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

Erik Vernet; Jørgen Sauer; Peter Andreas Andersen; Knud Jørgen Jensen; Bjørn Gunnar Rude Voldborg

doi:10.1016/j.ab.2011.03.015

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

Erik Vernet, Jørgen Sauer, Peter Andreas Andersen, Knud Jørgen Jensen, Bjørn Gunnar Rude Voldborg

3 Citations (Scopus)

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.

Original language	English
Journal	Analytical Biochemistry
Volume	414
Issue number	2
Pages (from-to)	312-314
Number of pages	3
ISSN	0003-2697
DOIs	https://doi.org/10.1016/j.ab.2011.03.015
Publication status	Published - 15 Jul 2011

Keywords

Amino Acid Sequence
Cloning, Molecular
Endopeptidases
Genetic Vectors
Molecular Sequence Data
Mutagenesis
Plasmids
Polyethylene Glycols
Recombinant Proteins
Serine

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10.1016/j.ab.2011.03.015

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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.",

keywords = "Amino Acid Sequence, Cloning, Molecular, Endopeptidases, Genetic Vectors, Molecular Sequence Data, Mutagenesis, Plasmids, Polyethylene Glycols, Recombinant Proteins, Serine",

author = "Erik Vernet and J{\o}rgen Sauer and Andersen, {Peter Andreas} and Jensen, {Knud J{\o}rgen} and Voldborg, {Bj{\o}rn Gunnar Rude}",

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doi = "10.1016/j.ab.2011.03.015",

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journal = "Analytical Biochemistry",

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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

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 -

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

Abstract

Keywords

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