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By using the selectively infective phage (SIP) technology, we selected non-repetitive linkers for a single-chain Fv fragment to have genes more robust against deletions in PCR-based gene assembly and directed evolution experiments than is the case for the classical (Gly4Ser)3 linker. We designed linkers encoding turns at both ends and random positions in the middle where glycines and polar and charged residues were allowed to occur. After only a single round of SIP, all clones obtained were fully functional. Properties such as antigen binding constants, urea denaturation curves and expression of soluble scFv fragments were identical with those of the parental fragment with the (Gly4Ser)3 linker. This demonstrates that SIP is a very fast and powerful technique to remove rapidly sequences of poor functionality, exclusively yielding sequences of the desired overall property in a single round.

Non-repetitive single-chain Fv linkers selected by selectively infective phage (SIP) technology