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Background  Single chain Fvs (scFvs) are widely applied in research, diagnostics and therapeutic settings. Display and selection from combinatorial libraries is the main route to their discovery and many factors influence the success of this process. They exhibit low thermodynamic stability, resulting in low levels of premature cytosolic folding or aggregation which facilitates  sec  YEG-mediated translocation and phage in  E. coli . However, there is little data analysing how this is related to and influenced by scFv protein expression.    Results  We characterised the relationship between overall scFv expression and display propensity for a panel of 15 anti-tetanus toxin scFvs and found a strong positive correlation (Rho = 0.88, p < 0.005) between the two parameters. Display propensity, overall expression and soluble localisation to the periplasm and extracellular fractions were clone specific characteristics which varied despite high levels of sequence homology. There was no correlation between display of scFv or its expression in non-fused (free) form with soluble scFv localisation to the periplasm or culture supernatant. This suggests that divergence in the fate of scFv-pIII and non-fused scFv after translocation to the periplasm accounts for the observed disparity. Differential degrees of periplasmic aggregation of non-fused scFv between clones may affect the partitioning of scFv in the periplasm and culture supernatant abrogating any correlation. We suggest that these factors do not apply to the scFv-pIII fusion since it remains anchored to the bacterial inner membrane as part of the innate phage packaging and budding process.    Conclusion  We conclude that in the absence of premature cytosolic aggregation or folding, the propensity of a scFv to be displayed on phage is directly related to its overall expression level and is thus indirectly influenced by factors such as codon bias, mRNA abundance or putative DNA motifs affecting expression. This suggests that scFvs capable of high overall expression and display levels may not produce high yields of non phage-fused soluble protein in either the periplasmic or extracellular fractions of  E. coli . This should be considered when screening clones selected from combinatorial libraries for further study.  The nucleotide and amino acid sequences of the anti-tetanus toxin scFvs have been deposited in the EMBL data base: accession numbers-C1:  AM749134 , C2:  AM749135 , C3:  AM749136 , C4:  AM749137 , C5:  AM749138 , N1:  AM749139 , N2:  AM749140 , N3:  AM749141 , N4:  AM749142 , N5:  AM749143  J1;  AM749144 , J2:  AM749145 , J3:  AM749146 , J4:  AM749147 , J5:  AM749148 .

Single-chain Fv phage display propensity exhibits strong positive correlation with overall expression levels