Scientists made a version of the immune‑boosting peptide thymosin‑alpha‑1 that’s attached to a big protein (human serum albumin) and added a tiny chemical tag (N‑acetyl) using a bacterial enzyme. This tag keeps the peptide stable, and putting the peptide at the start of the fusion keeps its activity, unlike previous designs where it was at the end.

Thymosin alpha 1 (T&#x3b1;1) is an important immunomodulating agent with various clinical applications. The natural form of T&#x3b1;1 is <i>N^&#x3b1;</i> -acetylated, which was supposed to be related to <i>in vivo</i> stability of the hormone. In this study, fusion protein T&#x3b1;1-HSA was constructed and expressed in <i>Pichia pastoris</i>. RimJ, a <i>N^&#x3b1;</i> -acetyltransferase from <i>E.coli</i>, was also overexpressed and purified to homogeneity. <i>In vitro</i> acetylation of T&#x3b1;1-HSA in the presence of RimJ and acetyl coenzyme A resulted in <i>N^&#x3b1;</i> -acetyl T&#x3b1;1-HSA. The <i>N^&#x3b1;</i> -acetylation was determined by LC-MS/MS. Kinetic assay indicated that RimJ had a higher affinity to desacetyl T&#x3b1;1 than to T&#x3b1;1-HSA. Bioactivity assay revealed fully retained activity of T&#x3b1;1 when the hormone was connected to the N-terminus of the fusion protein, while the activity was compromised in our previously constructed HSA-T&#x3b1;1. With fully retained activity and N-terminal acetylation, <i>N^&#x3b1;</i> -acetyl T&#x3b1;1-HSA was expected to be a more promising pharmaceutical agent than T&#x3b1;1.