Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

TY - JOUR

T1 - Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

AU - Vernet, Erik

AU - Sauer, Jørgen

AU - Andersen, Peter Andreas

AU - Jensen, Knud Jørgen

AU - Voldborg, Bjørn Gunnar Rude

N1 - Copyright © 2011 Elsevier Inc. All rights reserved.

PY - 2011/7/15

Y1 - 2011/7/15

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Cloning, Molecular

KW - Endopeptidases

KW - Genetic Vectors

KW - Molecular Sequence Data

KW - Mutagenesis

KW - Plasmids

KW - Polyethylene Glycols

KW - Recombinant Proteins

KW - Serine

U2 - 10.1016/j.ab.2011.03.015

DO - 10.1016/j.ab.2011.03.015

M3 - Journal article

C2 - 21414287

SN - 0003-2697

VL - 414

SP - 312

EP - 314

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

ER -