Protein J (2014) 33:253–257
DOI 10.1007/s10930-014-9558-x
Preliminary Crystallographic Analysis of a Cruciferin Protein
from Seeds of Moringa oleifera
Ahmed Akrem • Nasser Yousef • Afshan Begum •
Amr Negm • Arne Meyer • Markus Perbandt •
Friedrich Buck • Christian Betzel
Published online: 6 April 2014
Ó Springer Science+Business Media New York 2014
Abstract A 55 kDa cruciferin protein has been purified
and characterized from seeds of Moringa oleifera plant.
Protein blast of N-terminal amino-acid sequence showed
60 % sequence similarity with cruciferin from Brassica
napus. The M. oleifera protein has been crystallized
applying the sitting drop method using 5 % polyethylene
glycol 8,000, 38.5 % 3-methyl-1,5-pentanediol and 0.1 M
sodium cacodylate pH 6.5. The crystals belonged to the
P6322 hexagonal space group with cell dimensions, a =
b = 98.4, c = 274.3 Å. Initial diffraction data have been
collected to a resolution of 6 Å.
Keywords Brassica napus
Cruciferin
Crystallization

Moringa oleifera
Sitting drop method
Sodium
cacodylate
Abbreviations
PEG Polyethylene glycol
MPD 3-Methyl-1,5-pentanediol
A. Akrem
N. Yousef
A. Begum
A. Meyer
M. Perbandt

C. Betzel
Laboratory for Structural Biology of Infection and Inflammation,
Department of Chemistry, c/o DESY, University of Hamburg,
Notkestrasse 85, 22603 Hamburg, Germany
A. Akrem (&)
Department of Botany, Institute of Pure and Applied Biology,
Bahauddin Zakariya University, 60800 Multan, Pakistan
e-mail: ahmedakrem@hotmail.com
A. Negm
Biochemistry Department, Faculty of Science, Mansoura
University, Mansoura, Egypt
F. Buck
Institute for Clinical Chemistry, University Medical Centre
Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
MO Moringa oleifera
DESY Deutsches Elektronen Synchrotron
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
DLS Dynamic light scattering
1 Introduction
The horseradish tree or Moringa oleifera Lam. is tropical
plant species and belongs to Moringaceae family [1]. This
plant can survive poor soils, drought conditions and grows
equally well in both humid tropic as well as the hot dry
lands [2]. Earlier studies showed the plant to be nontoxic
[3]. Almost all parts of this tree are used as highly nutritive
vegetables in areas like Hawaii, Philippines, Pakistan, India
and in many African regions [4–6]. During development,
plant seeds accumulate large amount of proteins, which are
used as a main nitrogen source for seed germination and
early seedling growth [7].
Cruciferin is one of the major storage rapeseed proteins,
which comprise about 60 % of the total proteins in mature
seeds [8]. During the expansion phase of the embryo
development, these storage proteins are synthesized as
precursor forms on membrane bounded ribosomes and then
they are transported to vacuole from endoplasmic reticulum
through Golgi apparatus [9]. Inside of the vacuoles, the final
processing of these precursors ultimately produces the
mature cruciferin [10]. Cruciferin (12S globulin) is a large,
neutral and oligomeric protein [11]. Up to now several
reports about the coagulating cationic peptides of low
molecular weight ranging from 6 to 16 kDa and isoelectric
point of 10 have been published [12]. However, only few
studies about the high molecular weight proteins from MO
123
254
seeds have been reported e.g., a 66 kDa protein has been
isolated from MO seeds with coagulation activity compa-
rable with that of basic peptides and alum [13]. Also a new
26.5 kDa lectin was reported recently from MO seeds
showing hemagglutinating activities [14]. The present study
is about the purification, characterization and preliminary
X-ray crystallographic analysis of a high molecular weight
(55 kDa) protein identified as cruciferin from MO seeds.
2 Materials and Methods
2.1 Protein Purification
Moringa oleifera seeds used in this study were obtained
from a local seed supplier and seed shells were removed
prior to purification. 10 g of seeds were crushed to fine
powder and suspended in 100 ml of 95 % ethanol for
30 min by continuous stirring at room temperature for
delipidation. White precipitates, recovered by centrifuga-
tion at 1,500 rpm for 15 min, were dried completely. The
precipitate was re-dissolved in 300 ml of buffer A (20 mM
Tris, pH 7.5) and stirred for several hours, after which the
solution was centrifuged at 12,000 rpm for 30 min. The
supernatant was collected and the pellet was discarded.
Supernatant was made 35 % (w/v) in ammonium sulphate
to precipitate high molecular weight proteins which sedi-
ment in the pellet after centrifugation at 2,000 rpm for
15 min and re-dissolved in 50 ml of the same buffer. All
steps were carried out at room temperature. The partially
purified solution was used for a cationic exchange chro-
matography via a pre-packed Mono SÒ HR 10/10 column
pre-equilibrated with buffer A. The protein was eluted with
a linear gradient of 0–30 % (v/v) buffer B (20 mM Tris, pH
7.5, 1 M NaCl). The observed peaks on the chromatogram
were collected, desalted and analyzed on 15 % sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) stained with Coomassie Brilliant Blue dye. Highly
purified fractions were pooled and concentrated to
10 mg ml-1 using Amicon Ultra-15 (10,000 Da cutoff,
Millipore) as shown in Fig. 1. The N-terminal amino acid
sequence was determined by use of an Applied Biosystems
Edman sequencer 476. Purified protein analyzed by
dynamic light scattering (DLS) measurements with the
Spectroscatter 201 (Molecular Dimensions, UK) confirmed
the monodispersity of the solution (Fig. 3).
2.2 Protein Crystallization
The PCTTM Pre-Crystallization Test (Hampton Research,
USA) was used to optimize the protein concentration
(15 mg ml-1) for crystallization experiments. In total 384
crystallization conditions (ComPAS, JCSG?, Classics and
123
A. Akrem et al.
Fig. 1 Purified Moringa cruciferin protein under non-reduced con-
ditions on 15 % SDS-PAGE, lane M, protein marker, L1 and L2,
purified 55 kDa proteins
Cryos Suites, Qiagen, Germany) were screened using the
Honeybee 961 crystallization robot (Zinsser Analytic
GmbH, Germany) at 293 K in 96-well sitting-drop vapour-
diffusion crystallization plates (NeXtal QIA1 lplates,
Qiagen). Initial crystals were obtained from condition No.
17 of JCSG? Suite Composition kit (Qiagen, Germany)
containing 5 % PEG 8,000, 40 % (v/v) MPD and 0.1 M
sodium cacodylate pH 6.5. Drops consisting of 2 ll of the
protein solution and 2 ll of the reservoir solution were
equilibrated against 1 ml of reservoir solution. Single
crystals of approx. (0.1 9 0.05 9 0.025 mm3) size were
appeared after 2 weeks, as shown in Fig. 4.
2.3 Data Collection and Analysis
To characterize the crystals and to obtain information about
the unit cell parameters, a single crystal was fished out
within a nylon loop and was dipped in same reservoir
solution containing 15 % glycerol solution as cryoprotec-
tant and was flash-cooled in a cold nitrogen-gas stream at
100 K. Initial diffraction data were recorded at the con-
sortium beam line X13 at Deutsches Elektronen Synchro-
tron (DESY), with a 165 mm MAR CCD detector and a
wavelength of 0.8123 Å to characterize the crystals. The
exposure time was approx. 1.5 min per frame with crystal-
to-detector distance of 120 mm. The entire data was col-
lected with 0.6° oscillation range per frame. All intensity
data were processed and scaled with the HKL-2000 pack-
age [15]. The resulting R merge is moderate due to the
relative low diffraction power of the small crystals. The
final statistics of the diffraction data processing are pre-
sented in Table 1.
Preliminary Crystallographic Analysis of a Cruciferin Protein
Table 1 Statistics of data processing with digits in parentheses are of
the highest resolution shell
Space group P6322
Unit-cell parameters (Å,°) a = b = 98.4 c = 274.3
a = b = 90, c = 120
VM (Å3 Da-1) 3.5
Solvent content (%) 65
Resolution range (Å) 30.0–6.0 (6.14–6.0)
Total reflections 88,707
Unique reflections 2,189
Redundancy 3.1 (2.8)
Average I/r (I) 5.8 (3.5)
R mergea (%) 16.2 (35.6)
Data completeness (%) 96.1 (98.0)
a P P P P
R merge = hkl i | Ii {hkl} - {I (hkl)} |/ hkl i Ii (hkl),
where {I (hkl)} is the mean intensity of the observations Ii (hkl) of
reflection hkl
3 Results
Cruciferins have been reported in different plant species
like Sinapsis alba, Raphanus sativus, Arabidopsis thaliana
and Brassica napus. These cruciferins are the major seed
storage proteins in these plants serving as Nitrogen source
during seed germination. Some storage proteins have
allergic properties which confers some clinical importance
to these proteins as well. The cruciferin protein from MO
seeds has been successfully purified by ion-exchange
chromatography. Oily nature of the seeds was removed by
using 95 % ethanol. 35 % ammonium sulphate precipita-
tion helped in segregation of higher molecular weight
proteins (including cruciferin) from proteins of lower
molecular weight. An optimized and reproducible combi-
nation of cation and gel filtration chromatography provided
ample quantities of highly purified protein fractions. Puri-
fied protein was subjected to size exclusion chromatogra-
phy using Hi LoadTM 16/60 SuperdexTM 75 column to
know the correct molecular weight of the protein. The
SDS-PAGE analysis of the purified protein bands under
both reduced and non-reduced conditions indicated the
presence of inter chain disulfide linkage. The Edman
degradation methodology was used to get the partial
N-terminal amino acid sequence as given below:
GFEETVCTMRMLEHIDDPLR
Homology comparison of N-terminal amino acid
sequence suggested a high similarity of cruciferin to
already known seed storage protein. Dynamic Light Scat-
tering signal confirmed the monodisperse nature of the
concentrated (10 mg ml-1) protein solution. However, the
cruciferin quantity was further concentrated to 15 mg ml-1
for crystallization experiments. The initially identified
255
Fig. 2 Highly concentrated purified cruciferin protein under non-
reduced condition (L1) and reduced condition (L2) on 15 % SDS-
PAGE, Lane M protein marker
crystallization recipe was further optimized by the sitting
drop method using 5 % PEG 8,000, 38.5 % 3-methyl-1,5-
pentanediol (MPD) and 0.1 M sodium cacodylate pH 6.5
and single crystal diffracted to 6 Å resolution applying
synchrotron radiation. The crystals belong to the space
group P6322 with cell dimensions a = b = 98.4 Å,
c = 274.3 Å.
4 Discussion
A seed storage protein named as cruciferin has been iso-
lated and characterized from the seeds of M. oleifera, a
member of the Moringaceae family. The purified protein
was identified and characterized as cruciferin using the
N-terminal amino acid sequence. Analysis of this sequence
via the PROTEIN BLAST (ExPasy Proteomics Server)
[16] indicates a 60 % homology with a cruciferin-type
protein from B. napus, [17] which is the most abundant
storage protein in the seeds of A. thaliana and other cru-
cifers, sharing structural similarity with the cupin super
family of proteins [7]. On SDS-PAGE, under reducing
conditions (*1 mM b-mercaptoethanol), the purified pro-
tein produced two smaller subunits. However, under non-
reducing conditions; it produced a single high molecular
weight protein band of approx. 55 kDa. On reducing SDS-
PAGE, the two smaller bands are approx. 22 and 33 kDa as
shown in Fig. 2. This observation is well in agreement with
already reported molecular weights of cruciferin proteins.
Similarly, it has been reported that under reducing condi-
tions, cruciferin protein produced two major bands which
corresponds to basic (MW 22,000–24,000 Da) and acidic
(MW 30,000–37,000 Da) polypeptides [18, 19]. The two
subunits of a cruciferin are composed of a- and b-
123
256
Fig. 3 Dynamic light scattering
measurement showing a
monodispersive solution and a
hydrodynamic radius (RH) of
approx. 5.7 nm
Fig. 4 UV-microscopic photo of crystals applying the DUVI light
source (Molecular Dimensions, UK). The largest dimension of the
crystal is approx. 100 lm. Scale bar, 0.05 mm
polypeptides linked through a disulfide bond [20]. It has
been reported that the electrophoretic pattern of cruciferin
protein under reducing condition shows typical bands
corresponding to approx. 30 and 20 kDa, characteristic for
a- and b-polypeptides, while under non-reducing condi-
tions, a major protein band of 55 kDa corresponding to
cruciferin subunits is found [21].
Similarly the monodisperse cruciferin solution showed a
hydrodynamic radius of approx. 5.7 nm (Fig. 3) which
indicated that protein molecules are not monomeric, rather
forming either trimers or hexamers in the solution. Cru-
ciferins are believed to be oligomeric molecules composed
of six subunits where each individual subunit (50 kDa) is
composed of a light b chain and a heavy a chain, which are
produced from a common precursor and remained joined
by a disulfide-bridge [22]. It has also been reported that the
gel filtration chromatography of procruciferin 2/3a corre-
sponds to a molecular size of 130 kDa [23], suggesting the
123
A. Akrem et al.
trimeric structure for this procruciferin 2/3a, like in the
case of soybean proglycinin [22].
Initial diffraction data were recorded only to 6 Å due to the
relative small size of the crystals, which have presently
maximum dimensions of approx. (0.1 9 0.05 9 0.025 mm3).
The recombinant rapeseed procruciferin was also crystallized
and crystals diffracted applying synchrotron radiation to
5 Å [23]. The Matthews coefficient [24] suggests six mol-
ecules in the asymmetric unit corresponding to a packing
parameter VM of 3.5 Å3 Da-1 and a corresponding solvent
content of approx. 65 %. Optimization of growth conditions
to obtain larger crystals is presently underway and data
collection at a micro-focus beam line will be considered as
well (Fig. 4).
5 Conclusion
A 55 kDa protein predicted as cruciferin from seeds of M.
oleifera have been isolated and purified. N-terminal amino
acid sequencing showed more than 60 % homology to the
cruciferin protein from B. napus. Dynamic light scattering
confirmed the monodispersity of the purified protein solution.
Small crystals with maximum dimensions of 0.1 mm have
been grown under the sitting drop vapor diffusion methodol-
ogy. Preliminary X-ray diffraction data indicated that the
crystals correspond to the hexagonal, space group P6322, with
a = b = 98.4, c = 274.3 Å, and diffracted to 6 Å resolution.
Efforts are underway to increase the size of the crystals in
order to collect high resolution diffraction data.
Acknowledgments The work was supported by Higher Education
Commission (HEC), Pakistan and Deutscher Akademischer Austausch
Dienst (DAAD), Germany under the collaborative project ‘‘Ph.D
scholarships for Engineering & Sciences in Germany’’ as a part of
Human Resource Development (HRD) plan of Pakistan government.
Preliminary Crystallographic Analysis of a Cruciferin Protein
References
1. Ndabigengesere A, Narasiah KS, Talbot BG (1995) Active agents
and mechanism of coagulation of turbid waters using Moringa
oleifera. Water Res 29:703–710
2. Morton JF (1991) The horseradish tree, Moringa pterygosperma
(Moringaceae). A boon to arid lands. Econ Bot 45:318–333
3. Grabow WOK, Slabert JL, Morgan WSG, Jahn SAA (1985)
Toxicity and mutagenicity evaluation of water coagulated with
Moringa oleifera seed preparations using fish, protozoan, bacte-
rial, coliphage, enzyme, and Ames Salmonella assays. Water
11:9–14
4. D’souza J, Kulkarni AR (1993) Comparative studies on nutritive
values of tender foliage of seedlings and mature plants of Mo-
ringa oleifera Lam. J Econ Taxon Bot 17:479–485
5. Anwar F, Ashraf M, Bhanger MI (2005) Interprovenance varia-
tion in the composition of Moringa oleifera oilseeds from Paki-
stan. J Am Oil Chem Soc 82:45–51
6. Anwar F, Bhanger MI (2003) Analytical characterization of
Moringa oleifera seed oil grown in temperate regions of Pakistan.
J Agric Food Chem 51:6558–6563
7. Wan L, Ross AR, Yang J, Hegedus DD, Kermode AR (2007)
Phosphorylation of the 12 S globulin cruciferin in wild-type and
abi1-1 mutant Arabidopsis thaliana (thale cress) seeds. Biochem
J 404:247–256
8. Crouch ML, Sussex IM (1981) Development and storage-protein
synthesis in Brassica napus L. embryos in vivo and in vitro.
Planta 153:64–74
9. Hoeglund AS, Roedin J, Larsson E, Rask L (1992) Distribution of
napin and cruciferin in developing rape seed embryos. Plant
Physiol 98:509–515
10. Simon AE, Tenbarger KM, Scofield SR, Finkelstein RR, Crouch
ML (1985) Nucleotide sequence of a cDNA clone of Brassica
napus 12S storage protein shows homology with legumin from
Pisum sativum. Plant Mol Biol 5:191–201
11. Roedin J, Rask L (1990) The relationship between mature chains
and their precursors of cruciferin, the 12S storage protein of
Brassica napus. Plant Sci 70:57–63
257
12. Gassenschmidt U, Jany KD, Tauscher B (1991) Chemical prop-
erties of flocculent—active proteins from Moringa oleifera lam.
Biol Chem Hoppe-Seyler 372:659
13. Agrawal H, Shee C, Sharma AK (2007) Isolation of a 66 KDa
protein with coagulation activity from seeds of Moringa oleifera.
Global J Biotechnol Biochem 2:36–39
14. Santos AFS, Luz LA, Argolo ACC, Teixeira J, Paiva PMG,
Coelho LCBB (2009) Isolation of a seed coagulant Moringa ol-
eifera lectin. Process Biochem 44:504–508
15. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction
data collected in oscillation mode. Methods Enzymol 276:307–326
16. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang 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
17. Roedin J, Sjoedahl S, Josefsson LG, Rask L (1992) Character-
ization of a Brassica napus gene encoding a cruciferin subunit:
estimation of sizes of cruciferin gene families. Plant Mol Biol
20:559–563
18. Dalgalarrondo M, Robin JM, Azanza JL (1986) Subunit com-
position of the globulin fraction of rapeseed (Brassica napus L.).
Plant Sci 43:115–124
19. Robin JM, Inquello V, Mimouni B, Azanza JL (1991) Relation-
ship between immunological properties and structural model of
11 s rapeseed globulin. Phytochemistry 30:3511–3513
20. Bérot S, Compoint JP, Larré C, Malabat C, Guéguen J (2005)
Large scale purification of rapeseed proteins (Brassica napus L.).
J Chromatography B 818:35–42
21. Derbyshire E, Wright DJ, Boulter D (1976) Legumin and vicilin,
storage proteins of legume seeds. Phytochemistry 15:3–24
22. Tandang MRG, Adachi M, Utsumi S (2004) Cloning and expression
of rapeseed procruciferin in Escherichia coli and crystallization of
the purified recombinant protein. Biotechnol Lett 26:385–391
23. Adachi M, Takenaka Y, Gidamis AB, Mikami B, Utsumi S
(2001) Crystal structure of soybean proglycinin A1aB1b homo-
trimer. J Mol Biol 305:291–305
24. Matthews BW (1968) Solvent content of protein crystals. J Mol
Biol 33:491–497
123