title: 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.
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, United States.
authors.forename: Ayako
authors.initials: A
authors.lastname: Kamei
authors.forename: Paul S
authors.initials: PS
authors.lastname: Hauser
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Beckstead
authors.forename: Paul M M
authors.initials: PM
authors.lastname: Weers
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine; D006801:Humans; D047668:Inteins; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D019791:Guanidine