Expressed protein ligation-mediated template protein extension

Abstract Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1–172, was expressed and isolated from Escherichia coli . Compared to full length apoA-I (243 amino acids), apoA-I(1–172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1–172) by EPL, creating apoA-I Milano (1–192). The EPL product displayed biophysical properties similar to full-length apoA-I Milano . The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size.