Chapter 9
Lactoferricin-Derived L5a Cell-Penetrating Peptide
for Delivery of DNA into Cells
Natalie J. Holl, Moumita Dey, Yue-Wern Huang, Shiow-Her Chiou,
and Han-Jung Lee
Abstract
Cell-penetrating peptides (CPPs) are small peptides which help intracellular delivery of functional macromolecules, including DNAs, RNAs, and proteins, across the cell membrane and into the cytosol, and even
into the nucleus in some cases. Delivery of macromolecules can facilitate transfection, aid in gene therapy
and transgenesis, and alter gene expression. L5a (RRWQW), originally derived from bovine lactoferricin, is
one kind of CPPs which can promote cellular uptake of plasmid DNA and enters cells via direct membrane
translocation. The peptide complexes noncovalently with DNA over a short incubation period. DNA
plasmid and L5a complex stability is confirmed by a decrease in mobility in a gel retardation assay, and
successful transfection is proven by the detection of a reporter gene in cells using fluorescent microscopy.
Here, we describe methods to study noncovalent interactions between L5a and plasmid DNA, and the
delivery of L5a/DNA complexes into cells. L5a is the one of the smallest CPPs discovered to date,
providing a small delivery vehicle for macromolecules in mammalian cells. A small vehicle which can
enter the nucleus is ideal for efficient gene uptake, transfer, and therapy. It is simple to complex with
DNA plasmids, and its nature allows mammalian cells to be easily transfected.
Key words Cell-penetrating peptide, Electrophoretic mobility shift assay, Fluorescent microscopy,
Gel retardation, Gene therapy, Intracellular delivery, N/P ratio, Protein transduction domain,
Transgenesis
1 Introduction
The internalization of exogenous materials into cells through the
plasma membrane is an important function of all eukaryotic cells.
The use of cell-penetrating peptides (CPPs) has recently gained
attention as a bright field to deliver therapeutic and diagnostic
molecules into the cell in a nontoxic manner [1]. CPPs are peptides
that are able to translocate across cellular membranes and to deliver
diverse cargos into cells [2]. Most synthetic or natural CPPs are
shorter than 30 amino acids [3, 4]. In 1988, the first CPP, transactivator of transcription (TAT) of human immunodeficiency virus,
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
113
Lactoferricin-Derived L5a Cell-Penetrating Peptide
for Delivery of DNA into Cells
Natalie J. Holl, Moumita Dey, Yue-Wern Huang, Shiow-Her Chiou,
and Han-Jung Lee
Abstract
Cell-penetrating peptides (CPPs) are small peptides which help intracellular delivery of functional macromolecules, including DNAs, RNAs, and proteins, across the cell membrane and into the cytosol, and even
into the nucleus in some cases. Delivery of macromolecules can facilitate transfection, aid in gene therapy
and transgenesis, and alter gene expression. L5a (RRWQW), originally derived from bovine lactoferricin, is
one kind of CPPs which can promote cellular uptake of plasmid DNA and enters cells via direct membrane
translocation. The peptide complexes noncovalently with DNA over a short incubation period. DNA
plasmid and L5a complex stability is confirmed by a decrease in mobility in a gel retardation assay, and
successful transfection is proven by the detection of a reporter gene in cells using fluorescent microscopy.
Here, we describe methods to study noncovalent interactions between L5a and plasmid DNA, and the
delivery of L5a/DNA complexes into cells. L5a is the one of the smallest CPPs discovered to date,
providing a small delivery vehicle for macromolecules in mammalian cells. A small vehicle which can
enter the nucleus is ideal for efficient gene uptake, transfer, and therapy. It is simple to complex with
DNA plasmids, and its nature allows mammalian cells to be easily transfected.
Key words Cell-penetrating peptide, Electrophoretic mobility shift assay, Fluorescent microscopy,
Gel retardation, Gene therapy, Intracellular delivery, N/P ratio, Protein transduction domain,
Transgenesis
1 Introduction
The internalization of exogenous materials into cells through the
plasma membrane is an important function of all eukaryotic cells.
The use of cell-penetrating peptides (CPPs) has recently gained
attention as a bright field to deliver therapeutic and diagnostic
molecules into the cell in a nontoxic manner [1]. CPPs are peptides
that are able to translocate across cellular membranes and to deliver
diverse cargos into cells [2]. Most synthetic or natural CPPs are
shorter than 30 amino acids [3, 4]. In 1988, the first CPP, transactivator of transcription (TAT) of human immunodeficiency virus,
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
113