibody gene amplification (PCR). In the construction of scFv (single-chain fragment variable), the order of the domains can be VH-linker-VL or VL-linker-VH. There are several strategies for expressing recombinant antibodies in E. coli:

(1) One method is to express single-chain antibodies directly in the cytoplasm of E. coli. Use an E. coli signal peptide. The results showed that the antibody was expressed in large quantities in the reducing environment of the bacterial cytoplasm and subsequently formed insoluble aggregates called inclusion bodies. The inclusion bodies must be regenerated in vitro.

(2) A signal peptide is used to direct the secretion of scFv (single-chain fragment variable) antibodies into the periplasmic space between the inner and outer membranes of Gram-negative bacteria. This periplasmic space has been identified as containing proteins, such as chaperones and disulfide isomerases, which aid in the proper folding of the recombinant protein. During the transfer across the inner membrane into the oxidizing environment of the cytoplasm, the signal peptide attached to the N-terminus is cleaved, allowing the chain to fold and assemble, resulting in the formation of intra-domain and inter-domain sulfides.

III. Detection of Single-chain Antibodies

Detection of scFv (single-chain fragment variable strain) can be accomplished using a secondary antibody that recognizes a specific tag that has been fused to the C- or N-terminus of the scFv. Currently, many tags have been used to fuse scFv, such as c-myc or E-tag (Pharmacia). Since soluble scFv (single chain fragment variable) fold improperly, they are easily inactivated when coated on microtiter plates, which is caused by the lack of constant domains of heavy and light chains.

IV. Application of Single Chain Antibodies

Antibodies in the form of scFv (single chain fragment variable) retain complete antigen binding ability, so it can promote a potential unique molecule, especially for use in cancer treatment. Another important application of single chain antibodies is as diagnostic reagents. In the past few years, specially made recombinant single chain antibodies produced in bacteria have emerged as potential alternatives to these "traditional" immunodiagnostic reagents. The function of recombinant antibody fragments (scFv) as immunoreagents has been discovered in several different detection methods.

TekBiotech has established a complete recombinant antibody preparation and production system based on a mature protein expression platform. It has multiple systems suitable for antibody expression, including prokaryotic expression systems (including but not limited to BL21, Nissle 1917, Rosetta and other prokaryotic expression host systems), mammalian expression systems (including but not limited to Expi 293F, freestyle 293F, CHO-S, CHO-K1, 293T and other mammalian host systems), yeast expression systems (including but not limited to GS115, X33 and other Pichia pastoris expression systems and Saccharomyces cerevisiae expression systems), etc., to meet your various needs. Welcome to consult!

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