Production and stabilization of the trimeric influenza
hemagglutinin stem domain for potentially broadly
protective influenza vaccines
Yuan Lua, John P. Welsha, and James R. Swartza,b,1
Departments of aChemical Engineering and bBioengineering, Stanford University, Stanford, CA 94305
Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved November 15, 2013 (received for review May 7, 2013)
The rapid dissemination of the 2009 pandemic H1N1 influenza
virus emphasizes the need for universal influenza vaccines that
would broadly protect against multiple mutated strains. Recent
efforts have focused on the highly conserved hemagglutinin (HA)
stem domain, which must undergo a significant conformational
change for effective viral infection. Although the production of
isolated domains of multimeric ectodomain proteins has proven
difficult, we report a method to rapidly produce the properly
folded HA stem domain protein from influenza virus A/California/
05/2009 (H1N1) by using Escherichia coli-based cell-free protein
synthesis and a simple refolding protocol. The T4 bacteriophage
fibritin foldon placed at the C terminus of the HA stem domain
induces trimer formation. Placing emphasis on newly exposed pro-
tein surfaces, several hydrophobic residues were mutated, two
polypeptide segments were deleted, and the number of disulfide
bonds in each monomer was reduced from four to two. High pH
and Brij 35 detergent emerged as the most beneficial factors for
improving the refolding yield. To stabilize the trimer of the HA
stem-foldon fusion, new intermolecular disulfide bonds were fi-
nally introduced between foldon monomers and between stem
domain monomers. The correct immunogenic conformation of
the stabilized HA stem domain trimer was confirmed by using
antibodies CR6261, C179, and FI6 that block influenza infection
by binding to the HA stem domain trimer. These results suggest
great promise for a broadly protective vaccine and also demon-
strate a unique approach for producing individual domains of com-
plex multimeric proteins.
V accination is the most effective way to protect against in-
fluenza virus infection, with most of the neutralizing anti-
bodies recognizing the hemagglutinin (HA) protein on the
surface of the virus. As of 2007, all commercial influenza vac-
cines were produced in embryonated chicken eggs, but the
generation of a new vaccine takes six to eight months, making it
ineffective for combating potential pandemics (1). In 2007, to
partially address this concern, the European Union approved
Optaflu, a vaccine produced by Novartis using a mammalian cell
line (2). In 2013, the recombinant HA vaccine (Flublok) manu-
factured in insect cells by Protein Sciences was also licensed in
the United States (3). However, the new processes would still
likely only improve production times by several weeks (4).
Hence, rapid production (for example in 1–2 mo) of large
amounts of vaccine to stop an epidemic/pandemic remains an
important objective. This challenge can be addressed in two
ways, either by producing a more broadly protective vaccine to
lower the probability of a pandemic occurrance or by developing
new, rapid, and scalable technologies for vaccine production.
This work seeks to address both.
The trimeric HA ectodomains consist of a head domain and
a stem domain (Fig. 1A). The assembled HA ectodomain also
consists of HA1 and HA2 domains, which are defined by a spe-
cific proteolytic cleavage (5). The majority of protective anti-
bodies bind to the HA globular head domain. Consequently, new
viral strains arise with the most common mutations occurring in
the head domain, thereby avoiding initial antibody suppression.
www.pnas.org/cgi/doi/10.1073/pnas.1308701110
Because the HA head domain evolves with a high mutation rate,
the influenza vaccine has to be regularly updated. The stem
domain, on the other hand, is much more conserved (SI Ap-
pendix, Fig. S1) (6, 7). At low pH (5∼6), as the influenza virus is
trafficking in endosomal vesicles, the trimeric HA stem domain
undergoes a conformational change to trigger fusion to the
endosomal membrane, thereby enabling RNA release and suc-
cessful infection (8). Several research groups have devised con-
structs consisting of portions of this stem domain in hopes of
producing a broadly protective influenza vaccine (1, 9, 10).
However, none have produced properly folded stem trimer.
Recent work suggests that antibodies generated against this stem
domain can cross-react between different virus subtypes, lending
hope toward using this domain as an antigen for broadly pro-
tective influenza vaccines (10–13). A vaccine, multivalent against
several stem subtypes, would not need annual updates and could
dramatically reduce the risk of influenza pandemics.
The natural production mechanisms suggest that HA stem
production would be technically challenging. The first challenge
is that the HA stem domain has not evolved to fold and trimerize
as an independent unit. Further, its contemporaneous folding
along with that of the head domain most likely occurs cotrans-
lationally as first part of the stem, then the head, and finally the
rest of the stem domain are extruded from the ER membrane
and are orientated by this association. During this process,
disulfide bonds must form within each monomer while avoiding
intermonomer linkage. Finally, the absence of the head domain
exposes internal HA polypeptides that may now be disordered,
hydrophobic, or otherwise inappropriate as surface epitopes for
Significance
The discovery of neutralizing antibodies that block influenza
APPLIED BIOLOGICAL
infection by binding to the hemagglutinin (HA) stem domain
raised the hope for broadly protective vaccines. These could
SCIENCES
avoid the need for annual vaccinations and reduce pandemic
threats, and the stem subdomain of the trimeric HA ectodo-
main would be an ideal antigen. However, its production has
proven extremely difficult. Here, we describe a simple pro-
cedure resulting in high yields of HA stem trimer recognized
identically by a panel of neutralizing antibodies as compared
with recognition of the full-length HA ectodomain. Cell-free
protein synthesis is followed by a simple refolding procedure
to produce a rationally mutated stem in which newly exposed
protein surfaces are modified and trimerization is induced and
covalently stabilized.
Author contributions: Y.L., J.P.W., and J.R.S. designed research; Y.L. and J.P.W. performed
research; Y.L. contributed new reagents/analytic tools; Y.L., J.P.W., and J.R.S. analyzed
data; and Y.L., J.P.W., and J.R.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: jswartz@stanford.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1308701110/-/DCSupplemental.
PNAS | January 7, 2014 | vol. 111 | no. 1 | 125–130
Fig. 1. (A) Structural features of the influenza virus HA trimer and its head and stem domains. HA1 regions are shown in blue, and HA2 regions are in cyan.
The figure was derived from the X-ray structure of HA from the (H1N1) isolate A/California/05/2009 (H1N1) (PDB ID code 3LZG) and was drawn by using the
program PyMOL. Protein yields (B) and autoradiograms (C) of nonreducing SDS/PAGE gels of CFPS products. The arrow indicates the monomer. Stem: no
trimerization domain, 32 kDa; Stem-foldon: with the foldon trimerization domain, 36 kDa; Stem-CAT: with the CAT trimerization domain, 58 kDa; S, soluble;
T, total protein. Autoradiogram from nonreducing SDS/PAGE gels of refolded proteins (D) and pI analysis of HA protein fragments (E). Polypeptides were
refolded with different pH environments (pH 6.0, 8.0, or 10.5) and with (+) or without (−) Tween 20 (0.05%). The theoretical pI values were calculated by
using the program ProtParam. All of the residue numbers were in H3 numbering.

stable and soluble protein. Because of these complications, in
vivo Escherichia coli expression produced low yields of only 2 mg/L,
and the HA stem polypeptides accumulated as inclusion bod-
ies (1). Although some refolding methods have been attempted
(14–16), the recovery yields of soluble products have been
low, and properly folded stable trimeric assembly has not been
confirmed.
In this study, we demonstrated the production of HA stem
domain from the 2009 pandemic H1N1 strain by using E. coli-
based cell-free protein synthesis (CFPS) production methods
followed by protein refolding. For inducing the trimerization of
the HA stem domain, either a CAT or foldon domain was fused
to the C terminus. To reduce the chance of the formation of
intermolecular disulfide bonds, several HA stem domain muta-
tions were introduced, including removal of hydrophobic and
positively charged residues and decreasing the number of disul-
fide bonds. Protein refolding methods were also optimized. The
trimer was further stabilized by introducing disulfide bonds be-
tween foldon domains and in the distal region of the stem trimer.
The correct immunogenic conformation of this stabilized HA
stem trimer was confirmed by using antibodies CR6261, C179,
and FI6 that block influenza infection by binding to the HA stem
trimer at a site that inhibits the structural changes required for
membrane fusion. Production consistency and high recovery
yields were also demonstrated.
Results and Discussion
Production and Refolding of HA Stem Domain Trimer. The stem
domain of the HA protein is considerably more conserved than
the head domain (1, 9). HA is classified into 17 subtypes (H1–
H17) based on antigenicity and sequence diversity (17), and, in
particular, the stem domains in the same subtype are highly
conserved (SI Appendix, Fig. S1). Two subtypes of influenza A,
H1N1 and H3N2, most commonly infect humans. Therefore, for
example, a vaccine presenting these two stem domains could
serve as a broadly protective vaccine.
In historical records of previous influenza pandemics, H1N1
influenzas occurred most often and were severe, so the HA stem
domain from the influenza virus A/California/05/2009 (H1N1)
was chosen as the first target. It is highly likely that the HA stem
domains from other subtypes could also be produced by using
the same protocol described in this work. Because HA is active
as a trimer on the viral surface, two different sequences were
fused to the C terminus of the stem domain to induce trimeri-
zation. The first was a 29-aa “foldon” sequence that forms a β-
propeller structure comprising the C terminus of the fibritin
domain of the T4 bacteriophage (18). This domain has been used
as a trimerization domain for HA (12, 19). The second candidate
trimerization domain was chloramphenicol acetyl transferase
(CAT), which expresses and folds well in typical CFPS reactions
and also naturally forms a trimer (20). Both domains were linked
to the HA sequence by a cleavable tobacco etch virus (TEV)
protease site and also contained a His6 tag at the C terminus.
The E. coli-based CFPS system was used for this study because
it has many advantages for rapidly screening protein constructs
compared with traditional E. coli expression systems (21). First,
direct use of plasmids or PCR templates as expression tem-
plates avoids time-consuming molecular cloning steps. Second,

126 | www.pnas.org/cgi/doi/10.1073/pnas.1308701110 Lu et al.

milligrams of protein can be produced in a few hours in multiwell
plates and then directly purified without a cell-lysis step. Third,
the lack of a cell wall barrier allows for easy manipulation of
reaction conditions, including the incorporation of isotopically
labeled amino acids. The E. coli-based CFPS system is linearly
scalable from micro-scale (20 μL) to large-scale reactions (100 L)
(22, 23) with nearly identical process performance. The system
has also been shown to produce active pharmaceutical proteins
(22–24).
Initial microscale (20 μL) CFPS reactions tested expression of
the HA stem construct either alone or with the trimerization
domains. These reactions were conducted at 30 °C for 6 h and
produced high total yields but much lower soluble yields (Fig.
1B). Nonreducing SDS/PAGE with autoradiography (Fig. 1C)
indicated no significant accumulation of soluble monomer, and
the higher molecular weight species indicated the formation of
incorrect intermolecular disulfide bonds.
Refolding of the inclusion bodies was then attempted, using
a dialysis refolding method (25–27) under different pH con-
ditions. Eight molar urea was used to solubilize the inclusion
bodies and the HA stem protein was purified by using the His6
tag. Oxidized (GSSG) and reduced glutathione (GSH) (molar
ratio 1:4) were added to establish a sulfhydryl/disulfide redox
environment for the formation and potential isomerization of
correct disulfide bonds. L-arginine (0.5 M) and the detergent,
Tween 20 (0.05% wt/vol), were also added to assist in refolding.
Three different pH values (6.0, 8.0, and 10.5) were tested (Fig.
1D). At pH 6.0, most of the protein was lost, apparently adhering
to the dialysis membrane. At pH 8.0, most reaggregated, whereas
at pH 10.5, less aggregation occurred. However, the soluble
fraction from the pH 10.5 procedure also aggregated when the
pH was reduced to 8.0. These observations prompted us to ex-
amine regional isoelectric point (pI) values for the polypeptide
calculated by using the program ProtParam (28) (Fig. 1E).
The theoretical pI values of Stem-HA1-Fragment1 (pI 8.85),
Stem-HA1-Fragment2 (pI 9.10), and Stem-HA2-Fragment1
(pI 8.60) were above 8.5, whereas the pI values of other stem
domain fragments were less than 5. The divergent pIs suggested
the potential for significant interdomain ionic attractions at
neutral pH that would largely be avoided at pH 10.5. Fur-
thermore, because the detergent, Tween 20, reduced aggre-
gation, inappropriate hydrophobic interactions were also
suggested. Finally, significant aggregation resulted at all three
pHs with the CAT trimerization fusion and this folding partner
was abandoned.
Fig. 2. (A) HA stem constructs with different mutations in the exposed hydrophobic region. (B) Size-exclusion HPLC profiles of refolded proteins (wild-type,
mutants M1, M2, M3, M4, and M5). Dotted red traces indicated 14C radioactivity after 14C leucine incorporation during CFPS. (C) Three-dimensional structures
of mutants M5 and M6. (D) Autoradiogram of nonreducing SDS/PAGE gel of refolded HA stem domain mutants M5 and M6.
Lu et al.
Mutations of Hydrophobic Regions. Based on these considerations,
five sets of mutations were designed (Fig. 2A) to either mitigate
newly exposed hydrophobicity (SI Appendix, Fig. S2), reduce the
potential for intermolecular ion pairing, or both. Five different
groups of mutations were evaluated, M1 (I69T + I72E + I74T +
C77T), M2 (I69T + I72E + I74T + C77T + F164D), M3 (I69T +
I72E + I74T + C77T + F164D + L174D), M4 (F164D), and M5
(F164D + L174D). The mutations decreased the pIs of two
newly exposed stem domain fragments (Stem-HA1-Fragment1
and Stem-HA2-Fragment1) (SI Appendix, Table S1). It should be
noted that the targeted mutations are distant from the surface
recognized by the neutralizing antibody (12). However, the CFPS
soluble yields of these mutants were still low (SI Appendix, Fig.
S3). The insoluble inclusion bodies were then refolded by using
the dialysis refolding protocol at pH 10.5 (SI Appendix, SI
Materials and Methods), and the products were evaluated us-
ing size-exclusion HPLC (Fig. 2B). Mutants M3 and M5 produced
much fewer aggregates than the wild-type or other variants. The
most influential mutations appeared to be F164D + L174D.
Therefore, mutant M5 (F164D + L174D) was used for further
development.
Improving Protein Refolding at pH 8.0. Although the high pH (10.5)
folding conditions and the M5 mutation increased the soluble
fraction after refolding, when the pH was then lowered to 8.0,
the product still became insoluble. We therefore focused on
adjusting components in the dialysis buffer to improve folding at
pH 8.0, thereby avoiding the need for subsequent pH change.
Many factors can affect protein refolding, such as pH, chaotropic
agent choice, -S-H/S-S redox ratio, ionic strength, buffer choice,
and low molecular weight additives such as arginine and deter-
gents. Detergent Brij 35 was more effective than Tween 20 as
judged by SDS/PAGE and size-exclusion HPLC analysis (SI
Appendix, Fig. S4). Because the HA stem domain protein con-
tains four disulfide bonds, the choice of sulfhydryl/disulfide redox
buffer was next examined. The cystamine/cysteamine system was
more effective than GSSG/GSH (SI Appendix, Fig. S5). Cyst-
amine and cysteamine are smaller molecules than GSSG and
GSH and possibly can more readily diffuse into the protein
structure to assist in disulfide formation and interchange.
To identify the best refolding conditions while evaluating
seven different factors, an orthogonal (design of experiment)
procedure was designed with the seven factors evaluated at two
levels (SI Appendix, Table S2). The seven factors are buffer
choice (50 mM Tris or 50 mM histidine), [Brij 35] (0% or 0.03%),
APPLIED BIOLOGICAL
SCIENCES
PNAS | January 7, 2014 | vol. 111 | no. 1 | 127
Fig. 3. Introduction of disulfide bonds between foldon monomers. (A) Three-dimensional structures of foldon trimer with six possible disulfide bond
positions. Autoradiogram of a nonreducing SDS/PAGE gel (B) and size-exclusion HPLC profiles (C) of refolded HA stem construct mutant M6 (O) and constructs
with the six different foldon mutations.
[arginine] (0 mM or 500 mM), [NaCl] (0 mM or 150 mM), [su-
crose] (0% or 10%), [glycerol] (0% or 10%) and [urea] (0 M or
0.5 M). The data were analyzed by using IBM SPSS Statistics
software. The results showed that Brij 35 was the most important
factor for improving the refolding yield (SI Appendix, Table S3).
Arginine (500 mM) was also marginally beneficial for suppres-
sion of protein aggregation in the refolding process and was in-
cluded in the refolding buffer.
Furthermore, two different refolding protocols, dialysis refold-
ing and dilution refolding, were compared. These two protocols
produced similar refolding results (SI Appendix, Fig. S6). The di-
lution refolding protocol is much faster and more convenient and
was used in the subsequent experiments. Although the preceding
changes improved protein refolding results, SDS/PAGE analysis
indicated that intermolecular disulfide bonds were still impeding
proper folding.
Targeted Deletions for Further Improvement. To avoid the forma-
tion of undesired intermolecular S-S bonds, further reduce sur-
face hydrophobicity and pI, and avoid regions with possibly
disordered structure, mutant M5 was further modified by de-
leting two polypeptide regions (H38 to C43 and C49 to N61)
containing hydrophobic residues, two positively charged lysines,
and three cysteines (Fig. 2C). Cysteine 77 was also mutated to
threonine to remove a cysteine that was then unpaired. Again,
the deleted regions are far away from the neutralizing antibody
binding region. The number of disulfide bonds in each monomer
was thereby decreased from 4 to 2. This mutant was named M6.
SDS/PAGE results (Fig. 2D) showed that these modifications
greatly decreased the formation of undesired intermolecular
disulfide bonds.
Stabilization of the Stem-Foldon Trimer. The HA stem trimer may
be used as an independent vaccine antigen or could possibly be
conjugated to virus-like particles (VLPs) or other carriers. In ei-
ther case, it will be important for the trimeric form to be stable
during manufacturing, storage, and administration. Reliable sta-
bility is particularly important during the Cu(I) catalyzed click
conjugation reaction. Although the foldon domain proved effec-
tive in forming the trimers, we reasoned that the limited number
of intermolecular hydrogen bonding attractions in the foldon may
not sufficiently stabilize this unnatural ectodomain trimer.
To stabilize the HA stem trimer, we introduced new cysteine
residues into the foldon domain to potentially form intermolecular
disulfide bonds between foldon monomers. Beginning with mu-
tant M6, we introduced cysteines at the six following locations to
evaluate which pairs could most effectively form intermolecular
disulfide bonds (A: G12-D19; B: R10-D19; C: R10-G20; D: P9-
R20; E: A14-K18; and F: Y15-R17), as shown in Fig. 3A. SDS/
PAGE results (Fig. 3B) showed that mutant E formed the most
disulfide-linked trimer. Size-exclusion HPLC results were con-
sistent with this interpretation (Fig. 3C).
128 | www.pnas.org/cgi/doi/10.1073/pnas.1308701110
To further confirm the formation of intermolecular S-S bonds
between foldon monomers, we used the TEV protease cleavage
site between the HA stem domain and foldon domain to release
the foldon trimer after the refolding procedure. Nonreducing
SDS/PAGE (SI Appendix, Fig. S7) of the released foldon
domains also showed that version E enabled the best formation
of disulfide-linked trimers. This mutant was used in subsequent
development.
Further Polypeptide and Folding Protocol Adjustments. To increase
the proportion of trimer and the completeness of intermolecular
disulfide bond formation within the foldon trimer, the refolding
conditions were further examined. Using cystamine and cyste-
amine as the “oxido-shuffling” reagent, we compared several
ratios, 1:4, 1:10, and 1:20. During such procedures, oxygen-
driven oxidation can modify the sulfhydryl/disulfide redox po-
tential over time, and a higher fraction of trimer was obtained
anaerobically. We therefore conducted refolding procedures in
an anaerobic glove box (SI Appendix, Fig. S8), and the cystamine
to cysteamine 1:20 ratio was best (SI Appendix, Fig. S9). The final
composition of the modified refolding buffer was as follows: 50
mM Tris·HCl, 600 mM arginine, 2 mM EDTA, 0.5 mM cyst-
amine/10 mM cysteamine, and 0.05% Brij35 at pH 8.
Although a higher proportion of trimer was obtained after
improving refolding conditions with the M6E mutant, we still
observed dimer and monomer bands. In the initial design of the
HA stem domain antigen, the foldon domain is separated from
the HA stem sequence by a cleavable TEV protease site and the
fusion protein also has a C-terminal His6 tag. We hypothesized
that the TEV protease site and the direct linkage to the His6 tag
could have impeded the trimer formation or formation of the
intermolecular S-S bonds in the foldon trimer. To structurally
isolate the foldon domain, stem trimer mutant M6 with foldon
mutation E (M6E) was modified by replacing the TEV protease
Fig. 4. Newly designed HA stem construct (M6EL) based on mutant M6 with
foldon mutation E (M6E). (A) Schematic diagram of new HA stem construct
design. (B) Autoradiogram of nonreducing SDS/PAGE gel of refolded M6EL
HA stem trimer.
Lu et al.
Fig. 5. Introduction of intermolecular disulfide bonds between stem monomers in the stem trimer. (A) The position of newly introduced intermolecular S-S
bonds between stem monomers. (B) Protein yields and overall recovery yields of stem construct mutants (M6L and M6EL with the S-S mutations SS1, SS2, SS3,
or SS4) after protein expression, purification, and refolding. (C) Autoradiogram of a nonreducing SDS/PAGE gel of refolded HA stem construct mutants. (D)
Size-exclusion HPLC profiles of refolded HA stem construct mutant M6EL and M6EL(SS2).

target sequence with a (GS)3 linker, and another (GS)3G linker
was added between the foldon domain and the His6 tag, as shown
in Fig. 4A. This HA stem design was named “M6EL.” Non-
reducing SDS/PAGE results (Fig. 4B) showed that the addition
of the (GS)n linkers increased the proportion of trimer to ap-
proximately 80%. Repeated production batches indicated ex-
cellent reproducibility (SI Appendix, Table S4).
Additional Stabilization of the Stem Trimer. Although the HA stem
trimer had been stabilized with disulfide bonds between foldon
monomers (Fig. 3) at the membrane proximal end of the mole-
cule, we reasoned that stability could be further improved by also
introducing disulfide bonds between stem monomers at the distal
end of the protein structure. Cysteines were introduced in the
Stem-HA2-Fragment3 (Fig. 1E) at the following locations (SS1:
I192C-W193C; SS2: G188C-F189C; SS3: K184C-V185C; SS4:
L181C-N182C), as shown in Fig. 5A. Cysteine adjacency was
chosen to discourage intramolecular bonds in favor of bonds
between monomers. Sites were also chosen based on natural
intermolecular proximity. Disulfide bond formation at these sites
could both encourage and help confirm proper trimer formation.
SDS/PAGE results (Fig. 5C) showed that all four designs formed
intermolecular disulfide bonds between stem monomers in the
absence of foldon mutation E. The SS2 mutant of M6EL showed
the highest protein yield (363 μg/mL CFPS reaction volume;
27% higher than M6EL) after protein expression, purification,
and refolding (Fig. 5B). The mutant M6EL(SS2) was used in
subsequent studies. Size-exclusion HPLC results (Fig. 5D) fur-
Conclusion
In this work, we describe an E. coli-based CFPS method for
producing an HA stem domain trimer assembly derived from the
2009 pandemic H1N1 strain. The CFPS production of this antigen
potentially provides a rapid, scalable, and cost-effective means to
combat pandemic and epidemic threats when a rapid response
and large quantities of vaccine are required. Although the HA
stem antigen by itself may not be an effective vaccine, it can now
be tested with adjuvants or by attaching it to virus-like particles to
enhance its immunogenicity and increase protective efficacy.
We also developed an effective refolding method to produce
the correctly folded trimeric HA stem domain protein. We suggest
this stepwise procedure may provide a valuable paradigm for pro-
ducing properly folded multimeric domains of membrane-associated
cell and viral surface proteins. To reduce the aggregation and the
formation of incorrect intermolecular disulfide bonds, several
newly exposed hydrophobic residues were replaced, the isoelectric
points of newly exposed protein fragments were decreased, and the
number of cysteines in the HA stem domain was reduced from eight
to four. The HA stem trimer was further stabilized by introducing
intermolecular disulfide bonds between foldon monomers and
between stem domain monomers. The refolded and stabilized tri-
meric HA stem domain protein was recognized identically com-
pared with full-length HA ectodomain by neutralizing antibodies
CR6261, C179, and FI6, suggesting that the neutralizable epitope is

APPLIED BIOLOGICAL
ther confirmed better trimer formation for M6EL(SS2).

SCIENCES
Confirming Neutralizing Antigen in Stabilized HA Stem Trimer. The
correct immunogenic conformation of this stabilized HA stem
trimer M6EL(SS2) was confirmed by using antibodies CR8020,
CR6261, C179, and FI6 known to be specific for neutralizing
epitopes located in the HA stem domain. An ELISA was used
with green fluorescence protein (GFP) as the negative control.
The full-length H1 HA monomer [A/California/07/2009(H1N1)]
and the full-length H1 HA trimer [A/South Carolina/1/1918
(H1N1)] (with foldon trimerization domain) were used as posi-
tive controls. ELISA binding results (Fig. 6) showed that the new
H1 HA stem trimer and the full-length H1 HA trimer control
were recognized with nearly identical affinity by all four anti-
bodies. As expected, recognition of the full-length HA monomer
differed significantly from that of the trimers. The FI6 antibody
bound HA monomer more strongly than the trimer, whereas
Fig. 6. ELISA of HA stem construct M6EL(SS2) using antibody CR8020 (A),
CR6261 and C179 bound the trimer more strongly. CR6261,
CR6261 (B), C179 (C), and FI6 (D). Green fluorescence protein (GFP) protein
C179, and FI6 all bind HAs belonging to the H1 subtype, but with a C-terminal His6 tag was used as the negative control. The full-length
CR8020 only can bind HAs belonging to H3 and H7 subtypes H1 HA monomer [A/California/07/2009(H1N1)] (with His6 tag) and the full-
(29). Together these results demonstrate that the M6EL(SS2) length H1 HA trimer [A/South Carolina/1/1918(H1N1)] (with foldon trimeri-
HA stem antigen is properly folded. zation domain and His6 tag) were used as the positive controls.

Lu et al. PNAS | January 7, 2014 | vol. 111 | no. 1 | 129

properly folded. This influenza HA stem trimer therefore is an
excellent candidate for the design and production of broadly pro-
tective influenza vaccines. Based on the insights gained, we believe
that similar procedures can be rapidly developed for the other in-
fluenza HA stem domains needed for universal vaccines.
As mentioned, this overall protocol may provide a paradigm for
the rational design and evaluation of stable properly folded
domains of other multimeric proteins. The protocol begins with
cell-free protein expression and then involves four main steps. Of
course, the development of any successful folding protocol ben-
efits when the starting material is relatively pure, and a rapid and
precise assessment method for protein folding is essential.
i) Analyze the pI of protein fragments particularly on newly
exposed protein surfaces. Significant pI differences between
fragments could result in significant intermolecular ionic
attractions and protein aggregation at neutral pH, which
could inhibit folding. Mutations to reduce or alter charges
on new surfaces may be beneficial.
ii) Analyze the hydrophobicity at the newly exposed protein surfa-
ces. Newly exposed hydrophobic regions could result in unde-
sired protein aggregation in the protein folding process. Mutating
these exposed hydrophobic residues to hydrophilic residues
could simultaneously reduce surface hydrophobicity and the pI
differences of polypeptide domains. If possible, targeted muta-
tions should be distant from the biologically relevant regions
of protein.
iii) Introduce intermolecular covalent disulfide bonds between
monomers to stabilize the multimeric protein. The disulfide
bonds can form where the distance between the two residues
is expected to be less than 8.5 Å. Again, if possible, these
modifications should be introduced at points distant from the
region of the protein with important functional properties.
iv) Optimize the refolding conditions, including pH, disulfide
exchange reagents, buffer additives (arginine, salts), divalent
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130 | www.pnas.org/cgi/doi/10.1073/pnas.1308701110
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Materials and Methods
Design and Construction of HA Stem Domain Construct. The HA stem domain
protein was from the influenza virus A/California/05/2009 (H1N1) hemag-
glutinin (accession no. ACP41926). The sequences of all HA stem constructs
are shown in SI Appendix, SI Materials and Methods.
CFPS. CFPS was conducted by using the PANOx-SP (phosphoenolpyruvate,
amino acids, nicotinamide adenine dinucleotide, oxalic acid, spermidine, and
putrescine) cell-free system as described (30, 31) (SI Appendix).
Protein Purification and Refolding. After CFPS reactions, the denatured pro-
teins were purified by a Ni-NTA column (Qiagen). Two different refolding
methods were used and compared: dialysis refolding and dilution refolding
(SI Appendix, SI Materials and Methods).
Size Exclusion HPLC. HA stem domain proteins (mutants M1, M2, M3, M4, and
M5) refolded at pH 10.5 were initially analyzed by using a Discovery Bio GFC
500 HPLC column (Sigma-Aldrich). Unless specifically stated otherwise, other
refolded HA stem domain proteins were tested by using an Ultrahydrogel
500 HPLC column (Waters). See SI Appendix for details.
ELISA Binding of HA Stem Constructs. Antibodies CR8020, CR6261, C179, or FI6
were coated on 96-well ELISA plates (NUNC MaxiSorp) for ELISA tests. Full-
length HA monomer [A/California/07/2009(H1N1) and full-length HA trimer
(A/South Carolina/1/1918(H1N1)] were used as the positive controls. GFP
protein was used as the negative control. See SI Appendix for details.
ACKNOWLEDGMENTS. We thank Prof. Mark M. Davis for help in providing
the initial funding during the 2009 influenza pandemic and Dr. Jeffrey
Boyington and the National Institutes of Health (NIH) Vaccine Research
Center for providing antibodies and HA proteins. This work was supported
through the NIH-funded Stanford U19 consortium Grants AI057229
and AI090019.
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