Computationally Designed Antibody Fragments

Antibodies were computationally designed as previously described [18 (link),37 (link)]. Complementary peptides designed to bind specific epitopes were grafted onto the CDR loops of domain antibodies [18 (link),37 (link)]. The expression and purification of the antibodies were carried out as previously described [18 (link)]. In brief, DesAb constructs were expressed and purified using pRSET-B vector in E. coli Rosetta-gami 2 (DE3, Merck Millipore, Burlington, MA, USA), cells were grown, harvested and lysed, and the antibodies (circa 18 kDa) were purified using a Ni²⁺-NTA Superflow column (Qiagen, Hilden, Germany) [18 (link)]. The His-tagged antibodies were eluted by increasing the concentration of imidazole, and were subsequently dialyzed thoroughly in PBS. Protein concentration was determined by absorbance at 280 nm [18 (link)]. DesAb aliquots were stored at −20 °C until use and never thawed more than once. The sequence of DesAb_18-24 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPKLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIGWVRRAPGKGEEWVASIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAAGSVFVGTEAEEEAAAWGQGTLVTVSSGT and that of DesAb_34-40 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPKLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIGWVRRAPGKGEEWVASIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAAGSIRETALLYEEEAAAWGQGTLVTVSSGT.