Single Domain Antibody (sdAb) Expression in Bacterial System
In recent years, small recombinant antibody fragments play important roles in the area of anticancer therapies and diagnostic. The fragment formats usually include fragments antigen binding (Fabs), single-chain variable fragments (scFvs), and also single domain antibodies (sdAbs).
Bacterial System for sdAb Production
The production of engineered antibody fragments in the bacterial system presents great potentials in scientific research and clinical trials. However, the most limitation is the antibody production in large amounts as soluble, stable, and functional proteins. Derived from camelid heavy-chain only antibodies, sdAbs consist solely of the antigen-specific domain. Compared with conventional antibodies and other fragment formats, single domain antibodies (sdAbs) offer multiple advantages including small size, great stability, and high solubility. Thanks to their simple structure, bacterial system presents a facile method for sdAb production.
Basic Strategies for Bacterial System
In general, there are two basic strategies for antibody fragments expression in bacterial system. The first entails directing the antibody fragment product to the reducing environment of the cytoplasm. The second refers to directing the antibody fragment product to the more oxidizing environment of the periplasmic space between the cytoplasmic and outer membranes. By reducing the complexity and the size of antibody fragments, many problems related to in vivo expression, correct folding, solubility, and stability can be avoided. This is the reason why sdAbs are expressed very well in bacterial system.
Brief Workflow for sdAb Production
- Clone
- Colony PCR
- Small-Scale Expression of sdAbs
- Large-Scale Expression of sdAbs
- Periplasmic Extraction of sdAbs
- Cytosolic Extraction of sdAbs
The VHH genes against the antigen of interest are isolated from the phage library. After a series of PCR, digestion, and purification, the VHH fragments are ligated into the expression vector and finally transformed into E. coli strain TG1 cells.
The colony PCR is performed in a total volume of 15 mL. After the PCR, 5 mL of the PCR mix is applied on 1% agarose gels to identify the clones if they contain a full insert of approximately 650 bp in size. The corresponding primers are used to verify the VHH sequences.
The expressed sdAbs are transported into the periplasmic space of E. coli. The osmotic shock method-based extraction protocol is able to increase the permeability of the outer membrane enabling the sdAbs to be released from the periplasm.
Use a single clone to inoculate 25 mL of LB/Amp and culture overnight. Then transfer the entire overnight culture into a large volume medium. After a series of centrifuge and re-suspend, the pellet can then be subjected to periplasmic or cytosolic protein extraction.
Resuspend the pellet in sucrose solution and then incubate at room temperature. After a series of centrifuging and resuspending, the fractions containing sdAbs are dialyzed overnight and the protein would be purified.
Resuspend the pellet in ice-cold lysis buffer and lyse the bacteria by adding freshly lysozyme solution. After a series of centrifuging and re-suspending, the final solution is dialyzed overnight and the protein would be purified.
Advantages of Bacterial System
- Simple fermentation conditions
- Ease of scale-up
- Short duration between transformation and expression
- Avoid viral contamination that is harmful to humans
- Low capital costs for fermentation
Case Study
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