ScFv

scFv (single chain fragment variable) are artificially produced antibodies fragments consisting of a variable region of the heavy (V_H) and light (V_L) chain.The two chains are covalently linked together with a flexible peptide linker. The main advantage is that expression in E. coli is possible and it is possible to improve or change the properties of the scFv's, such as opening or specificity, by protein engineering [1]
All cloning work of the scFv's was performed using the expression vector pTXB1 (figure 1)https://www.neb.uk.com/products/neb-catalogue/protein-analysis,-exp-purification/ptxb1-vector backbone. The vector pTXB1 was chosen due to its suitability for protein expression. By inserting the desired gene sequence upstream of the Mxe intein/Chitin binding domain, the gene of interest can be expressed as a fusion protein. Due to the chitin binding domain, affinity purification of the fusion protein can be performed using chitin bound to a column and the tag can be cleaved off the protein by a thiol-induced cleavage reaction [2],[3]

Figure 1: Plasmid map of the pTXB1 vector with inserted progesterone scfv fragment. The plasmid contains the selection marker Amp^R and Mxe intein/chitin binding domain, which is used for purification of the expressed scfv. The restriction sites of NdeI and SpeI are highlighted. The plasmid map was generated via SnapGene.

Figure 2 shows the self-designed scfv fragment for progesterone. In addition to the sequence for the light (V_L) and heavy chain (V_H), it also contains a linker, which is the connecting element between the two chains. The linker also provides a way to immobilize the expressed scfv on a gold surface. In addition, it contains hydrophilic amino acids such as glycine (G) and serine (S) as well as histidine (H), which mediates the connection between the gold surface and the scfv [4] .Furthermore, the fragment has a Gibson overhang, which was used for cloning into the pTXB1 vector. Additionally the restriction enzymes NdeI and SpeI are marked.

Figure 2 Schematic illustration of the used Scfv fragment. The 1312 bp gene fragment is shown with the respective Gibson overlaps and the heavy and light chains connected by a linker. Additionally marked are the restriction enzymes NdeI and SpeI.

References

[1] M. ARSLAN, D. KARADAĞ, und S. KALYONCU, „Protein engineering approaches for antibody fragments: directed evolution and rational design approaches“, Turk. J. Biol., Bd. 43, Nr. 1, S. 1–12, Feb. 2019, doi: 10.3906/biy-1809-28.
[2] E. S. Hosseini, R. Moniri, Y. D. Goli, und H. H. Kashani, „Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus“, Probiotics Antimicrob. Proteins, Bd. 8, Nr. 4, S. 202–210, Dez. 2016, doi: 10.1007/s12602-016-9236-8.
[3] N. H. Shah und A. J. Stevens, „Identification, Characterization, and Optimization of Split Inteins“, in Expressed Protein Ligation: Methods and Protocols, M. Vila-Perelló, Hrsg. New York, NY: Springer US, 2020, S. 31–54.
[4] Chain Fragment Variable Antibody for Immunosensors“, Anal. Chem., Bd. 77, Nr. 21, S. 6834–6842, Nov. 2005, doi: 10.1021/ac0507690.