Prokaryotic Expression of Nanobodies-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Prokaryotic Expression of Nanobodies

Camel serum contains both traditional heterotetrameric antibodies and unique functional heavy (H) chain antibodies. The H chain of these homodimeric antibodies consists of an antigen-binding domain VHH and two constant domains. Due to the loss of the first constant domain and the remodeling of the VHH side surface, HCAbs are unable to bind light (L) chains, which are usually associated with L chains in conventional antibodies. The genetic elements that make up HCAbs have been identified, but the research on the generation of true antibodies with antigen specificity and affinity maturation from their dedicated genes in vivo is still largely insufficient.

The advent of recombinant DNA technology has enabled the precise engineering and production of monoclonal antibodies with higher efficiency and specificity, and also reduced adverse reactions. Nanobodies are easily expressed in various expression systems, including Escherichia coli and yeast cells. For these reasons, nanobodies have become the preferred antibody fragment for protein engineering, disease diagnosis and treatment. To date, the U.S. Food and Drug Administration (FDA) has approved two nanobody-based therapeutics. Many candidate diseases, such as cancer, immune system diseases, infectious diseases and neurodegenerative diseases, are in preclinical and clinical research.

1. Nanobodies

Camelid single-domain antibodies (sabs, VHHs, or Nanobodies) are antibody fragments consisting only of the variable domain of the heavy chain. These VHHs have unique structural and functional characteristics due to their small size, thermal stability, and high solubility. Compared with traditional antibodies, VHHs can be manufactured in microorganisms, greatly saving cost, labor, and time, because VHHs lack the Fc domain containing N-linked oligosaccharides. So far, VHHs have been expressed in several production systems, from prokaryotic cells, yeast, fungi, insect cells, mammalian cell lines to plants.

Different forms of antibodies-Tekbiotech.png

Figure 1 Different forms of antibodies

II. Expression of Nanobodies in Escherichia coli

VHHs are still not easy to express in prokaryotic hosts such as E. coli because the non-productive interactions of the newly produced polypeptides lead to aggregation in the cytoplasm instead of the native protein. In addition, many VHHs contain an additional disulfide bond linking CDR3 to the end of CDR1 or the beginning of CDR2, which may hinder the correct folding and high expression of the VHHs structure.

Compared with the reducing cytoplasm that leads to non-functional aggregation, the periplasm is more suitable for the expression of VHHs because it is the only compartment with an oxidizing environment. In addition, the presence of many chaperones and isomerases, such as the Dsb system, can promote the correct formation and folding of disulfide bonds. Most proteins containing disulfide bonds are first produced in the cytoplasm of E. coli and then enter the periplasmic space through the Sec pathway with the help of the N-terminal leader sequence.

Other factors, such as the culture medium, also have an important impact on the expression of VHHs in E. coli. Considering the higher cell density that can be imagined, the enzyme-based glucose release system (EnPresso medium) and Terrific broth (TB) can produce 5 to 10 times more VHHs than the common medium Luria Bertani (LB). However, EnPresso medium is not widely used because it is much more expensive than TB or LB. The promoter is also one of the key parameters that affect the expression rate of VHH. For example, T7, lacUV5, and m/xylS promoters can achieve high product yields in some heterologous proteins.

III. Other Prokaryotic Expressions

Lactobacillus is a common intestinal bacterium that is generally considered safe for human consumption; their harmless effects on humans make them suitable for the production and delivery of therapeutic molecules. Lactobacillus expression systems are often used for surface display and secretory expression of VHHs antibodies; although these bacteria only produce 1-3μg/mL of VHHs, this range is much lower than the average yield of Escherichia coli.

Similar yields were obtained in another lactic acid bacteria platform, Bifidobacterium longum. In addition, for large-scale fermentation, up to 3g/L of VHHs can be obtained in a 3L fed-batch tank fermenter using the Brevibacillus choshinensex expression system.

TekBiotech uses the pIII protein of the M13 phage for VHH antibody (nanobody) surface display. Our scientists constructed a phage vector with dual tags of Flag and 6*His tag, and prepared TG1 and XL1-Blue host bacteria with a transfection efficiency of 10^8. After 2-3 transfections and amplifications, we can provide customers with a high-quality VHH antibody display library with an effective library capacity of 10^8-10^9 and a packaging capacity of 10^13-10^16/ml particles.

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