sdAb Library Construction

Isolate and amplify sdAb genes from an existing library and clone them into a suitable bacterial display vector, such as a plasmid. The vector should contain a signal peptide for secretion, a surface display scaffold protein (e.g., OmpA, PelB), and a gene encoding a selectable marker (e.g., antibiotic resistance) for screening and enrichment.

Transformation and Expression

Once the sdAb library is constructed, it is introduced into a host bacterium, such as Escherichia coli, through a process called transformation. The transformed bacteria then express the sdAbs on their cell surface under the control of a strong promoter.

Affinity Enrichment

This step aims to enrich the library for sdAbs with high affinity towards the target molecule. This process typically involves multiple rounds of binding and washing steps, where the bacteria displaying sdAbs are exposed to the target molecule. The non-binding bacteria are washed away, while those bacteria with bound sdAbs are collected and amplified for subsequent rounds of enrichment.

Screening and Isolation

Next, the bacterial library is subjected to screening assays to identify sdAbs with the desired binding specificity and affinity. Various screening methods can be employed, such as flow cytometry, magnetic bead-based assays, or fluorescence-activated cell sorting (FACS).

Characterization and Iterative Optimization

Once potential sdAb candidates are identified, they undergo thorough characterization and optimization to ensure their functionality and stability. Multiple parameters are evaluated, such as binding kinetics, thermal stability, chemical stability, and expression level. In addition, site-directed mutagenesis techniques can be employed to enhance the affinity or alter the binding specificity of selected sdAbs.