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Altering Phenylalanine at Position 67 to Serine
Using Site-Directed Mutagenesis to Examine
Furin Structure, Function, and Trafficking
Amber Lynne Anger
Department of Biology at University of Michigan-Flint
ABSTRACT: Furin is a ubiquitously expressed transmembrane protein involved in key aspects
of development and homeostasis. This proprotein convertase has been studied extensively (2). Its
main function appears to be processing precursors in the constitutive pathway (1). It has also been
implicated in the pathology of various diseases (2). Site-directed mutagenesis allowed further
examination of this protein by altering a single amino acid in the furin sequence. Namely,
phenylalanine at position 67 was changed to serine. This was accomplished by mutating a single
nucleotide in fur cDNA which was cloned into a pcDNA3 expression plasmid, and inserted into
E. coli cells. After undergoing replication in the E. coli cells, the plasmid DNA was isolated and
purified to be used for the purpose of transfection in RPE.40 cells. Findings of this study were
limited, and further experimentation is necessary to make any conclusions about the effects of the
F67S mutation on furin structure, function, and trafficking. Future research would involve
transfecting mutant and wild-type furin into RPE.40 cells and subjecting them to further analysis,
including immunofluorescence and PEA toxin assays.
1. Introduction
Furin activity is involved in critical aspects of
embryogenesis and homeostasis, as well as
certain diseases (3). This protein is capable of
cleaving a wide array of substrates in vivo
including: growth factors, receptors,
adhesion molecules, metalloproteinases, viral
glycoproteins, bacterial toxins, and the
proton pump V-ATPase accessory protein
Ac45 subunit (2). Its main function consists
of processing precursors in the constitutive
pathway (1). For example, furin cleaves the
transmembrane receptor Notch which is
necessary for the release of its intracellular
domain by γ-secretase proteolysis. This
intracellular domain binds to the
transcriptional regulator CSL, which
activates genes required for cell-to-cell
communication during development.
Alternatively, uncleaved Notch mediates a
distinct signaling pathway that inhibits cell
differentiation. The exact mechanism
through which furin regulates this process is
unknown. Another important function of
furin is catalyzing pro-β-nerve growth factors
that mediate the cell-survival pathway in
neurons allowing formation of synaptic
complexes (3).
Experimental disruption of furin activity in
mice has been associated with defects in the
heart and insufficient distribution of blood
throughout the body, resulting in premature
death of the subjects. Aside from embryonic
death, the inactivation of furin in salivary
cells of mice appears to cause overexpression
of an adenoma gene resulting in tumor

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growth (1). Unregulated furin can also
contribute to health problems; for example,
the processing of β- and γ-secretases by furin
leads to formation of plaques that alter the
structure of neurons causing neuro-
degenerative disorders such as Alzheimer’s
disease (2). In addition, furin’s endoprotease
activity is responsible for activating Pseudo-
monas exotoxin A, a bacterial toxin that
inhibits translation in eukaryotic cells by
enzymatically inactivating elongation factor
2, thereby interrupting translation resulting in
death of the cell/organism. Furthermore,
furin activates viral proteins necessary for
HIV infection, avian influenza, and Ebola
virus, as well as other bacterial toxins such as
the anthrax toxin and diphtheria toxin (2).
Furin’s role in pathology suggests it may
have important medical implications. This
protein is often upregulated in cancers and
has therefore been proposed as a potential
therapeutic target. It is also upregulated in the
cartilage of patients with osteoarthritis, so it
is thought that its inhibition may be useful in
treatment of this disorder. Furin inhibition
may also work to block bacterial toxicity (1).
Overall it is apparent that furin is a significant
protein with obvious importance in develop-
mental, homeostatic, and pathological
aspects of research.
Furin structure is of particular importance
when considering trafficking of the
transmembrane protein. It consists of an N-
terminal, a signal peptide and a domain
structure that contains a propeptide followed
by a catalytic domain, a P domain, a cysteine-
rich domain, a transmembrane domain, and a
cytoplasmic domain (2). The signal peptide is
responsible for directing furin to the secretory
pathway. Furin is found as an inactive
precursor in the rough endoplasmic
reticulum, and must be activated to be able to
proceed through the Golgi apparatus and
eventually end up in a vesicle that will
transiently fuse with the plasma membrane.
The propeptide domain acts as an
intramolecular chaperone, and aids in protein
folding and activation of the zymogen.
Cleavage is necessary for proper folding, and
occurs autocatalytically in the endoplasmic
reticulum; this initial cleavage is a
requirement for transport out of the ER. The
propeptide will remain associated with the
zymogen until a second cleavage occurs in
the trans-Golgi network/endosomal system.
The catalytic domain contains the active site
and the P domain is involved in the protein’s
enzymatic activity (2). The cysteine-rich
domain, as well as the transmembrane
domain, work to anchor secreted versions of
furin to the cell membrane. The cytoplasmic
domain mediates Golgi localization. The
carboxy-terminal domain has been shown to
be important for intracellular trafficking of
furin (2).
The goal of this research was to change a
single amino acid in the 794 amino acid
sequence for furin via site-directed
mutagenesis using the fur cDNA cloned into
pcDNA3 from the previous semester as a
template, and then introduce wild type and
mutant versions of furin into RPE.40 cells to
examine furin activity. To accomplish this, a
comparison of the sequences of the fur gene
in a many diverse species was performed to
determine which ones are highly conserved,
and of importance. Aside from selecting a
highly conserved amino acid to mutate, a
drastic change in the polarity and structure of
this highly conserved amino acid was
considered, as this seemed more likely to
alter furin’s normal activity. It was decided
that phenylalanine-67 would be changed to
serine, altering the composition from a non-
polar amino acid to a polar amino acid, and
eliminating the ring structure. A schematic of
the methods used can be seen in Figure 1.

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Figure 1| Schematic Overview of Methods Used
A) Cobalt was used to generate a multiple sequence alignment on ten diverse species to identify highly conserved regions in
amino acid sequences for furin. B) Primers were designed according to Agilent Technologies guidelines and ordered from Life
Technologies (Carlsbad, CA). C) A QuikChange II kit from Agilent Technologies was utilized for site-direct mutagenesis to
induce a mutation on a single nucleotide in fur cDNA. D) Mutagenesis reactions were transformed into super competent XL1-
blue E. coli cells by adding DpnI treated DNA to culture tubes containing the E. coli cells in a CaCl2 solution. E) Plasmid DNA
was isolated from the transformed XL1-blue E. coli cells using a QIA prep mini-kit (Qiagen; Valencia, CA). F) Purified plasmids
were digested using HindIII and Xba-I restriction enzymes and subjected to agarose gel electrophoresis using 1% agarose gel and
0.5% TBE buffer. G) Sanger sequencing was performed on the plasmid samples at University of Michigan Sequencing Core
(Ann Arbor, MI). H) Culture of RPE.40 cells was performed using the standard protocol“Transferring Mammalian Cells”
provided by theUniversity of Michigan-Flint Biology Department. I) Cell were counted according to theprocedure provided by
the University of Vermont Department of Microbiology and Molecular Genetics.
2. Materials and Method
2.1 Multiple Sequence Alignment
Furin protein sequences for ten different
diverse species were acquired from the
international DNA and protein database
GenBank. The genus and species names and
their corresponding accession numbers can
be seen in Table 1. A multiple sequence
alignment was performed using Cobalt, and
three amino acids were analyzed as potential
candidates for mutation based on their
chemical properties and location.
Phenylalanine at position 67 in the furin
amino acid sequence of wild-type Chinese
hamster ovarian cells (CHO-K1) cells was
selected to be mutated to serine (Figure 2).
Figure 2| Mutation Selection
The mutation under investigation can be seen here, as well
as the chemical structure of phenylalanine and serine. This
illustrates thepolarity change induced and the ring structure
that was eliminated.

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Species Used for Multiple Sequence Alignment (MSA)
2.2 Mutagenic Primer Design
Mutagenic oligonucleotide primers were
prepared according to guidelines provided by
Agilent Technologies (Santa Clara, CA).
These guidelines include a primer length
between 25 and 45 bases, a melting
temperature greater than 78 degrees Celsius,
and a minimum GC base content of 40%. In
addition, the mutation was required to be in
the middle of the primer sequence with 10-15
bases on both sides, and terminated on one or
more C or G bases. Primers were ordered
from Life Technologies (Carlsbad, CA), and
reconstituted using sterile water. Primer
sequences can be seen in Figure 3.
Figure 3| Mutagenic Primer Design
Primer Sequences containing the desired mutation ordered
from Agilent Technologies can be visualized here.
2.3 Site-Directed Mutagenesis
Site-directed mutagenesis was utilized to
change phenylalanine at position 67 of the
furin amino acid sequence to serine using a
QuikChange II site directed mutagenesis kit
from Agilent Technologies. The DNA
template that encodes for furin and the
mutagenic primers were quantified using a
nanodrop spectrometer prior to mutagenesis.
Double stranded DNA molecules were
combined with reaction buffer, dNTP mix,
experimentally designed mutagenic primers,
and PfuUltra high fidelity DNA polymerase,
and subjected to thermal cycling to denature
the dsDNA, and allow primers to be
annealed, and extended. The methylated and
hemi-methylated parental DNA strands were
degraded using DpnI, leaving the
unmethylated DNA that may contain the
mutation of interest.
2.4 Transformation
Mutagenesis reactions were transformed into
XL1-blue E. coli cells by adding the DpnI
treated DNA to culture tubes containing the
super competent cells. Transformation
reactions were heat pulsed at 42 degrees
Celsius for 45 seconds, and then placed on
ice; NZY+ broth was added. After plates
were incubated, and observed for growth,
single colonies were inoculated into 2
separate tubes containing LB-AMP broth in
preparation of plasmid purification. Super
competent XL1-blue E. coli cells were
obtained from Agilent Technologies.
2.5 Plasmid Purification
Plasmid DNA was isolated from the
transformed XL1-blue E. coli cells using a
QIA prep mini-kit (Qiagen; Valencia, CA).
Transformed cells were resuspended in P1
buffer, lysed using P2 buffer, and neutralized

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using buffer N3. The supernatant was added
to a QIA prep spin column, and centrifuged.
The negatively charged DNA bound to the
positively charged resin of the column. DNA
was eluted using water and the plasmid was
quantified using nanodrop spectrometer.
2.6 Restriction Digest and DNA Sequencing
Purified plasmids were digested using
HindIII and Xba-I restriction enzymes.
Restriction digests contained plasmid DNA
isolated from the transformed E. coli cells,
buffer M, BSA, and the restriction enzymes.
Digests were subjected to agarose gel
electrophoresis using 1% agarose gel and
0.5% TBE buffer and viewed on photodoc
(Figure 4). DNA sequencing was performed
on the plasmid samples at the University of
Michigan Sequencing Core (Figure 5).
2.7 Mammalian Cell Culture and Counting
Furin-null mutants derived from Chinese
hamster ovarian cells, known as RPE.40
cells, were cultured in T25 tissue culture
flasks with Dulbecco’s Modification of
Eagle’s Minimal Essential Medium (DMEM)
following the standard protocol “Transferring
Mammalian Cells”, provided by the
University of Michigan-Flint. Cells were split
using trypsin-EDTA solution twice a week
for a total of three weeks. After which, the
cells were diluted 1:10 in PBS and counted
using a hemocytometer according to the
standard protocol provided by the
Department of Microbiology and Molecular
Genetics at the University of Vermont. Cells
were plated on P35 dishes (25,000 cells per
dis) and incubated overnight, to be used for
transfection for further experimentation.
3. Results
Mutagenic primers ordered from Life
Technologies were calculated to have a
51.51% GC content meeting the minimum
requirement of at least 40%. Melting
temperature was calculated at 84.7 degrees
Celsius; this also met the minimum
requirement of 70 degrees Celsius. Results of
the pre-mutagenesis nanodrop spectrometry
indicated primer 1 had a concentration of
325.2 ng/μL and primer 2 had a concentration
of 206.8 ng/μL. The DNA template
concentration was found to be 173.1 ng/μL.
Figure 4| Gel Electrophoresis of
Restriction Digest Reactions
Images were captured using Photodoc technology. A)
Results of gel electrophoresis on the first restriction digest
reaction. Themolecular weight ladder can be seen at thetop
in well 1 of the first image. Samples were loaded onto well
2 and 7 of the gel. Bands were not observed at the2400 base
pair or 5400 basepair mark. B) Results of gel electrophoresis
on the second restriction digest reaction. The molecular
weight ladder can again be seen on the top of the second
image in well 1. Samples were loaded onto well 3 and 5 of
thegel. Bands were observed at the2400 and 5400 base pair
mark which suggests theplasmid samplecontains molecules
the same molecular weight or close to the same molecular
weight as theplasmid DNA (5400 bp) and cDNA containing
the fur mutation (2400 bp). Gene sequencing is required to
determine if thesebands actually correspond to thesuspected
DNA.

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Preliminary investigation of site-directed
mutagenesis success was analyzed by
growing the transformed E. coli cells on LB-
ampicillin plates to determine if they exhibit
ampicillin resistance. Resistance may suggest
the amp-resistant plasmid was successfully
inserted and the E. coli cells acquired this
resistance; however, the cell may have
mutated and developed this resistance
without taking up the plasmid. Plasmid
purification was analyzed using gel
electrophoresis as seen in Figure 4. Plasmid
digests inserted in well 2 and 7 of gel 1 did
not display bands in the expected area
corresponding to the plasmid DNA at 5400
base pairs or the fur cDNA at 2400 base pairs.
This may have been due to insufficient digest
time. The samples were subjected to gel
electrophoresis a second time to address this
possibility, and the reactions were incubated
10 minutes longer, for a total time of 70
minutes. Bands were seen in the expected
area the second time, suggesting the plasmid
sample contains molecules the same
molecular weight or close to the same
molecular weight as the plasmid DNA (5400
bp) and fur cDNA (2400 bp). Gene
sequencing revealed that the plasmid 1
mutagenesis reaction was unsuccessful, but
the plasmid 2 mutagenesis reaction
successfully mutated phenylalanine-67 to
serine. Sequencing results can be seen in
Figure 5.
Figure 5| Plasmid Sequencing Results for Plasmid 1 (2606355) and Plasmid 2 (2696356)
A) Sanger sequencing performed by the University of Michigan DNA Sequencing Core revealed that plasmid 1 sample number
2606355 was not a successful mutant because phenylalanine-67 coding sequence (TTC) was not altered to the serine coding
sequence (TCC). The mutagenesis reaction was unsuccessful for the first reaction. B) Plasmid 2 sample number 2606356 was
successfully mutated as seen in green, indicating the mutagenesis reaction was successful for this sample.
4. Discussion
In an effort to elucidate the activity of furin,
site directed mutagenesis was utilized to
change phenylalanine at position 67 to serine.
Findings of this study were limited, and
further experimentation is necessary to make
any conclusions about the effects of the F67S
mutation on furin structure, function and
trafficking. DNA sequencing revealed that
phenylalanine was successfully mutated to
serine in the plasmid sample 2, but not in the
plasmid sample 1. The sample was not
transfected into RPE.40 cells as originally
planned due to missing materials, namely the
mutated DNA. This was a major limitation of
this study, as it prevented further
experimentation that would otherwise
provide insight into the mutation’s effects on
furin activity.
Future research would involve using
liposome-mediated transfection, employing
TransIT reagent (Mirus Bio; Madison, WI),
to introduce the successfully mutated DNA
and wild-type fur DNA into mammalian
RPE.40 cells to observe any changes in furin

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activity resulting from the F67S mutation. An
immunofluorescence assay, following the
standard protocol provided by the University
of Michigan-Flint Biology Department,
would be performed to examine furin
trafficking and determine whether the
mutated furin is retained in the endoplasmic
reticulum, or if it is able to successfully move
through the secretory pathway. This would
provide insight into how the mutation may
have altered furin structure and trafficking. A
PEA toxin assay would also be implemented
to examine phenotypic changes that may
have occurred as a result of the mutation.
Mammalian cells containing the mutated
DNA would be exposed to Pseudomonas
Exotoxin A (PEA) to examine whether the
mutation altered furin’s endoprotease
activity.
Immunofluorescence results of the RPE.40
cells transfected with wild-type fur DNA
should show a juxtanuclear pattern indicating
localization to the Golgi, as per usual.
Immunofluorescence of the mutated furin
may show a perinuclear pattern, indicating it
was retained in the ER, or a juxtanuclear
pattern indicating it localized to the Golgi.
Retention in the ER may indicate the
mutation resulted in improper folding of
furin. PEA exposure to the RPE.40 cells
transfected with wild-type fur DNA should
result in cell death. PEA is a substrate of
furin; active furin will process PEA allowing
it to act on EF-2 and inhibit translation
causing the cells to die. PEA treatment to the
RPE.40 cells containing mutated furin would
allow the activation state of the mutated
protein to be determined. If the mutation
rendered furin inactive, PEA exposure should
not result in cells death due to furin’s inability
to process the toxin.
In summary, the F67S mutation may impact
furin structure, function, and trafficking,
however more research is necessary to
determine its effects. Identification of
significant mutations in furin may contribute
to a better understanding of its genetic
components, as well as furin’s role in various
diseases and developmental processes. This
could provide means to improve treatment
and diagnostic techniques for assorted
conditions related to abhorrent furin activity.
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2 Taylor, N. A. "Curbing Activation: Proprotein
Convertases in Homeostasis and Pathology." The
FASEB Journal 17.10 (2003): 1215-227. Web.
3 Thomas, Gary. "Furin at the Cutting Edge: From
Protein Traffic to Embryogenesis and
Disease." Nature Reviews Molecular Cell Biology Nat
Rev Mol Cell Biol 3.10 (2002): 753-66. Web.