sdAb Selection by Bacterial Two-Hybrid System
Intracellular single domain antibodies (sdAbs) refer to the recombinant proteins consisting of one variable region domain fragment. With the ability to bind intracellular molecules specifically and further interfere with their particular functions within various cellular compartments, intracellular sdAbs have been served as important tools for biological diagnosis and therapeutics. Here, Creative Biolabs describes the novel bacterial-two-hybrid selection method for sdAb generation.
Introduction of Intracellular Selection Methods
For monoclonal antibodies discovery, phage display and ribosome display are the most efficient screening techniques to obtain target-specific binders. However, both of them are ex vivo methods which means the selected binders can work well in the extracellular, oxidizing environment but not in reducing conditions as encountered in the cytoplasm. To avoid this problem, the novel intracellular selection methods were developed to obtain binders that perform equally well in oxidizing (extracellular) and reducing (intracellular) situations. Among them, the yeast-two-hybrid system is used to identify scFv scaffold with improved intracellular stability and solubility. Then this scaffold can be used for a synthetic library construction to select antigen-specific intrabodies.
Fig.1 Schematic representation of the bacterial-two-hybrid strategy and the phage display method.
Bacterial-Two-Hybrid System Introduction
Compared with the phage display or ribosome display, the yeast-two-hybrid system presents a great advantage while the antigen can be available in an unpurified form. However, the yeast-two-hybrid system is difficult to handle large and diverse libraries, so that the Intracellular Antibody Capture Technology (IACT) is necessary to narrow the library size. What’s more, the yeast-two-hybrid system is limited to obtain binders against non-homologous eukaryotic proteins and also lacks a distinction between high-affinity and low-affinity binders. In this case, the bacterial-two-hybrid system is developed while fast-growing bacteria can be used instead of yeast, moreover, single-chain antibodies (scFvs) can be replaced by sdAbs. Based on the excellent biological properties, such as high stability, great solubility, and good expression levels, the intracellular sdAbs may present huge potentials in biological diagnostics and therapeutics.
Main Steps for Bacterial-Two-Hybrid System
- Animal immunization
- sdAb library construction
- Preparation of electrocompetent cells
- Binders selection by bacterial-two-system
- sdAbs expression and purification
- Intracellular functionality of sdAbs retrieved by bacterial-two-system
It is worth mentioning that the bacterial-two-hybrid selection will not work for many post-translationally modified proteins. In theory, it is also possible to develop an epitope mapping strategy for the selected intrabodies by cloning subfragments of the target cDNA.
Case Study
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