一．Nanobody Recombinant Expression System

1. Prokaryotic Expression System: Using E. coli to express nanobodies has several advantages: the genetic background of E. coli is clear, gene manipulation is simple, and it is easy to culture. In addition, the E. coli expression system has mature operation methods and a large number of commonly used expression vectors and hosts. There are two expression strategies for expressing nanobodies in E. coli: signal peptide-mediated periplasmic expression and cytoplasmic expression. The expression level of nanobodies in the periplasm is usually low. In order to increase the expression level of nanobodies in the periplasm, nanobodies can be fused with tag proteins for expression. The commonly used expression hosts for expressing nanobodies in E. coli are shown in the following table:

2. Eukaryotic Expression System

2.1 Yeast Cells: Eukaryotic cells have advanced folding, post-translational modification and other functions, which can increase the secretion of antibodies (including full-length IgG). Yeast is a low-level unicellular eukaryotic organism. Compared with other eukaryotic cells, it has a short growth cycle, is easy to culture, and has simple genetic manipulation. Saccharomyces cerevisiae has a clear genetic background and has been used in the food and pharmaceutical industries for a long time. It is safe and reliable and does not produce toxins. Therefore, it has become one of the important expression systems for producing antibodies. Certain amino acids in the J region of nanoantibodies can participate in the recruitment of molecular chaperones in the endoplasmic reticulum, thereby narrowing the difference between the molecular chaperone mechanisms of Saccharomyces cerevisiae and mammalian cells, which is beneficial to the folding dynamics of nanoantibodies and further affects the yield of nanoantibodies in Saccharomyces cerevisiae.

2.2 Filamentous Fungi: Some filamentous fungi of the genera Trichoderma and Aspergillus have efficient protein secretion ability and can secrete a large amount of proteins and metabolites into the culture medium. In order to increase the expression of antibodies, antibody gene fragments can be inserted into expression vectors for fusion expression with glucoamylase promoter and glucoamylase N-terminus, and restriction sites such as KexB are introduced to obtain the target protein.

2.3 Insect Cells: Nanobodies can be expressed in high yields in insect expression systems, while IgG, scFv and Fab have low yields in insect expression systems. Complex proteins under the control of strong promoters may not have enough time to form correct folding and post-translational modifications, resulting in aggregation of target proteins and reduced antibody production.

2.4 Mammalian Cells: The most important cells for producing recombinant antibodies and antibody fragments are CHO, NS0, Sp2/0 and HEK293. Most literature uses CHO to produce antibodies/antibody fragments. To date, 50% of industrially produced therapeutic proteins are still produced by CHO.

二．Strategies to Improve Nanobody Production

2.1 Molecular level: Use gene synthesis design technology to optimize codons of gene coding sequences to maximize translation efficiency, which can effectively increase protein expression; expression rate control is an important factor related to recombinant antibody production, based on lac initiation The promoter and its derived promoters can achieve higher production of antibodies and antibody fragments; in E. coli, the solubility of recombinant proteins can be increased by improving protein folding efficiency, and molecular chaperones (GroEL/GroES, etc.) or folding enzymes (Dsb family proteins) can be overexpressed in cells (SHuffle), both can improve the production of recombinant antibodies.

2.2 Expression level: Prokaryotic expression system is suitable for expressing small molecule antibodies that do not require glycosylation, such as scFv, VHH, etc.; eukaryotic expression system can perform post-translational modifications such as glycosylation on recombinant proteins, and is suitable for expressing therapeutic antibodies; in nanometer During the antibody culture process, using a nutrient-rich medium and maintaining a stable pH will help cell growth, maintain a high density of cells, and increase antibody production.

Improving nanobody production levels will significantly reduce costs. Nanobody production can be improved from three aspects: at the gene level, modification of the target gene sequence and optimization of vector expression components; at the expression level, selection of expression systems, hosts, vectors, and optimization of culture conditions.

Tek Biotech focuses on providing customers with high-quality, cost-effective early-stage antibody drug discovery technology services. We have 10 years of project development experience and experience in drug antibody discovery, and have accumulated sufficient experience in antibody customization and recombinant antibody production. Based on a complete recombinant antibody expression platform, Tek Biotech can provide customers with monoclonal antibodies (including but not limited to VHH antibodies, scFv antibodies, Fab antibodies, chimeric antibodies) from different species including but not limited to camel, sheep, mouse and other species. Humanized antibodies and various types of Fc fusion recombinant antibodies) recombinant expression preparation services, and its supporting complete traceability document system. TekBiotech's CHO-K1, Expi 293F and other mammalian cell host systems, combined with independently designed secretory high expression vectors and amplification expression systems of various specifications, can provide customers with high-quality recombinant antibody expression services to meet the needs of various customers. Scientific research and production needs.