Affibody synthesis methods

The ability of Affibodies to specifically recognize their targets is achieved through combinatorial protein engineering. Approximately 13 amino acids on the surfaces of helices 1 and 2 are randomized, and the best-performing variants are subsequently selected using high-throughput methods such as phage, bacterial, or yeast display. After selection using these various methods, affibody can be produced by chemical synthesis or by expression in E. Coli [70]. Meanwhile, affibody molecules can be produced recombinantly in bacteria such as E. coli, either as single domains or as more complex fusion constructs. This approach makes their production more cost-effective and simpler compared to antibodies [22]. In addition to recombinant production, affibodies can also be chemically synthesized using SPPS in a more controlled manner [71].

Synthesis of affibody using E. Coli

E. coli is a bacterium frequently used to deliver recombinant proteins due to its rapid growth and the most studied biological characteristics [72]. Therefore, E. coli is a preferred host for affinity protein selection. In Gram-negative bacteria such as E. coli, a natural mechanism, the autotransporter system, provides an effective solution for displaying recombinant proteins on the cell surface [73]. This system has the primary advantage of efficiently transporting proteins across the inner and outer membranes sequentially, while simultaneously producing high levels of surface protein expression [74]. This high surface expression yields a strong signal when analysed by flow cytometry [75].

Recombinant affibody production in E. coli begins with inserting the gene encoding affibody into a suitable expression vector. This vector is then transformed into E. coli cells, converting the protein from a single gene. Once the plasmid containing the affibody gene is inside the cell, protein expression begins with transcription of DNA into mRNA, followed by translation of the mRNA into the affibody polypeptide chain by ribosomes in the cytoplasm. For surface display, an autotransporter system is used to carry affibodies from inside the cell out through the outer membrane and attach them to the cell surface [76].

Synthesis of peptide-based affibody

Affibodies can also be produced through chemical peptide synthesis [77]. In SPPS, affibody synthesis is carried out by linking the first amino acid to a solid support (resin) via a stable covalent bond. The growing peptide chain remains bound to the solid phase and insoluble throughout the synthesis process, facilitating washing and filtration after each reaction step. This makes the synthesis of long peptides, such as affibody, more time-, labour- and material-efficient. Synthesis proceeds stepwise through cycles of deprotection of the α-amine group and subsequent attachment of the protected amino acid until the entire affibody sequence is assembled. Upon completion of synthesis, the peptide is released from the resin and purified in the solution phase [78].

Affibody molecules can also be specifically labelled at defined sites using peptide synthesis [66]. Their small size and rapid folding properties allow the incorporation of specific functional groups, such as reporter agents. Even with the addition of these functional moieties, affibodies remain much smaller than antibodies. In a recent study, Lindgren et al. [77] reported that SPPS of affibodies conjugated with fluorophores resulted in high yield and purity, enabling the rapid and straightforward production of multiple affibody variants.

Overall, both methods can produce affibodies with comparable structure and function. However, SPPS offers important advantages, including significantly lower endotoxin levels and higher product purity, making it more suitable for contamination-sensitive in vivo applications [79,80]. In addition, SPPS provides greater design flexibility, such as the ability to generate affibodies without a His-tag, thereby reducing molecular mass and enabling higher degrees of hydrogel modification [85]. Meanwhile, the recombinant method remains highly useful for early-stage screening or for producing larger proteins, since SPPS generally becomes less efficient as peptide length increases [81,82].