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 mu M). 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.

Automated summary

Genetic fusion of human serum albumin with peptides is an important strategy to enhance the lifespan of peptides in the bloodstream. A study found that inserting a peptide into residue 363-364 of albumin can maintain the activity of the peptide. Internal fusion of the protein has a stronger inhibitory effect compared to fusion at the N-terminus, without affecting albumin expression, secondary structure, and inherent drug binding activity.