The cellulosomes from Clostridium cellulolyticum
Identification of new components and synergies between complexes
Imen Fendri
1
, Chantal Tardif
1,2
, Henri-Pierre Fierobe
1
, Sabrina Lignon
3
, Odile Valette
1
, Sandrine
Page
`
s
1,2
and Ste
´
phanie Perret
1,2
1 Laboratoire de Chimie Bacte
´
rienne, CNRS, UPR9043, IMM, Marseille, France
2 Universite
´
Aix Marseille, France
3 Centre de microse
´
quencage et d’analyse prote
´

cellulosome; Clostridium cellulolyticum;
diversity; new components; synergy
Correspondence
S. Perret, Laboratoire de Chimie
Bacte
´
rienne, CNRS, UPR9043, 31 chemin
Joseph Aiguier 13009, Marseille, France
Fax: +33 4 91 71 33 21
Tel: +33 4 91 16 43 40
E-mail:
(Received 18 January 2009, revised 24
March 2009, accepted 27 March 2009)
doi:10.1111/j.1742-4658.2009.07025.x
Cellulosomes produced by Clostridium cellulolyticum grown on cellulose
were purified and separated using anion-exchange chromatography.
SDS ⁄ PAGE analysis of six fractions showed variations in their celluloso-
mal protein composition. Hydrolytic activity on carboxymethyl cellulose,
xylan, crystalline cellulose and hatched straw differed from one fraction to
another. Fraction F1 showed a high level of activity on xylan, whereas
fractions F5 and F6 were most active on crystalline cellulose and carb-
oxymethyl cellulose, respectively. Several cellulosomal components specific
to fractions F1, F5 and F6 were investigated using MS analysis. Several
hemicellulases were identified, including three xylanases in F1, and several
cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein
protease inhibitor were identified in F5 and F6. Synergies were observed
when two or three fractions were combined. A mixture containing fractions
F1, F3 and F6 showed the most divergent cellulosomal composition, the
most synergistic effects and the highest level of activity on straw (the most
heterogeneous substrate tested). These findings show that on complex sub-

in: Cel5A binds to the most divergent cohesins with
similar affinities [18] and cohesin 1 shows a similar
affinity for Cel5A and Cel48F [19]. In addition, over-
production of a minor cellulosomal enzyme, the man-
nanase Man5K, resulted in mannanase-enriched
cellulosomes [20]. The data strongly suggest that the
composition of C. cellulolyticum cellulosomes is hetero-
geneous and may depend on the relative amounts of
dockerin-containing enzymes available.
The hydrolytic efficiency of cellulosomes has also
been studied in mini-cellulosomes assembled in vitro.
These mini-cellulosomes had a strictly controlled
enzyme composition and contained two or three engi-
neered cellulases [21,22]. Enzyme binding to scaffoldin
was found to enhance the activity of the enzymatic
components, particularly on recalcitrant substrates.
This enhancement was attributed to the physical prox-
imity of the enzymes in the mini-cellulosomes and to
cellulose targeting of the complexes via the CBM of
the mini-scaffoldin [21]. The most active mini-cellulo-
some on microcrystalline cellulose was composed of
the processive cellulase Cel48F combined with endo-
glucanase Cel9G. Adding the C. thermocellum bifunc-
tional esterase ⁄ xylanase Xyn10Z to this cellulase pair
yielded the most active mini-cellulosome on hatched
straw [22]. Compared with naturally occurring cellulo-
somes, however, the most active mini-cellulosomes are
fivefold less active on crystalline cellulose and 3.5-fold
less active on straw. Additional factors present in
naturally occurring cellulosomes may therefore account

sed from the beginning to the end of the large peak;
the peak was arbitrarily divided into six fractions
numbered F1–F6 (Fig. 1) and the protein composition
Fig. 1. Anion-exchange chromatography of the cellulosomal
fraction purified by gel filtration. Three milligrams of protein
were loaded onto the column. F1–F6 are the arbitrarily separated
fractions. The grey line gives the continuous NaCl gradient.
I. Fendri et al. Diversity of cellulosomes and their synergies
FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS 3077
and enzymatic properties of the cellulosomes present
in each fraction were analysed.
Protein analyses of the fractionated cellulosomes
The six fractions, separated as described above, were
first analysed using Native PAGE. In all fractions, a
single major diffuse band was observed, which showed
that the proteins present in all the fractions were in a
‘cellulosome state’ [20] and that anion-exchange chro-
matography did not dissociate the complexes (data not
shown). The subunit composition of these complexes
was therefore analysed further by SDS ⁄ PAGE (Fig. 2).
A control sample (C) was formed by pooling the elu-
tion fractions (F1–F6) corresponding to the entire
peak obtained using anion-exchange chromatography.
In this control sample, the proportions were those of
naturally occurring cellulosomes and the sample was
subjected to the same chromatographic procedures as
each of the fractions analysed separately. Each of the
fractions obtained by anion-exchange chromatography
showed numerous proteins, most of which had molecu-
lar masses in the range 30–160 kDa. As expected, the

such as carboxymethyl cellulose (CMC) and xylan,
Fig. 2. Composition of the cellulosome fractions (F1–F6). Five
micrograms of protein were separated on a 10% SDS ⁄ PAGE and
silver stained. C, control sample containing the unfractionated mix-
ture of cellulosomes; F1–F6, fractions separated by anion-exchange
chromatography. Major components CipC, Cel9E and Cel48F are
indicated; bands analysed using MS methods are numbered; the
asterisks indicate a band containing a nonsecreted protein identified
by ion-trap MS ⁄ MS as a ketol-acid reductoisomerase, which is not
a cellulosomal component (data not shown).
Fig. 3. Identification of several components in cellulosomes from
fractions F1–F6. Proteins (5 lg) were separated on 10% SDS ⁄
PAGE, transferred to a polyvinylidene fluoride membrane and
probed with anti-CelA, anti-CelC, anti-CelM and anti-CelG serum.
C, control sample corresponding to the unfractionated mixture
of cellulosomes; F1–F6, fractions separated by anion-exchange
chromatography.
Diversity of cellulosomes and their synergies I. Fendri et al.
3078 FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS
microcrystalline cellulose (Avicel) and hatched straw, a
heterogeneous natural substrate. As shown in Fig. 4,
fractions F1–F6 showed different patterns of activity.
On CMC, the most active fraction was F6, which was
45% more active than F5, and 70–90% more active
than the control sample and fractions F1–F4. On
xylan, rather weak activity was measured with the con-
trol sample and all the fractions, except for F1, which
was found to be approximately fivefold more active
than the others. The cellulosomes present in fractions
F1 and F6 therefore have the most efficient enzymes

fraction. However, GH10 (protein 12) is also present
in noticeable quantities in the other five fractions.
All the proteins present in F5 were also present in
F6. Among these, we detected protein 2 (Cel9P) [14]
and proteins 5a (Cel9G) [25] and 5b (Cel9H) [28].
Cel9P and Cel9H show the same modular organization
as the endoglucanase Cel9G (GH9-CBM3c-Doc) char-
acterized previously [25]. Although Cel9P and Cel9H
have not yet been characterized, they are expected to
show enzymatic properties similar to those of Cel9G.
Four enzymes belonging to the GH5 family were also
identified: proteins 7a and 11 correspond to the endo-
cellulases Cel5D [30] and Cel5A [31], respectively, and
protein 8 corresponds to the carboxymethyl cellulase
Cel5N [14], and protein 4, in which the GH5 catalytic
domain shows 33% identity with a mannanase from
Bacillus circulans (accession no. BAA25878.1) [31].
Protein 7b was identified as the mannanase Man26A
[14]. Lastly, protein 13 was identified as an N-terminal
dockerin-borne chagasin domain. Chagasin belongs to
Fig. 4. Enzymatic activities of the cellulosome fractions on various
substrates. Specific activities were measured at 37 °C after 30 min
on 0.8% CMC and xylan, and after 24 h on 0.35% microcrystalline
cellulose Avicel and hatched straw at final protein concentrations of
2, 3, 20 and 6 lgÆmL
)1
, respectively.
I. Fendri et al. Diversity of cellulosomes and their synergies
FEBS Journal 276 (2009) 3076–3086 ª 2009 The Authors Journal compilation ª 2009 FEBS 3079
a family of cystein protease inhibitors found in lower

indicates that on this natural substrate, this combina-
tion of different fractions results in synergistic activity.
Because straw is a complex substrate composed mostly
of cellulose (40% w ⁄ w) and hemicelluloses (15% w ⁄ w),
we analysed its degradation using the following combi-
nation of fractions showing complementary activities:
the xylanase F1 fraction combined with either the
Table 1. Identification of specific components detected in fractions F1, F5 and F6 using MS analysis. All identifications were based on pep-
tide mass fingerprint analyses using the MALDI-TOF technique, except for the proteins 6, 7a and 7b, and 10 which were identified using the
MS ⁄ MS technique. The modular structure of new proteins was determined by performing PFAM and BLAST analyses. S, signal sequence;
GH, glycoside hydrolase; CBM, carbohydrate binding module, GH and CBM numbers are those of the carbohydrate active enzyme database
classification (); Doc, dockerin domain; Ig, immunoglobulin-like domain of cellulase; X2, Ig-like module of unknown func-
tion; M
r
, theoretical molecular mass of the mature protein. The cleavage site was determined using />Cov, percentage of amino acid coverage in the matched proteins; M
pep
, the number of unique matched peptides; U
pep
, the number of
unmatched peptides in the MALDI-TOF experiments. The function of new proteins is based on the GH family of the catalytic module and
the modular organization of the protein, or on the identity of the catalytic domain with another characterized protein (see text).
Protein GI number
a
Modular structure
M
r
(kDa) M
pep
⁄ U
pep

7a 121824 S-GH5-doc 63.4 5 9.8 50.2
b
Cellulase Cel5D 25
7b 110588924 S-GH26-doc 61.8 2 10.5 20.2
b
Mannanase Man26A 9
8 220928189 S-GH5-doc 56.6 6 ⁄ 7 10.0 68.0
c
Cellulase Cel5N 9
11 121802 S-GH5-doc 50.7 29 ⁄ 39 45.0 249.0
c
Cellulase Cel5A 19
13 220929230 S-doc-Chagasin 31.0 5 ⁄ 18 16.0 60.0
c
Unknown function This study
F6
1 220929070 S-GH9-CBM3-doc 102.3 12 ⁄ 23 13.0 74.0
c
Cellulase Cel9R This study
3 220928185 S-GH9-CBM3-doc 81.3 23 ⁄ 28 26.0 165.0
c
Cellulase Cel9J 22
10 220928973 S-GH18-doc 51.1 4 9.5 40.2
b
Putative chitinase This study
a
Accession numbers of new components are those of the newly released complete genome (; GI:220927459).
b
Scores obtained using using BioworksBrowser search engine (MS ⁄ MS data).
c

ulosomes from C. cellulovorans may be partly due to
the existence of various categories of cohesins and
dockerins which determine the composition of the cell-
ulosomes [23,34]. Despite the enzymatic diversity of
the cellulosomes from C. cellulolyticum (there are 62
ORFs encoding dockerin-containing protein versus 8
enzymatic units per cellulosome), anion-exchange chro-
matography gave a single peak followed by a long tail.
This suggests a random assembly of enzymes on the
scaffoldin, leading to a large number of enzyme combi-
nations.
In this study, a GH11 xylanase and a GH5 ⁄ 30 puta-
tive xylanase were identified. In the genome sequence of
C. cellulolyticum, only one gene encoding a cellulosomal
GH11 was found. To date, GH11 modules have been
found in modular bifunctional cellulosomal enzymes,
such as XynA from C. cellulovorans (GH11-Doc-
CE4) [35] and XynA from C. thermocellum strain
ATCC27405 (GH11-CBM6-Doc-CE4) [33], or associ-
ated with a CBM6 module such as in XynB (GH11-
CBM6-Doc) from C. thermocellum strain F1 ⁄ YS [36].
In the C. cellulolyticum enzyme, the GH11 catalytic
module had no such catalytic or CBM partner, which
suggests that the catalytic behaviour of the enzyme may
differ from that of previously described enzymes con-
taining GH11. To date, no GH5 ⁄ GH30 enzymes have
been found in C. cellulovorans cellulosomes, whereas a
bifunctional GH30-a-l-arabinofuranosidase B has been
detected in C. thermocellum cellulosomes [33]. The cata-
lytic domain of C. cellulolyticum GH5⁄ GH30 shows

protease cellulosomal system reported in C. thermocel-
lum [39,40]. A cellulosomal protease inhibitor ⁄ protease
system may, therefore, be more widespread than
expected and have a common and important function
in cellulosome regulation, displacement from the cell
surface, degradation and ⁄ or protection of the cellulo-
somes [40].
All the fractions showed a substantial level of activity
on crystalline cellulose. In previous studies, mini-cellulo-
somes reconstituted in vitro, in which the endocellulase
Cel9G (GH9-CBM3c-Doc) was combined with the
processive enzyme Cel48F, were found to hydrolyse
crystalline cellulose the most efficiently [21,22]. In this
study, all the cellulosome fractions contained Cel48F
and several GH9-CBM3c-Doc (Cel9P, Cel9G, Cel9H,
Cel9J). This enzyme combination may be essential for
efficient degradation of crystalline cellulose by the
cellulosome. The most active fraction on Avicel (F5)
was found to contain a small amount of Cel9J, but large
amounts of Cel9P and Cel9G ⁄ Cel9H. Because the least
active fractions, F3 and F4, contained large amounts of
Cel9G ⁄ Cel9H, but lower amounts of Cel9P, it seems
likely that Cel9P might contribute to the high level of
activity on crystalline cellulose seen in F5. Although F6
contained all the proteins present in F5, it showed lower
levels of activity on Avicel and higher levels on CMC
than F5. This may be because of the presence of addi-
tional proteins such as proteins 1 and 3 (which were
identified as GH9-CBM3c-Doc enzymes) and protein 10
(which was identified as a chitinase), and ⁄ or to varia-

were detected by performing proteomic analyses on
cellulosomes produced by C. cellulolyticum on cellulose
[14]. The enzyme diversity they contain and their heter-
ogeneous composition are inherent characteristics of
cellulosomes. Our data suggest that these characteristics
give rise to synergistic effects between diverse com-
plexes, which may account for the great efficiency of
plant cell-wall degradation processes.
Experimental procedures
Bacterial strain and cell culture conditions
C. cellulolyticum ATCC35319 [41] was grown anaerobically
at 32 °C on basal medium [42] supplemented with cellobi-
ose (4 gÆL
)1
; Sigma-Aldrich, St Louis, MO, USA) or
MN300 cellulose (5 gÆL
)1
; Serva, Heidelberg, Germany).
Purification of the cellulose-adsorbed cellulolytic
system from C. cellulolyticum
C. cellulolyticum cultures were inoculated with a cellobiose
culture at D
450
= 0.7, and grown in 800 mL of cellulose-
supplemented basal medium for 6 days. The cell culture
was filtered through a 3-lm pore size GF ⁄ D glass filter
(Whatman, Maidstone, UK). The residual cellulose was
subsequently washed with 50 and 12.5 mm Na
2
HPO

2
buffer before loading into a Resource Q column
(6 mL) (Amersham Biosciences) equilibrated with 20 mm
Tris ⁄ HCl (pH 8.0) and 2 mm CaCl
2
buffer. Elution was per-
formed with a linear NaCl gradient of 0–1 m, in the same
buffer. Fractions were concentrated using microconcentators
(30 kDa cut-off; Vivaspin, Vivasciences, Palaiseau, France).
Protein concentration was determined as described by Lowry
et al. [43], using bovine serum albumin as the standard.
Enzyme activity
Avicel microcrystalline cellulose (PH101; Fluka, Buchs,
Switzerland), CMC (medium viscosity; Sigma, St Louis,
MO, USA), oat spelt xylan (Sigma) and hatched straw
(Valagro, Poitiers, France) were used as substrates.
Hatched straw was prepared as described by Fierobe et al.
[22]. Insoluble xylan was washed four times in distilled
water and the concentration of the residual material was
estimated from the dry weight. Enzymatic assays were per-
formed in 20 mm Tris-maleate (pH 6.0) at 37 °C. A suitable
amount of protein (see legend to Figs 5 and 6) was mixed
with the substrate preparation at a final substrate concen-
tration of 0.8% (CMC or xylan) or 0.35% (Avicel or
straw). After incubating for 30 min (CMC and xylan) or
24 h (Avicel and straw), aliquots were analysed to deter-
mine the soluble reducing sugar content using the method
of Park & Johnson [44] with d-glucose as the standard.
SDS
⁄

MALDI-TOF MS analyses
Complete experimental procedures of MALDI-TOF MS
analysis are described in Doc. S1. Digested peptides were
treated using MALDI-TOF Voyager DE-RP apparatus
(Applied Biosystems, Foster City, CA, USA) in the positive
reflectron mode. Contaminant peaks were removed prior to
a peptide mass fingerprint search against the nonredundant
NCBI database (20080210), restricted to ‘Other Firmicutes’
(445 464 sequences) using the freely available MASCOT
search engine (). Searches
were performed using a maximum peptide mass tolerance
of 150 p.p.m., one missing cleavage allowed, a fixed modifi-
cation of cysteines by iodoacetamide (carbamidomethyl),
a variable modification of methionines (oxidation) and
N-term glutamine (pyro-glutamine).
Proteins were taken to have been identified only when
they had at least five matching peptides and scores > 60
(P < 0.05). When identification scores < 60 were obtained,
we assessed their reliability using the search engine MS-FIT
v4.27.2Basic (). In the case of
peptides matching multiple members of a protein family,
the proteins selected were those with the largest number of
matching peptides. When several proteins were identified
with equal numbers of matching peptides we checked that
they corresponded to the same gene product and selected
the database entry that was the best annotated.
Ion-trap MS
⁄
MS analyses
Complete experimental procedures of ion-trap MS ⁄ MS anal-

was assessed by a cross-correlation number (Xcorr) versus
charge state, as follows: Xcorr > 1.5 for singly charged ions,
Xcorr > 2.0 for doubly charged ions and Xcorr > 2.5 for
triply charged ions, peptide probability was £ 5 · 10
)3
. Pro-
tein identification required maximum coverage or at least
two rank one unique peptides.
Protein sequence analyses
The amino acid sequences of the new proteins were com-
pared with those in the NCBI sequence databases using the
blast program [46]. Protein domain compositions were
analysed using the PFAM database (ger.
ac.uk) [47]. Signal peptide position was determined using
the server [48].
Acknowledgements
Imen Fendri received a doctoral fellowship from the
Tunisian Ministry of Higher Education and Scientific
Research. We are very grateful to Danielle Moinier and
Re
´
gine Lebrun (Centre de microse
´
quencage et d’analyse
prote
´
omique, IMM, Marseille, France) for performing
the MS analysis. Financial support from the Marseille-
Nice Ge
´

the walls come crumbling down: recent structural
biochemistry of plant polysaccharide degradation. Curr
Opin Plant Biol 11, 338–348.
6 Coutinho PM & Henrissat B (1999) Carbohydrate-
active enzymes: an integrated database approach. In
Recent Advances in Carbohydrate Bioengineering
(Gilbert HJ, Davies G, Henrissat B & Svensson B, eds),
pp. 3–12. Royal Society of Chemistry, Cambridge.
7 Rincon MT, Ding SY, McCrae I, Martin JC, Aurilia V,
Lamed R, Shoham Y, Bayer EA & Flint HJ (2003)
Novel organization and divergent dockerin specificities
in the cellulosome system of Ruminococcus flavefaciens.
J Bacteriol 185, 703–713.
8 Rincon MT, Cepeljnik T, Martin JC, Lamed R, Barak
Y, Bayer EA & Flint HJ (2005) Unconventional mode
of attachment of the Ruminococcus flavefaciens cellulo-
some to the cell surface. J Bacteriol 187, 7569–7578.
9 Ding SY, Bayer EA, Steiner D, Shoham Y & Lamed R
(2000) A scaffoldin of the Bacteroides cellulosolvens cell-
ulosome that contains 11 type II cohesins. J Bacteriol
182, 4915–4925.
10 Tardif C, Be
´
laı
¨
ch A, Fierobe HP, Page
`
s S, de Philip P &
Be
´

`
s S (2007)
Enzyme diversity of the cellulolytic system produced by
Clostridium cellulolyticum explored by two-dimensional
analysis: identification of seven genes encoding new
dockerin-containing proteins. J Bacteriol 189, 2300–
2309.
15 Maamar H, Abdou L, Boileau C, Valette O & Tardif C
(2006) Transcriptional analysis of the cip-cel gene clus-
ter from Clostridium cellulolyticum. J Bacteriol 188,
2614–2624.
16 Maamar H, Valette O, Fierobe HP, Be
´
laı
¨
ch A, Be
´
laı
¨
ch
JP & Tardif C (2004) Cellulolysis is severely affected in
Clostridium cellulolyticum strain cipCMut1. Mol Micro-
biol 51 , 589–598.
17 Perret S, Maamar H, Be
´
laı
¨
ch JP & Tardif C (2004) Use
of antisense RNA to modify the composition of cellulo-
somes produced by Clostridium cellulolyticum. Mol

some chimeras. Substrate targeting versus proximity of
enzyme components. J Biol Chem 277, 49621–49630.
20 Perret S, Be
´
laı
¨
ch A, Fierobe HP, Be
´
laı
¨
ch JP & Tardif C
(2004) Towards designer cellulosomes in Clostridia:
mannanase enrichment of the cellulosomes produced by
Clostridium cellulolyticum. J Bacteriol 186, 6544–6552.
21 Fierobe HP, Mechaly A, Tardif C, Be
´
laı
¨
ch A, Lamed
R, Shoham RY, Be
´
laı
¨
ch JP & Bayer EA (2001) Design
and production of active cellulosome chimeras. Selective
incorporation of dockerin-containing enzymes into
defined functional complexes. J Biol Chem 276, 21257–
21261.
22 Fierobe HP, Mingardon F, Mechaly A, Be
´

25 Gal L, Gaudin C, Be
´
laı
¨
ch A, Page
`
s S, Tardif C &
Be
´
laı
¨
ch JP (1997) CelG from Clostridium cellulolyticum:
a multidomain endoglucanase acting efficiently on crys-
talline cellulose. J Bacteriol 179, 6595–6601.
26 Fierobe HP, Bagnara-Tardif C, Gaudin C, Guerlesquin
F, Sauve P, Be
´
laı
¨
ch A & Be
´
laı
¨
ch JP (1993) Purification
and characterization of endoglucanase C from Clostrid-
ium cellulolyticum. Catalytic comparison with endoglu-
canase A. Eur J Biochem 217, 557–565.
27 Be
´
laı

bacterial pathogens. FEBS Lett 542, 12–16.
33 Gold ND & Martin VG (2007) Global view of the
Clostridium thermocellum cellulosome revealed by
quantitative proteomic analysis.
J Bacteriol 189, 6787–
6795.
34 Park JS, Matano Y & Doi RH (2001) Cohesin–docker-
in interactions of cellulosomal subunits of Clostrid-
ium cellulovorans. J Bacteriol 183, 5431–5435.
35 Kosugi A, Murashima K & Doi RH (2002) Xylanase
and acetyl xylan esterase activities of XynA, a key sub-
unit of the Clostridium cellulovorans cellulosome for
xylan degradation. Appl Environ Microbiol 68, 6399–
6402.
36 Hayashi H, Takehara M, Hattori T, Kimura T, Karita
S, Sakka K & Ohmiya K (1999) Nucleotide sequences
of two contiguous and highly homologous xylanase
genes xynA and xynB and characterization of XynA
from Clostridium thermocellum. Appl Microbiol Biotech-
nol 51, 348–357.
37 St John FJ, Rice JD & Preston JF (2006) Characteriza-
tion of XynC from Bacillus subtilis subsp. subtilis strain
168 and analysis of its role in depolymerisation of
glucuronoxylan. J Bacteriol 188, 8617–8626.
38 Rawlings ND, Morton FR, Kok CY, Kong J & Barrett
AJ (2008) MEROPS: the peptidase database. Nucleic
Acids Res 36 (database issue), D320–D325.
39 Kang S, Barak Y, Lamed R, Bayer EA & Morrison M
(2006) The functional repertoire of prokaryote cellulo-
somes includes the serpin superfamily of serine protein-

Z, Miller W & Lipman DJ (1997) Gapped BLAST and
PSI-BLAST: a new generation of protein database
search programs. Nucleic Acids Res 25, 3389–3402.
47 Finn RD, Mistry J, Schuster-Bo
¨
ckler B, Griffiths-Jones
S, Hollich V, Lassmann T, Moxon S, Marshall M,
Khanna A, Durbin R et al. (2006) PFAM: clans, web
tools and services. Nucleic Acids Res 34, 247–251.
48 Dyrlov Bendtsen J, Nielsen H, von Heijne G & Brunak
S (2004) Improved prediction of signal peptides: SignalP
3.0. J Mol Biol 340, 783–795.
Supporting information
The following supplementary material is available:
Fig. S1. MS spectrum of protein 9, fraction 1.
Fig. S2. MS spectrum of protein 12, fraction 1.
Fig. S3. MS spectrum of protein 14, fraction 1.
Fig. S4. MS spectrum of protein 2, fraction 5 ⁄ fraction 6.
Fig. S5. MS spectrum of protein 4, fraction 5 ⁄ fraction 6.
Fig. S6. MS spectrum of protein 5, fraction 5 ⁄ fraction 6.
Fig. S7. MS spectrum of protein 8, fraction 5 ⁄ fraction 6.
Fig. S8. MS spectrum of protein 11, fraction 5 ⁄ fraction 6.
Fig. S9. MS spectrum of protein 13, fraction 5 ⁄ fraction 6.
Fig. S10. MS spectrum of protein 1, fraction 6.
Fig. S11. MS spectrum of protein 3, fraction 6.
Table S1. Masses and peptide assignments.
Doc. S1. Complete experimental procedures of mass
spectrometry analyses.
This supplementary material can be found in the
online version of this article.