A versatile insertion point on albumin to accommodate peptides and maintain their activities

Genetic fusion of human serum albumin to peptides is an important strategy to enhance the plasma half-life of the peptide. An inherent challenge of such method is the reduction of specific activity of the cargo peptides upon connecting at N- or C-termini of albumin. Here, we report a finding that residue 363-364 of albumin can be inserted with a peptide while maintaining the peptide activities. We insert a peptide inhibitor into this site, and at the N-terminus of albumin, for comparison. The chimeric protein displays potent inhibition (IC₅₀ value of 30 nM) to its target (uPAR), but not the N-terminally fused construct. We also study the chimera of HSA with a cyclic peptide inhibitor of murine urokinase-type plasminogen activator grafted at either the internal site or the N-terminus. The internally peptide-grafted protein possesses a much more potent inhibition compared to the N-terminally located fusion (IC₅₀ value of 32 nM vs 19 μM). We further demonstrate that such internal fusion does not affect albumin expression, secondary structure, and inherent drug binding activity. Thus, this work identifies a versatile insertion point inside albumin for maintaining fusion peptide activity, and opens a new avenue to expand the applications of albumin fusion technology.