Schematic diagram of the coding sequence in a phage antibody construct in a pComb3 family phagemid vector (Andris-Widhopf et al. 2000). The coding sequence for a chicken single-chain variable fragment (scFv) is inserted between the SfiI cloning sites. The colored blocks together constitute the coding sequence for a recombinant protein in which a chicken scFv is fused to the C domain of pIII (including TM) through the last 27 amino acids of pIII's flexible linker, L2. The N-terminal domains N1, L1, and N2 are absent. The gray blocks are spacers with no specified function in the protein. The entire coding sequence spans 463 amino acids if Vλ and VH are the germline genes (the number can be slightly different if Vλ or VH have been modified by somatic gene conversion, as explained in the next section). The 21-amino-acid ompA signal peptide (yellow), like pIII's natural signal peptide, targets the protein to the Sec secretion system, and is cleaved off as the recombinant polypeptide passes through the inner membrane into the periplasmic space. The 6×His and HA tags allow virions to be specifically purified by immobilized metal affinity chromatography (IMAC) or affinity chromatography with an antibody against influenza hemagglutinin protein, respectively.
Figure 7.

Schematic diagram of the coding sequence in a phage antibody construct in a pComb3 family phagemid vector (Andris-Widhopf et al. 2000). The coding sequence for a chicken single-chain variable fragment (scFv) is inserted between the SfiI cloning sites. The colored blocks together constitute the coding sequence for a recombinant protein in which a chicken scFv is fused to the C domain of pIII (including TM) through the last 27 amino acids of pIII's flexible linker, L2. The N-terminal domains N1, L1, and N2 are absent. The gray blocks are spacers with no specified function in the protein. The entire coding sequence spans 463 amino acids if Vλ and VH are the germline genes (the number can be slightly different if Vλ or VH have been modified by somatic gene conversion, as explained in the next section). The 21-amino-acid ompA signal peptide (yellow), like pIII's natural signal peptide, targets the protein to the Sec secretion system, and is cleaved off as the recombinant polypeptide passes through the inner membrane into the periplasmic space. The 6×His and HA tags allow virions to be specifically purified by immobilized metal affinity chromatography (IMAC) or affinity chromatography with an antibody against influenza hemagglutinin protein, respectively.

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  1. Published in Advance October 22, 2025, doi: 10.1101/pdb.over107753 Cold Spring Harb Protoc 2026: pdb.over107753-
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