title: Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation.
abstract: Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5' LIC site of expression constructs and report on the construction of expression vectors with N-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (TEV) protease cleavage. In a practical application, the N-terminal serine was oxidized to an aldehyde, subsequently reacted with an amino-oxy functionalized polyethylene glycol (PEG) ligand under aniline catalysis to provide a protein selectively modified at the N-terminus.
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. erik.vernet@cpr.ku.dk
authors.forename: Erik
authors.initials: E
authors.lastname: Vernet
authors.forename: Jørgen
authors.initials: J
authors.lastname: Sauer
authors.forename: Andreas
authors.initials: A
authors.lastname: Andersen
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Bjørn
authors.initials: B
authors.lastname: Voldborg
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012694:Serine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D012694:Serine; D010450:Endopeptidases; C099488:TEV protease