Expression of four mutant fibrinogen cC domains in Pichia pastoris confirms them
as causes of hypofibrinogenaemia
Campbell R. Sheen*,1, Amy Dear, Stephen O. Brennan
Molecular Pathology Laboratory, Department of Pathology, University of Otago, Christchurch, New Zealand
a r t i c l ei n f o
Received 1 April 2010
and in revised form 4 May 2010
Available online 16 May 2010
a b s t r a c t
Mutations in the fibrinogen gene cluster can cause low plasma fibrinogen concentrations, known as hyp-
ofibrinogenaemia. It is important to verify whether a detected sequence variant in this cluster is delete-
rious or benign and this can be accomplished using protein expression systems. In this study, four
mutations in the fibrinogen cC domain that had previously been described in patients with hypofibrino-
genaemia were introduced into a cC construct and expressed in a Pichia pastoris yeast system to inves-
tigate their effects on protein stability and secretion. These experiments showed that the fibrinogen
Middlemore (N230D), Dorfen (A289V), Mannheim II (H307Y), and Muncie (T371I) mutations were not
secreted, supporting their causative role in hypofibrinogenaemia. Overexpression of the N230D, A289V
and H307Y mutants revealed that the majority of the synthesised protein was retained in the endoplas-
mic reticulum, with only a minor proportion reaching the trans-Golgi network. Regardless, none of this
protein was secreted which confirms that the four mutations investigated are indeed responsible for
? 2010 Elsevier Inc. All rights reserved.
Fibrinogen is a large dimeric glycoprotein that is activated by
thrombin to form an insoluble fibrin matrix in the terminal step
of the coagulation cascade. It is comprised of three pairs of poly-
peptide chains denoted (Aa Bb c)2and displays a symmetrical tri-
nodular structure with a central globular region formed by the
amino terminals of all six chains. This central ‘E’ region is laterally
connected by helical coiled-coils to two ‘D’ regions formed by the
highly structured C-terminals of the Bb and c chains .
Hypofibrinogenaemia is a disorder that is characterised by low
physical plasma fibrinogen concentrations (less than 1.5 g L?1) and
can be caused by mutations in any of the three fibrinogen genes
(FGA, FGB and FGG). Homozygosity or compound heterozygosity
for fibrinogen gene mutations may cause complete lack of the cir-
culating protein, known as afibrinogenaemia. Quantitative fibrino-
gen mutations may affect transcription, mRNA translation/
processing, protein assembly/folding, secretion or plasma stability
but all ultimately reduce circulating concentrations of the mature
protein (reviewed in [2,3]). While null mutations, such as deletions
and non-sense mutations are obviously sufficient to abolish
expression of fibrinogen chains, the effects of missense mutations
are often less clear. Therefore, it is important to verify the patho-
genic effects of these mutations.
Expression systems have previously been used to investigate
the effects of fibrinogen mutations on function, assembly and
secretion (for example ). These are usually carried out in mam-
malian cell lines, where a mutant gene can be co-transfected with
normal copies of the two other genes. Pichia pastoris is a methylo-
trophic yeast that is widely used to produce recombinant protein.
The system combines prokaryote-like simplicity of culture and
manipulation with eukaryotic protein folding, processing and
post-translational modification machinery [5,6]. The promoter of
the P. pastoris alcohol oxidase 1 gene (AOX1) is so highly inducible
that a protein under its control can comprise up to 30% of soluble
protein in cells cultured in methanol-supplemented media . In
addition, the presence of an a-factor secretion signal can also tar-
get recombinant protein for export from the cell. P. pastoris has
been used to express fibrinogen cC domains for X-ray structure
analyses  and to locate functional sites .
While the fibrinogen cC domain comprises only 18% of the
molecular weight of fibrinogen, 57% of missense mutations that
cause a quantitative defect are located in this region . Here
we present our investigation of four further mutations in the cC
domain (fibrinogens Middlemore (c N230D) , Dorfen (c
A289V) , Mannheim II (c H307Y)  and Muncie (c T371I)
) expressed in the P. pastoris expression system to determine
their effects on protein stability and secretion.
1046-5928/$ - see front matter ? 2010 Elsevier Inc. All rights reserved.
* Corresponding author. Address: University of Otago, Christchurch, PO Box 4345,
Christchurch, New Zealand.
E-mail address: email@example.com (C.R. Sheen).
1C.R.S. is the recipient of a Bright Futures Enterprise Scholarship from the New
Zealand Tertiary Education Commission and a Bayer Haemophilia Awards Special
Protein Expression and Purification 73 (2010) 184–188
Contents lists available at ScienceDirect
Protein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep
Methods and materials
The pPIC9 fusion expression vector (Invitrogen, Carlsbad, CA),
with a coding region spanning amino acid residues 143–411 of
the human fibrinogen c chain (pPIC9-cC) under the control of
the AOX1 promoter was generously supplied by Dr. Tatiana
Ugarova. This vector contains the cC fragment cloned in-frame
with the a-factor secretion signal and the AOX1 transcription
termination (TT2) signal of pPIC9.
Site-directed mutagenesis and DNA sequencing
performed using the QuikChange Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA) and HPLC-purified custom primers
according to the manufacturer’s instructions. Mutagenised plas-
mids were transformed into competent Escherichia coli TOP10 cells
according to the manufacturer’s instructions (Invitrogen, Carlsbad,
CA). Plasmids were extracted and purified using a QIAPrep Spin kit
(Qiagen GmbH, Hilden, Germany). The cC insert, a-factor secretion
signal and AOX1 transcription termination signal of the plasmids
were sequenced with BigDye terminator chemistry v. 3.1 using a
3100xl Avant genetic analyser (Applied Biosystems, Foster City, CA).
mutagenesisof the pPIC9-cC vectorwas
Pichia pastoris expression
Mutant and wild-type pPIC9-cC construct DNA was linearised
by restriction digestion with SalI (New England Biolabs) and used
for spheroplast transformation. Transformation of P. pastoris strain
GS115 with wild-type pPIC9 and mutant pPIC9-cC was performed
using a Pichia expression kit (Invitrogen) according to the manufac-
turer’s instructions. Colonies that grew on histidine-deficient med-
ia (His+) were subjected to methanol utilisation analysis and those
that employed methanol as their sole carbon source (Mut+) were
used in subsequent experiments.
Transformants were cultured for 48 h in Buffered Minimal Glyc-
erol complex medium (BMGY; 2% (w/v) peptone, 1.34% (w/v) YNB,
1% (w/v) yeast extract, 100 mM potassium phosphate (pH 6.0),
400 ng mL?1biotin, 1% (v/v) glycerol). Ten AU600of yeast cells were
subsequently incubated in Buffered Minimal Methanol complex
medium (BMMY; 2% (w/v) peptone, 1.34% (w/v) YNB, 1% (w/v)
yeast extract, 100 mM potassium phosphate (pH 6.0), 400 ng mL?1
biotin, 1% (v/v) methanol) for 96 h to induce cC domain protein
expression, with supplementary addition of 1% methanol every
24 h. Culture supernatants were concentrated by precipitation
with 55% (w/v) ammonium sulphate in order to purify the cC do-
main. Cell pellets were lysed by addition of an equal volume of
2? reducing SDS–PAGE sample buffer and incubation at 99 ?C for
5 min. Cellular debris was pelleted by centrifugation and the
supernatant was used for SDS–PAGE.
Genomic DNA was extracted from yeast cultured in Minimal
Glycerol medium (MGY; 1.34% (w/v) yeast nitrogen base (YNB),
1% (v/v) glycerol, 400 ng mL?1biotin) as previously described
. Southern hybridisation analysis was performed to determine
the copy number of vectors integrated into the P. pastoris genome
. Genomic DNA was digested with Hind III and a 367 bp PCR
product, corresponding to a region of the AOX1 TT sequence, was
used as a hybridisation probe.
Colony spot immunoassay
Colony spot immunoassays were performed as previously de-
scribed . Briefly, 0.03 AU600of yeast cultured in BMMY was
spotted in duplicate onto nitrocellulose membrane (Millipore,
Billerica, MA) and overlaid onto a Minimal Methanol (MM; 1.34%
YNB, 1% (v/v) methanol, 400 ng mL?1biotin) agar plate. The plate
was incubated at 30 ?C for 72 h before lysing the yeast by placing
the nitrocellulose membrane on filter paper saturated with 0.2 M
NaOH, 0.1% (w/v) SDS and 0.05% (v/v) b-mercaptoethanol for 1 h.
After lysis, 0.2 lg of purified human fibrinogen was spotted onto
the membrane as a positive control. The membrane was blocked
in blocking solution (10% (w/v) low fat milk powder dissolved in
1? Tris-buffered saline) supplemented with 0.02% (v/v) Tween-
20 solution before incubation in a rabbit polyclonal antibody to
human fibrinogen, diluted 1:1000 in blocking solution. Following
primary antibody incubation, the membrane was incubated with
a peroxidase labelled anti-rabbit secondary antibody diluted
1:10,000 in blocking solution. Protein was detected using
Biotechnology, Rockford, IL, USA) and exposure to radiographic film.
Western blot analysis of the culture fractions was carried out
by reducing samples in SDS–PAGE buffer containing 10% (v/v)
b-mercaptoethanol and denaturation at 99 ?C for 5 min. Samples
were run on a 10% Bis-acrylamide gel, with normal human fibrin-
ogen as a positive control. After protein transfer to Hybond-C extra
nitrocellulose membrane (GE Healthcare) by electroblotting in
Tris–glycine buffer with 10% methanol the membrane was sub-
jected to chemiluminescent detection as described above.
Electrospray ionisation mass spectrometry
Electrospray ionisation mass spectrometry (ESI MS) of isolated
recombinant proteins was performed using a Platform II mass
spectrometer (VG Instruments Ltd., Manchester, UK) operating in
positive ion mode according to the method of Brennan .
E. coli transformants for each mutagenesis reaction were se-
lected for liquid culture followed by plasmid extraction and
sequencing to confirm the introduction of the mutation. Mutant
plasmid DNA was transformed into P. pastoris GS115 and resulting
His+colonies (indicating successful transformation) with wild-type
levels of methanol utilisation (Mut+) were used for subsequent
experiments. Genomic DNA was extracted from each transformant
and was used as a template for PCR amplification of the integration
locus. DNA sequencing of these PCR products showed that each
transformant had the expected mutation and that the rest of the
pPIC9-cC vector was intact (not shown).
Southern hybridisation was used to detect multiple-copy tan-
dem integration events. The 2322 bp fragment detected by the
AOX1 transcription termination region probe represented the na-
tive P. pastoris AOX1 locus (present at a single copy per genome)
and the 1232 bp fragment identified multiple integrations of the
pPIC9-cC vector per genome. The copy number was determined
by dividing the intensity of the pPIC9-cC band by that of the
AOX1 band. Non-specific cross-hybridisation of the probe with a
fragment of approximately 1300 bp present at one copy per
2Abbreviations used: SS, secretion signal; TT, transcription termination; YNB, yeast
nitrogen base; ESI MS, electrospray ionisation mass spectrometry; ER, endoplasmic
reticulum; TGN, trans-Golgi network.
C.R. Sheen et al./Protein Expression and Purification 73 (2010) 184–188
genome was observed, and may represent the homologous AOX2
gene. Efforts to eliminate this cross-hybridisation by changing
the hybridisation buffer and increasing the hybridisation and wash
temperatures were unsuccessful, but in any event it did not inter-
fere with the densitometric analysis. For each of the four mutants,
at least two of the four transformants were present at one copy,
while the remaining transformants were present at between two
and seven copies (Fig. 1). Expressed cC proteins are referred to
with a subscript of their mutation, or WT for wild-type, and a suffix
denoting their copy number.
Reverse-phase HPLC ofcCWT.1supernatant concentrated 60-fold
by ammonium sulphate precipitation showed a major peak that
eluted at 18 min (not shown). Subsequent ESI MS analysis showed
that the major component of this had a mean molecular mass of
30,472 Da (not shown), consistent with the theoretical mass of
30,471 Da for the Y-V-cC protein following signal peptidase,
Kex2 and Ste13 cleavage of the yeast a-factor pre-pro-peptides.
ESI MS of peptides derived by tryptic digestion of the cC domain
identified peaks with m/z values corresponding to the N- and C-ter-
minal peptides, as well as numerous other representative peptides
Colony spot immunoassay was used to determine the total
expression of the cC domain for each transformant (that is, the
sum of intracellular and extracellular cC protein) as it is a rapid
method capable of simultaneously analysing several transfor-
mants. This showed that for the cC307Y, cC230Dand cC371Isingle-
copy-transformants, nocC protein could be detected (Fig. 2). While
some protein was detected for cC289V.1, this was only 2% of wild-
type levels. Multiple-copy-transformants were also assayed and
showed the different mutations apparently exert different degrees
of instability on the cC domain. For example, a cC289V.4transform-
ant showed over twice the expression of cC307Y.4and was approx-
imately 25% greater than cC230D.7(Fig. 2).
The presence of some mutantcC in the high copy number trans-
formants was validated by Western blots of concentrated culture
supernatants and whole cell lysates (Fig. 3). For each of the four
introduced mutations, one single-copy-transformant and the
transformant with the highest vector copy number (cC230D.7,
cC307Y.4, cC289V.4and cC371I.2) were analysed. For on each of the
single-copy-transformant blots, the culture supernatant from 1
AU600of cCWT.1cells was readily detected, but no protein was de-
tected in samples of concentrated supernatant corresponding to 50
AU of mutant cells. This indicates that secretion of mutant cC do-
mains is less than 2% of wild-type (Fig. 3). Overexposure of each of
the blots to the film for at least 2 h did not show any further spe-
cies for any of the lanes (not shown). Similarly, for the mutant mul-
supernatant (Fig. 3, right blots), indicating that even at high
expressions levels, the mutant cC modules could not be secreted.
While no protein was detected in the cell lysate of any of the sin-
gle-copy-transformants, the 289V, 307Y and 230D multiple-copy-
transformants all displayed intracellular cC domains. The amount
of protein detected in cC289V.4and cC230D.7was higher than for
cC307Y.4with both of the former showing detectable levels of pro-
tein at 0.1 AU, while trace amounts were detected only at 1 AU for
cC307Y.4. The predominant species identified in all three of these
transformants was a high molecular weight protein of ?48 kDa.
This protein has been characterised and corresponds to the cC do-
main that has undergone signal peptidase cleavage in the endo-
plasmic reticulum (ER), but still has the a-factor pro-sequence
attached as it has not been exposed to Kex2 or Ste13 peptidase
cleavage in the trans-Golgi network (TGN) . Thus, the occur-
rence of the majority of protein in this form indicates it had not
progressed past the ER to the TGN. The presence of small amounts
of 30 kDa material in three of the mutants indicates that a minor
fraction of the protein reached the TGN. No intracellular protein
was detected in cC371I.2, even at a loading of 10 AU600, perhaps
due to it possessing only two copies of the pPIC9-cC371Ivector.
wasdetected in the
The cC domain was the first region of the fibrinogen molecule
to have its structure determined at high resolution and this was
facilitated by expression of the natively folded functional domain
in P. pastoris . In this study we expressed four cC domain muta-
tions that were originally discovered in patients with hypofibrino-
genaemia in P. pastoris to confirm the causal relationship between
the mutations and the phenotype. ESI MS had previously been used
to show that heterozygotes for the Dorfen (A289V), Mannheim II
(H307Y) and Muncie (T371I) mutations lacked the variant chain
in their circulating fibrinogen , and ESI MS was uninformative
in the case of fibrinogen Middlemore (N230D) .
The expression studies performed here unambiguously show
that all four mutations prevent export of the cC module into the
cell supernatants, confirming the link between mutation and low
fibrinogen expression levels. Failure of normal fibrinogen export
Fig. 1. Southern hybridisation analysis of vector copy number. Genomic DNA from
untransformed P. pastoris GS115 (G) and P. pastoris GS115 transformed with the
empty pPIC9 vector (p), wild-type pPIC9-cC (c) and each of the mutant pPIC9-cC
vectors was digested with Hind III. The probe hybridised to the AOX1 transcription
termination (TT) signal in both the pPIC9-cC vector and the native AOX1 locus. Copy
numbers indicated are the mean of three separate hybridisations. (M), 1 kb DNA
Fig. 2. Colony spot immunoassay of wild-type and mutant fibrinogencC domain expression in Pichia pastoris. The intensity of each spot corresponds to the total amount ofcC
expressed. The expression level, relative to the wild-type control, was calculated by densitometry and is displayed beneath each duplicate immunoassay. Transformant copy
numbers are shown beside each panel. The displayed results are representative of three separate analyses.
C.R. Sheen et al./Protein Expression and Purification 73 (2010) 184–188
from hepatocytes could result from either polypeptide chain insta-
bility or failure of assembly of the mature six-chain (Aa Bb c)2
structure. As there is no assembly step involved in the yeast sys-
tem, the lack of expression in the culture media indicates the
mutations cause an intrinsic defect in polypeptide folding and/or
No intracellular material was detected in any of the single-copy-
transformants showing that the variant protein was turned over
(Fig. 3). However, with over expression from the multi-copy-trans-
formants significant intracellular accumulation was seen, particu-
larly for cC289V.4and cC230D.7. The 230D and 307Y transformants
both showed less intracellular accumulation per copy number than
the 289V mutant indicating that they are more efficiently turned
over, and that cC289V is more liable to escape the degradation
machinery, which may render it susceptible to aggregation.
In all cases the dominant intracellular form of the over ex-
pressed protein was the 48 kDa ER-specific form. Only a small por-
tion (5–15%) of the intra cellular material showed evidence of
having reached the TGN and being cleaved (by Kex2 protease) to
the 30 kDa protein . The absence of this 30 kDa mature form
from the culture supernatant indicated that it too lacked the struc-
tural integrity necessary for secretion and it that was targeted for
quality control degradation. Studies with mutant yeast strains that
lack different components of ‘‘housekeeping” pathways have
shown that the 30 kDa form is targeted to vacuoles to be degraded,
while the 48 kDa form is degraded by ER-associated degradation
(ERAD) after translocation to the cytosol and proteosome .
In conclusion the findings here confirm the causal role of the
Middlemore (N230D), Dorfen (A289V), Mannheim II (H307Y) and
Muncie (T371I) mutations in hypofibrinogenaemia, and suggest
P. pastoris may prove to be relatively inexpensive expression sys-
tem for analysing the effects of cC domain mutations on fibrinogen
 C.E. Hall, H.S. Slayter, The fibrinogen molecule: its size, shape, and mode of
polymerization, J. Biophys. Biochem. Cytol. 5 (1959) 11–16.
 G.J. Maghzal, S.O. Brennan, V.M. Homer, P.M. George, The molecular
mechanisms of congenital hypofibrinogenaemia, Cell Mol. Life Sci. 61 (2004)
 D. Vu, M. Neerman-Arbez, Molecular mechanisms accounting for fibrinogen
deficiency: from large deletions to intracellular retention of misfolded
proteins, J. Thromb. Haemost. 5S1 (2007) 125–131.
 D. Vu, P.H. Bolton-Maggs, J.R. Parr, M.A. Morris, P. de Moerloose, M. Neerman-
Arbez, Congenital afibrinogenemia: identification and expression of a missense
mutation in FGB impairing fibrinogen secretion, Blood 102 (2003) 4413–4415.
 J.M. Cregg, D.R. Higgins, Production of foreign proteins in the yeast Pichia
pastoris, Can. J. Bot. 73 (1995) S891–S897.
 G. Sberna, R. Cappai, A. Henry, D.H. Small, Advantages of the methylotrophic
yeast Pichia pastoris for high-level expression and purification of heterologous
proteins, Australas. Biotechnol. 6 (1996) 82–87.
 R. Couderc, J. Baratti, Oxidation of methanol by the yeast, Pichia pastoris.
Purification and properties of the alcohol oxidase, Agric. Biol. Chem. 44 (1980)
 V.C. Yee, K.P. Pratt, H.C. Cote, I.L. Trong, D.W. Chung, E.W. Davie, R.E. Stenkamp,
D.C. Teller, Crystal structure of a 30 kDa C-terminal fragment from the c chain
of human fibrinogen, Structure 5 (1997) 125–138.
 H.C. Cote, K.P. Pratt, E.W. Davie, D.W. Chung, The polymerization pocket ‘‘a”
within the carboxyl-terminal region of the gamma chain of human fibrinogen
is adjacent to but independent from the calcium-binding site, J. Biol. Chem.
272 (1997) 23792–23798.
 M. Hanss, F. Biot, A database for human fibrinogen variants, Ann. N. Y. Acad.
Sci. 936 (2001) 89–90.
 S.O. Brennan, C.R. Sheen, P.M. George, Novel gamma230 Asn?Asp substitution
in fibrinogen Middlemore associated hypofibrinogenaemia, Thromb. Haemost.
93 (2005) 1196–1197.
 A. Dear, S.O.Brennan, C.E.Dempfle,
Hypofibrinogenaemia associated with a novel heterozygous c289 AlaVal
substitution (fibrinogen Dorfen), Thromb. Haemost. 92 (2004) 1291–1295.
 A. Dear, C.E. Dempfle, S.O. Brennan, W. Kirschstein, P.M. George, Fibrinogen
Mannheim II: a novel c307 His –>Tyr substitution in the cD domain causes
hypofibrinogenemia, J. Thromb. Haemost. 2 (2004) 2194–2199.
 S.O. Brennan, J.M. Wyatt, A.P. Fellowes, J.S. Dlott, D.A. Triplett, P.M. George,
c371 Thr –>Ile substitution in the fibrinogen gammaD domain causes
hypofibrinogenaemia, Biochim. Biophys. Acta 1550 (2001) 183–188.
W. Kirschstein,P.M. George,
Fig. 3. Anti-fibrinogen Western blots of 10% reducing SDS–PAGE of fibrinogen cC mutant domains. (P), Normal plasma corresponding to 60 ng fibrinogen for cC289Vand
cC230D, 70 ng forcC371Iand 80 ng forcC307Y; 1? corresponds to 1 AU600of cells. LMW and HMW represent low molecular weight and high molecular weight variants of thecC
domain and the Aa, Bb and c chains of plasma fibrinogen are indicated. Blots were exposed to film for 2 min. SN indicates culture supernatant and Lys indicates cell lysate.
C.R. Sheen et al./Protein Expression and Purification 73 (2010) 184–188
 C.S. Hoffman, Preparation of yeast DNA, in: F.M. Ausubel, R. Brent, R.E. Download full-text
Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 1997.
 J.J. Clare, F.B. Rayment, S.P. Ballantine, K. Sreekrishna, M.A. Romanos, High-
level expression of tetanus toxin fragment C in Pichia pastoris strains
containing multiple tandem integrations of the gene, Biotechnology (NY) 9
 K.B. Kruse, A. Dear, E.R. Kaltenbrun, B.E. Crum, P.M. George, S.O. Brennan,
reticulum via endoplasmic reticulum-associated protein degradation and
autophagy: an explanation for liver disease, Am. J. Pathol. 168 (2006)
 S.O. Brennan, Electrospray ionisation analysis of human fibrinogen, Thromb.
Haemost. 78 (1997) 1055–1058.
C.R. Sheen et al./Protein Expression and Purification 73 (2010) 184–188