| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | DNA manipulation was performed according to standard techniques. Primers were synthesized by GenScript, China. All DNA constructs were sequenced (GenScript, China). The DARPin was composed of an N-terminal cap, three designed internal ARs, and a C-terminal cap. The library was constructed as previously described using isocaudamer ligation [22] (Fig.2). Oligo nucleotides containing random sites were assembled by PCR to generate the internal repeat sequences. After ligation and PCR amplification, the DNA library (N-terminal cap and three designed internal ARs) was digested with KpnI and BbsI endonucleases and purified. The fragment was inserted into the pBAD-SP-Lpp-OmpA-C-terminal cap expression vector digested with the same enzymes (the pBAD-SP-Lpp-OmpA plasmid was a kindly gifted by Prof. Wei wei, University of Nanjing [23]) and electro-transformed into E. coli MegaX DH10B cells. The size of constructed library was confirmed by colony counting on agar plate. The quality of constructed libraries was tested by sequencing. | Get A Quote |
Designed ankyrin repeat proteins (DARPins) are a kind of proteins containing the similar characteristics to antibodies. Which can specifically bind the target with high affinity. Here, we described an efficient method to select of DARPins effectively against the programmed death-ligand 1(PD-L1), a prominent target for cancer immune therapy, by E. coli surface display. E. coli surface display combined with magnetic cell sorting (MACS) were easy to manipulate with the advantages of solubility selected, antigen conformation kept and easily unbinding DARPins washed. The selected DARPin bound specifically to PD-L1 overexpressing cell CT26 and had a micromolar affinity for PD-L1. Animal experiments with tumor-bea... More
