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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 14, Number 5, 2007
Contents
High-Level Bacterial Expression and Purification
of Apicomplexan Micronemal Proteins for Structural Studies
Pp. 411-415
S. Saouros, T.M.A. Blumenschein, K. Sawmynaden, J. Marchant,
T. Koutroukides, B. Liu, P. Simpson, E.P. Carpenter and S.J.
Matthews
[Abstract]
Expression, Purification and Antibody Binding Activity
of Human Papillomavirus 16 L1 Protein Fused to Maltose Binding
Protein Pp. 417-424
H.-J. Cho, M.S. Hahm, M.K. Kim, I.-K. Han, W.-W. Jung,
H.-G. Choi, J.A. Kim and Y.-K. Oh
[Abstract]
SAXS Studies of Human Protein HC (α1-Microglobulin)
Pp. 425-429
M. Kozak and A. Grubb
[Abstract]
Molecular Cloning and the Allergenic Characterization
of Tropomyosin from Tyrophagus putrescentiae
Pp. 431-436
K.Y. Jeong, H. Lee, J.S. Lee, J. Lee, I.-Y. Lee, H.-I.
Ree, C.-S. Hong and T.-S. Yong
[Abstract]
Inhibition of Proliferation of ECV304 Cells by a Disintegrin
from Chinese Green Tree Viper Pp. 437-441
Y.-P. Han, X.-Y. Lu, J.-R. Xu and Y.-P. Zhang
[Abstract]
The Surface Plasmon Resonance Imaging Sensor for Papain
Based on Immobilized Cystatin Pp. 443-445
E. Gorodkiewicz
[Abstract]
Anti-Free-Radical Properties of the Peptide Fractions
Isolated from String Bean by Immobilized Metal Ion Affinity
Chromatography Pp. 447-454
M. Karas
[Abstract]
Identification of Immunogenic MHC Class II Tyrosinase-Derived
Peptides Using HLA-DR1 and HLA-DR4 Transgenic Mice
Pp. 455-460
R.B.V. Horton, S.A.S. Laversin, S.P. Reeder, R.C. Rees
and S.E.B. McArdle
[Abstract]
Variation in Resistance to Benzimidazole in Different
Biocontrol Agents Based on Protein Sequence Homology
Pp. 461-464
B.M.S. Jarullah, R.B. Subramanian and M.J. Jummanah
[Abstract]
Improvement of Prediction of Mutation Positions in
H5N1 Hemagglutinins of Influenza A Virus Using Neural Network
with Distinguishing of Arginine, Leucine and Serine
Pp. 465-470
G. Wu and S. Yan
[Abstract]
Atomic Force Microscopy Study of Peptides Homologous
to Beta Domain of Alpha-Lactalbumins Pp. 471-474
V.V. Egorov, K.V. Solovyov, N.A. Grudinina, D.V. Lebedev,
V.V. Isaev-Ivanov, O.I. Kiselev and M.M. Shawlovsky
[Abstract]
Purification of Native and Recombinant Cobra Venom
Factor Using Thiophilic Adsorption Chromatography
Pp. 475-480
J. Kölln, I. Braren, R. Bredehorst and E. Spillner
[Abstract]
Structural and Conformational Analysis of Scorpion
(Buthus sindicus) Hemocyanin Using Low Resolution
Techniques Pp. 481-488
S.A. Ali, J.G. Grossmann, A. Abbasi and W. Voelter
[Abstract]
Enrichment of Multiphosphorylated Peptides by Immobilized
Metal Affinity Chromatography Using Ga(III)- and Fe(III)-Complexes
Pp. 489-496
C. Sykora, R. Hoffmann and P. Hoffmann
[Abstract]
Variation of pKa in the N-Terminal Tyrosine Side Chain
in Octapeptide Analogs of Tendamistat Influences α-Amylase
Inhibition Pp. 497-501
D.L. Heyl, B. Sethi, A. Rogalski, C.E. Bowen, M. Lawrence,
L. Beitler, E. Harning, A. Hancer, S. Sreekumar and S. Fernandes
[Abstract]
Crystallization Reports
Crystallization and Preliminary X-Ray Crystallographic
Studies of SMU.134 Protein from Caries Pathogen Streptococcus
mutans Pp. 503-504
H. Li, L.-F. Li, X.-D. Su, X. Zhao and Y.-H. Liang
[Abstract]
Crystallization and Preliminary X-Ray Analysis of
Sau3AI/E64A Mutant Protein Pp. 505-506
C. Xu, J. Song, Y. Ding, F. Yu, L. Sun, L. Tang, X. Hu,
Z. Zhang and J. He
[Abstract]
Abstracts
[Back to top]
High-Level Bacterial Expression and Purification of
Apicomplexan Micronemal Proteins for Structural Studies
S. Saouros, T.M.A. Blumenschein, K. Sawmynaden, J. Marchant,
T. Koutroukides, B. Liu, P. Simpson, E.P. Carpenter and S.J.
Matthews
The cysteine-rich N-terminal domain of the micronemal adhesive
protein MIC1 (MIC1-NT) from Toxoplasma gondii was
cloned, expressed in Escherichia coli and purified.
MIC1-NT is amenable to structural studies as shown by preliminary
NMR and X-ray analysis. Positive results with two further
micronemal proteins indicate that our strategy has wider application.
[Back to top]
Expression, Purification and Antibody Binding Activity
of Human Papillomavirus 16 L1 Protein Fused to Maltose Binding
Protein
H.-J. Cho, M.S. Hahm, M.K. Kim, I.-K. Han, W.-W. Jung,
H.-G. Choi, J.A. Kim and Y.-K. Oh
Genetic human papillomavirus type 16 L1 (HPV16 L1) has been
widely studied for cervical cancer vaccine development. For
the enzyme-linked immunosorbent assay (ELISA) screening of
these vaccines, HPV16 L1 protein, which is required as a coating
protein, has previously been expressed from costly and laborious
recombinant baculovirus-infected insect cells. For a novel
HPV16 L1 expression system characterized by a high yield of
soluble form with simple purification steps, we have cloned
and expressed two different types of HPV16 L1, both fused
to maltose binding protein (MBP) or glutathione-S-transferase
(GST) in Escherichia coli. The yield of soluble HPV16
L1 was influenced by the cultivation temperature. The yield
of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16
L1) was 35% at 37°C, but increased to 85% at 22°C.
Among the fusion partners, MBP provided higher yields of total
and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold
higher yield of the soluble form over insoluble inclusion
bodies under optimized culture conditions. The soluble form
of MBP-HPV16 L1 was purified via MBP affinity chromatography
in a recovery yield of 9.7%. After fusion with MBP, HPV16
L1 showed binding activity to HPV16 L1-specific monoclonal
antibody comparable to HPV16 L1 from the insect cells in ELISA
tests. These results demonstrate that the use of MBP as a
fusion partner may generate a high yield of soluble HPV16
L1 under optimized temperature conditions, and that MBP-fused
HPV16 L1 might be applied further in evaluations of the immune
responses of HPV16 L1-based cervical cancer vaccines.
[Back to top]
SAXS Studies of Human Protein HC (α1-Microglobulin)
M. Kozak and A. Grubb
Protein HC is a low molecular weight heterogeneous glycoprotein
widely distributed in human body fluids and belonging to the
lipocalin superfamily. The monomer contains a single (183
amino acid residues long) peptide chain with 3 cysteine residues
(2 of which form a disulfide bridge) and is glycosylated.
The molecular mass of the glycosylated protein is about 27
kDa.
Native gel electrophoresis results revealed partial oligomerisation
of protein HC, which therefore was analysed by gel filtration.
Two forms (monomer and dimer) of the protein HC were isolated.
The SAXS data were recorded on an X33 camera using synchrotron
radiation (λ=0.15
nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg,
Germany). Solution scattering results permitted determination
of the structural parameters of both forms of the protein
studied. The monomer of protein HC is characterised by a radius
of gyration RG= 2.20 nm and
Dmax=6.3 nm and the dimer
by RG=2.99 nm and Dmax=9.5
nm.
[Back to top]
Molecular Cloning and the Allergenic Characterization
of Tropomyosin from Tyrophagus putrescentiae
K.Y. Jeong, H. Lee, J.S. Lee, J. Lee, I.-Y. Lee, H.-I.
Ree, C.-S. Hong and T.-S. Yong
Storage mites have been recognized as a cause of asthma and
rhinitis. Studies from several countries have shown that the
IgE-mediated allergy to storage mites is of considerable importance,
especially in rural populations. This study aimed to identify
and characterize new allergens from Tyrophagus putrescentiae.
A partial cDNA sequence encoding tropomyosin was isolated
from the cDNA library by immunoscreening using anti-mouse
IgG1 sera raised against T. putrescentiae whole body
extract. The deduced amino acid sequence shares 64-94% identity
with previously known allergenic tropomyosins. Its recombinant
protein was produced by using a pET 28b expression system
and purified by affinity chromatography using Ni-NTA agarose.
The IgE reactivities of tropomyosins from T. putrescentiae
and Dermatophagoides farinae were compared
by enzyme linked immunosorbent assay (ELISA). Recombinant
Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas
recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity
against the same sera from storage mite-sensitized and house
dust mite-sensitized subjects. Both recombinant Tyr p 10 and
Der f 10 showed little inhibition of IgE binding to T.
putrescentiae crude extract by ELISA. Tropomyosin seems
to contribute only a small portion of the cross-reactivity
with house dust mites.
[Back to top]
Inhibition of Proliferation of ECV304 Cells by a Disintegrin
from Chinese Green Tree Viper
Y.-P. Han, X.-Y. Lu, J.-R. Xu and Y.-P. Zhang
A novel disintegrin, stejnin, was purified from the Trimeresurus
stejnegeri venom by gel filtration and reverse phase
high performance liquid chromatography. The molecular weight
of stejnin was determined to be 7428 Da by MALD-TOF MS analysis.
The cDNA encoding the precursor of stejnin was cloned from
the venom gland. From the deduced amino acid sequence, stejnin
is composed of 71 amino acid residues contains the tripeptide
sequence Arg-Gly-Asp (RGD), a well-known characteristic of
the disintegrin family. Stejnin strongly inhibited ADP- and
ristomycin-induced human platelet aggregation with IC50 of
45 and 50 nM, respectively. Stejnin also possessed potent
inhibited cell proliferation of ECV304 cells.
[Back to top]
The Surface Plasmon Resonance Imaging Sensor for Papain
Based on Immobilized Cystatin
E. Gorodkiewicz
The Surface Plasmon Resonance Imaging (SPRI) sensor for papain
determination based on the interaction between the immobilized
cystatin and aqueous papain solution has been developed. The
development of the sensor is a stage in the development of
the sensor for the determination of cystein proteinases of
the papain group. Cystatin was immobilized onto a gold chip
by means of a thiol underlayer (cysteamine) and EDS/NHS reaction.
Conditions of cystatin- papain interaction were optimized
(pH, time of interaction and cystatin concentration). The
developed sensor works within the range of 1 – 10 ng
ml-1. However, the precision
of measurements (RSD equal to 45%) should be improved. The
response of the sensor to papain is specific. This was checked
using BSA as a reference.
[Back to top]
Anti-Free-Radical Properties of the Peptide Fractions
Isolated from String Bean by Immobilized Metal Ion Affinity
Chromatography
M. Karas
In this study, peptides and proteins extracted from string
bean with 1% TCA were separated by chromatography on columns
with copper ions immobilized through IDA and o-phosphoserine
OPS. Protein and peptide concentrations and the anti-free-radical
properties of the isolated fractions were determined. Identification
was obtained using mass spectrometry. The anti-free- radical
activity of all analyzed samples was determined using the
DPPH. test, and was found to depend on reaction
time, choice of chelating agent and the order in which the
fractions were eluted from the column.
[Back to top]
Identification of Immunogenic MHC Class II Tyrosinase-Derived
Peptides Using HLA-DR1 and HLA-DR4 Transgenic Mice
R.B.V. Horton, S.A.S. Laversin, S.P. Reeder, R.C. Rees
and S.E.B. McArdle
The immunogenicity of “novel” MART-1 and Tyrosinase
class-II peptides was assessed in transgenic mice. Tyrosinase141-161
peptide was found to be immunogenic and endogenously processed
in the HLA-DRβ1*0101
and HLA-DRβ1*0401
transgenic mice with peptide specific production of IFNγ
or IL-5 respectively. The MART-129-43
peptide was only found immunogenic in HLA-DRβ1*0101
mice.
[Back to top]
Variation in Resistance to Benzimidazole in Different
Biocontrol Agents Based on Protein Sequence Homology
B.M.S. Jarullah, R.B. Subramanian and M.J. Jummanah
Benzimidazole resistance in thirty isolates of the biocontrol
agents was studied with reference to specific mutations in
the β-tubulin
genes. Our results suggest that apart from the correlation
between specific mutations in the β-tubulin
gene and variation in resistance, the overall ratio of polar
and non-polar amino-acids may also play a vital role in conferring
benzamidazole resistance to these fungi.
[Back to top]
Improvement of Prediction of Mutation Positions in
H5N1 Hemagglutinins of Influenza A Virus Using Neural Network
with Distinguishing of Arginine, Leucine and Serine
G. Wu and S. Yan
In this study, we attempt to improve the prediction of mutation
position in H5N1 hemagglutinin from influenza A virus using
the neural network model with distinguishing of arginine,
leucine and serine. In comparison with previous logistic regression
prediction model, the 3-6-1 feedforward backpropagation neural
network model reduced independents from seven to three without
compromising the predictability, and the prediction is made
according to the most similar pa-rameterised hemagglutinin
rather than the population means.
[Back to top]
Atomic Force Microscopy Study of Peptides Homologous
to Beta Domain of Alpha-Lactalbumins
V.V. Egorov, K.V. Solovyov, N.A. Grudinina, D.V. Lebedev,
V.V. Isaev-Ivanov, O.I. Kiselev and M.M. Shawlovsky
Symmetrical peptide GYDTQAIVENNESTEYG (WT, Wild Type) identical
to 35-51 aminoacid residues of human alpha-lactalbumin (HLA)
and peptide GYDTQTVVNNNGHTDYG (ID, IDeal symmetry) homologous
to beta-domain of mammalian alpha-lactalbumins can form amyloid-like
fibrils in conditions required for fibrillogenesis of HLA.
The latter peptide can also form fibrils in deionized water.
Fibrils formed by these peptides can cause forming of HLA
amyloid-like aggregates in physiological conditions. These
results provide an evidence for presence of amyloidogenic
determinant in beta-domain of alpha-lactalbumin. Thus, symmetry
in the primary structure may play the role in fibrillogenesis
of proteins.
[Back to top]
Purification of Native and Recombinant Cobra Venom
Factor Using Thiophilic Adsorption Chromatography
J. Kölln, I. Braren, R. Bredehorst and E. Spillner
The complement activating venom component Cobra Venom Factor
(CVF) forms a stable CVF-dependent C3 convertase complex,
which initiates continuous activation of the complement system,
consumes all downstream complement components and obliterates
functional complement. Therefore, native CVF is routinely
used as decomplementing agent in vivo and in
vitro. However, in most countries, CVF and even unfractionated
cobra venom are now becoming unavailable due to the CITES
agreement. Although CVF is a complex molecule with three disulfide
linked polypeptide chains and pronounced glycosylation, recombinant
expression of the active molecule in eukaryotic host cells
may provide an alternative source. In this study we describe
a strategy for the production and efficient isolation of recombinant
CVF from supernatant of mammalian cells. Thiophilic adsorption
chromatography (TAC), an efficient procedure for purification
of the human homologue C3, was evaluated for its suitability
regarding purification of both native as well as recombinant
CVF. Native CVF could be purified by TAC in a one-step procedure
from cobra venom with yields of 92% compared to 35% by conventional
approaches. After establishment of stably transfected mammalian
cells recombinant CVF could be obtained and enriched from
CHO supernatants by TAC to a purity of 73%, and up to 90%
if an additional affinity chromatography step was included.
Subsequent characterization revealed comparable hemolytic
and bystander lysis activity and of rCVF and nCVF. These data
demonstrate that the functional expression in mammalian cells
in combination with TAC for purification renders rCVF a highly
attractive substitute for its native counterpart.
[Back to top]
Structural and Conformational Analysis of Scorpion
(Buthus sindicus) Hemocyanin Using Low Resolution
Techniques
S.A. Ali, J.G. Grossmann, A. Abbasi and W. Voelter
Blue oxygen binding protein hemocyanin from scorpion Buthus
sindicus has been investigated using low resolution techniques.
The native protein is a polymer of eight different types of
subunits arranged in four cubic hexameric form (4x6-mers)
as previously annotated using a combination of various types
of chromatographic and electrophoretic techniques. In addition,
both “top face” as well as the “side view”
of the native assembly has also been identified from the negatively
stained specimens using transmission electron microscopy confirming
the overall structural features of arthropodan hemocyanins.
These results are also supported from data obtained from another
low resolution technique i.e. Small Angle X-ray scattering
(SAXS). SAXS results under oxygenated and deoxygenated states
represent a validation case for this technique with key conformational
changes of Rg 88.0 →
86.0 Å ± 1% (Dmax 280.0 →
290.0 Å ± 2%), respectively suggesting that the
oxygenated hemocyanin is longer then the deoxygenated hemocyanin
by almost 2 Å. Likewise, active conformations of the
purified structural and functional subunit Bsin1 under oxygenated
and deoxygenated states also determined by SAXS measurements
revealed a Rg value of 25.2 →
25.7 Å ± 1% (Dmax 75.0 →
75.5 Å ± 2%), respectively suggesting very little
or no contribution of the individual subunit in the overall
conformational change in the native assembly during molecular
breathing. Preliminary molecular shapes for the oxy-molecules,
calculated directly from the scattering profile-alone
in a model-independent procedure, superimpose well on other
closely related known three-dimensional structures of the
same size. Structural and functional aspects of the native
as well as purified subunit and the application of these low
resolution techniques like transmission electron microscopy
and Small Angle X-ray scattering have been discussed.
[Back to top]
Enrichment of Multiphosphorylated Peptides by Immobilized
Metal Affinity Chromatography Using Ga(III)- and Fe(III)-Complexes
C. Sykora, R. Hoffmann and P. Hoffmann
The detection and identification of O-phosphorylation sites
in proteins with mass spectrometry remains a challenge. A
common approach to analyse these modifications is to enrich
phosphopeptides by immobilized metal affinity chromatography
(IMAC) prior to mass spectrometric analysis. In this study
two commercially available IMAC kits based on Fe(III)-ions
immobilized on magnetic beads and Ga(III)-ions immobilized
on a chelate-resin, have been investigated and the binding
efficiency of peptide mixtures containing non-phosphorylated,
singly, doubly and triply phosphorylated peptides have been
tested.
[Back to top]
Variation of pKa in the N-Terminal Tyrosine Side Chain
in Octapeptide Analogs of Tendamistat Influences α-Amylase
Inhibition
D.L. Heyl, B. Sethi, A. Rogalski, C.E. Bowen, M. Lawrence,
L. Beitler, E. Harning, A. Hancer, S. Sreekumar and S. Fernandes
Peptide analogs of tendamistat were synthesized and analyzed
for α-amylase
inhibitory activity. The pKa
of the N-terminal tyrosine was modified by incorporation of
ring-substituted analogs, which alters hydrogen bonding capacity.
Ki values ranging from 70
to 524 μM
generally increased with increasing pKa,
indicating a necessity for H-bond donor ability.
[Back to top]
Crystallization and Preliminary X-Ray Crystallographic
Studies of SMU.134 Protein from Caries Pathogen Streptococcus
mutans
H. Li, L.-F. Li, X.-D. Su, X. Zhao and Y.-H. Liang
The smu.134 gene encodes a putative transcriptional
regulator of 217 residues in Streptococcus mutans,
a major pathogen for human dental caries. The gene was cloned
into expression vector pET28α
and expressed in soluble form in E. coli strain BL21
(DE3) with a His tag at its N-terminus. The recombinant protein
SMU.134 was purified to homogeneity in a two step procedure
of Ni2+ chelating and size
exclusion chromatography. Crystals suitable for X-ray diffraction
were obtained by hanging-drop vapor diffusion method and diffracted
to 2.6 Å. The crystal belonged to space group P212121,
with unit-cell parameters a=55.03 Å, b=80.84
Å, c=107.96 Å.
[Back to top]
Crystallization and Preliminary X-Ray Analysis of
Sau3AI/E64A Mutant Protein
C. Xu, J. Song, Y. Ding, F. Yu, L. Sun, L. Tang, X. Hu,
Z. Zhang and J. He
Sau3AI is a type II restriction endonuclease that
recognizes the palindromic sequence 5'–GATC-3' and cleaves
5’ to G residue on each strand. The E64A mutant full
length protein was cloned and expressed in Escherichia
coli. The purified (His)6-tagged
protein has monomer and dimer fraction and was crystallized
by the hanging-drop vapor-diffusion technique. The dimer protein
crystals can diffract to 3.0Å resolution and the monomer
protein crystals can diffract to better than 2.8Å resolution.
One completed dataset has been collected and it shows that
the monomer orthorhombic Sau3AI/E64A crystal is in
space group C2221 with unit cell parameters (69.44, 197.60,
191.46, 90, 90, 90) and contains two molecules in one asymmetric
unit.
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