Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation
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Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation. / Vernet, Erik; Sauer, Jørgen; Andersen, Peter Andreas; Jensen, Knud Jørgen; Voldborg, Bjørn Gunnar Rude.
I: Analytical Biochemistry, Bind 414, Nr. 2, 2011, s. 312-314.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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
Y1 - 2011
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
VL - 414
SP - 312
EP - 314
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -
ID: 40289329