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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 14, Number 8, 2007
Contents
Regular Papers
Overexpression and Purification of the RyR1
Pore-Forming Region Pp. 742-746
G.B. Kang, H.-E. Song, D.-W. Song, M.-K. Kim, S.-H. Rho,
D.H. Kim and S.H. Eom
[Abstract]
Folding of the C-Terminal Fragment V111-D143 of Staphylococcal
Nuclease in Aqueous Solution Pp. 747-755
Y. Geng, M. Wang, T. Xie, Y. Feng and J. Wang
[Abstract]
Enhanced Soluble Production of Biologically Active
Recombinant Human p38 Mitogen-Activated-Protein Kinase (MAPK)
in Escherichia coli Pp. 756-766
S.K. Khattar, P. Gulati, P.K. Kundu, V. Singh, U. Bughani,
M. Bajpai and K.S. Saini
[Abstract]
Analysis of Monomeric Mutants of HSC70: A Possible
Relationship Between Oligomerization and Functional Properties
Pp. 761-765
M. Amor-Mahjoub, N. Gomez-Vrielyunck, J.P. Suppini, B.
Fouchaq, N. Benaroudj and M. Ladjimi
[Abstract]
Structural Characterization of Eight Cyclic Lipopeptides
Produced by Bacillus subtilis HSO121 Pp.
766-773
X. Liu, N.I.A. Haddad, S. Yang and B. Mu
[Abstract]
The Lysozyme from Insect (Manduca sexta)
Is a Cold-Adapted Enzyme Pp. 774-778
R.R. Sotelo-Mundo, A.A. López-Zavala, K.D. Garcia-Orozco,
A.A. Arvizu-Flores, E.F. Velázquez-Contreras, E.M.
Valenzuela-Soto, A. Rojo-Dominguez and M.R. Kanost
[Abstract]
Endothelin and Its Receptor Interactions: Role of
Extracellular Receptor Domain and Length of Peptide Ligands
Pp. 779-783
K. Saravanan, G. Hariprasad, O. Jitesh, U. Das, S. Dey,
S. Sharma, P. Kaur, T.P. Singh and A. Srinivasan
[Abstract]
Conformational Studies of Glutenin Polymers from Different
Wheat Cultivars by Circular Dichroism Spectroscopy
Pp. 784-787
S. Fisichella and A. Savarino
[Abstract]
Expression and Purification of Uniformly 15N-Labeled
Amyloid β
Peptide 1-40 in Escherichia coli Pp. 788-792
M. Nagata-Uchiyama, M. Yaguchi, Y. Hirano and T. Ueda
[Abstract]
Expression of a Lipid Transfer Protein in Escherichia
coli and Its Phosphorylation by a Membrane-Bound Calcium-Dependent
Protein Kinase Pp. 793-799
M.L. Martin, E. Espinosa Vidal and L. de la Canal
[Abstract]
A Novel Lectin Isolated from the Hemolymph of the
Marine Hair Crab Erimacrus isenbeckii
Pp. 800-803
Y.J. Na, Y.J. Kim, B.T. Park, B.W. Jung, K.W. Hwang and
H. Kim
[Abstract]
Overexpression in Escherichia coli, Purification
and Characterization of Thermoanaerobacter tengcongensis
Elongation Factor G with Multiple Rare Codons Pp.
804-810
L. Zhang, P. Xue and H. Zhang
[Abstract]
Prediction of Protein Structure Classes with Pseudo-Amino
Acid Composition and Fuzzy Support Vector Machine Network
Pp. 811-815
Y.-S. Ding, T.-L. Zhang and K.-C. Chou
[Abstract]
Screening of a Phage Display Library of Exendin-4
Mutants with the Extracellular Domain of Rat GLP-1 Receptor
Pp. 816-821
X. Yin, Y. Ma, M. Liu, W. Gao and D. Wei
[Abstract]
Expression, Purification and Characterization of Haemophilus
influenzae 3-Isopropylmalate Dehydrogenase (LeuB) Pp.
822-827
S. Martignon, F. Rossi and M. Rizzi
[Abstract]
Chitinase-Like Proteins with Antifungal Activity from
Emperor Banana Fruits Pp. 828-831
V.S.M. Ho and T.B. Ng
[Abstract]
Pigment Epithelium-Derived Factor (PEDF) Prevents
Advanced Glycation End Products (AGEs)-Elicited Endothelial
Nitric Oxide Synthase (eNOS) Reduction Through Its Anti-Oxidative
Properties Pp. 832-835
S.-I. Yamagishi, S. Ueda, T. Matsui, K. Nakamura, T. Imaizumi,
M. Takeuchi and S. Okuda
[Abstract]
Crystallization Report
Preparation, Crystallization and Preliminary X-Ray
Analysis of Threonine Synthase from Streptococcus mutans
Pp. 836-838
D.-W. Tang, L.-F. Li, Y.-M. Yu, X.-Y. Liu, X.-D. Su, X.
Zhao and Y.-H. Liang
[Abstract]
Abstracts
[Back to top]
Overexpression and Purification of the RyR1 Pore-Forming
Region Pp. 742-746
G.B. Kang, H.-E. Song, D.-W. Song, M.-K. Kim, S.-H. Rho,
D.H. Kim and S.H. Eom
Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium
channel that plays a pivotal role in skeletal muscle contraction.
Sequence comparison and mutagenesis studies indicate that
the pore architecture of RyR1, including the last two transmembrane
helices and the luminal loop linking them, is similar to that
of the bacterial KcsA K+
channel. Here, we describe the overexpression and purification
of the C-terminal polyhistidine-tagged RyR1 pore-forming region.
The nonionic detergent lauryldimethylamine oxide (LDAO) was
selected for solubilization of the protein based on its ability
to extract the protein from the membrane and to maintain it
in a monodisperse state. The protein was then purified using
nickel-affinity chromatography and gel filtration. Gel filtration
analysis confirmed that the RyR1 fragment containing the pore-forming
region (amino acids 4829-5037) is sufficient to form a tetramer.
[Back to top]
Folding of the C-Terminal Fragment V111-D143 of Staphylococcal
Nuclease in Aqueous Solution
Y. Geng, M. Wang, T. Xie, Y. Feng and J. Wang
Studies of conformational features of fragments SNase(111-143)
and SNase(118-143) and segment E122-K136 in 1-139 fragment
(SNase139) suggest that the high intrinsic helical propensity
can drive segment E122-K136 fold into a stable helix only
when the segments V111-H121 and L137-D143 flanked on segment
E122-K136 in staphylococcal nuclease (SNase) have stable folding.
[Back to top]
Enhanced Soluble Production of Biologically Active
Recombinant Human p38 Mitogen-Activated-Protein Kinase (MAPK)
in Escherichia coli
S.K. Khattar, P. Gulati, P.K. Kundu, V. Singh, U. Bughani,
M. Bajpai and K.S. Saini
The conditions were optimized for maximum soluble yield of
biologically active recombinant p38α
mitogen activated protein kinase (MAPK) vis-à-vis insoluble
fraction (inclusion body formation). This study reports a
rapid, economical and single step purification process for
the overproduction of GST tagged p38α
MAPK. A yield of 18 mg of highly purified and soluble protein
per liter of bacterial culture within 6 h timeframe was achieved.
The purified protein was found to be biologically suitable
for phosphorylation by upstream kinases and was catalytically
active. We further demonstrated that our in-house p38α
MAPK is more potent (>30%) than a commercially available
enzyme.
[Back to top]
Analysis of Monomeric Mutants of HSC70: A Possible
Relationship Between Oligomerization and Functional Properties
M. Amor-Mahjoub, N. Gomez-Vrielyunck, J.P. Suppini, B.
Fouchaq, N. Benaroudj and M. Ladjimi
Data of this study showed that αD-αE
helices and the conserved interdomain linker are two interfaces
essential not only for the self-association but also for the
functional properties of rat HSC70. Self-association which
is a conserved property of HSP70 seems to be important for
the activity of these proteins.
[Back to top]
Structural Characterization of Eight Cyclic Lipopeptides
Produced by Bacillus subtilis HSO121
X. Liu, N.I.A. Haddad, S. Yang and B. Mu
Lipopeptides are amphiphilic compounds which contain both
hydrophobic fatty acid moieties and amphiphilic peptide moieties.
From the cell-free broth of Bacillus subtilis HSO121,
eight cyclic lipopeptides were isolated by reversed-phase
high performance liquid chromatography (RP-HPLC). The peptide
part of each lipopeptide was elucidated according to electrospray
ionization quadruple-time-of-flight mass spectrometry (ESI
Q-TOF MS) and the fatty acid part was analyzed by electroionization
gas chromatography/mass spectrometry (EI GC/MS). It showed
that fractions 1-8 had molecular masses of 1007, 1021, 1021,
1035, 1035, 1035, 1063, and 1049, respectively. Analysis of
hydrolyzed lipopeptides revealed that they had invariant amino
acid compositions. The differences in molecular weights represent
changes in the number of methylene groups and different types
of branched chains in fatty acids. Peptide sequences of two
of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C,
which was different from previously reported lipopeptides.
The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C.
The fatty acid parts were found to be mixtures of iso
C12, iso C13,
anteiso C13, iso
C14, n C14,
iso C15, anteiso
C15, n C15,
anteiso C16 and
anteiso C17
β-hydroxy fatty acids. The structure of each
lipopeptide was determined to be the β-hydroxy
fatty acid bonded to the peptide chain.
[Back to top]
The Lysozyme from Insect (Manduca sexta)
Is a Cold-Adapted Enzyme
R.R. Sotelo-Mundo, A.A. López-Zavala, K.D. Garcia-Orozco,
A.A. Arvizu-Flores, E.F. Velázquez-Contreras, E.M.
Valenzuela-Soto, A. Rojo-Dominguez and M.R. Kanost
Enzymatic activity is dependent on temperature, although some
proteins have evolved to retain activity at low temperatures
at the expense of stability. Cold adapted enzymes are present
in a variety of organisms and there is ample interest in their
structure-function relationships. Lysozyme (E.C. 3.2.1.17)
is one of the most studied enzymes due to its antibacterial
activity against Gram positive bacteria and is also a cold
adapted protein. In this work the characterization of lysozyme
from the insect Manduca sexta and its activity at
low temperatures is presented. Both M. sexta lysozymes
natural and recombinant showed a higher content of α-helix
secondary structure compared to that of hen egg white lysozyme
and a higher specific enzymatic activity in the range of 5-30
0C. These results together
with measured thermodynamic activation parameters support
the designation of M. sexta lysozyme as a cold adapted
enzyme. Therefore, the insect recombinant lysozyme is feasible
as a model for structure-function studies for cold-adapted
proteins.
[Back to top]
Endothelin and Its Receptor Interactions: Role of
Extracellular Receptor Domain and Length of Peptide Ligands
K. Saravanan, G. Hariprasad, O. Jitesh, U. Das, S. Dey,
S. Sharma, P. Kaur, T.P. Singh and A. Srinivasan
Human endothelin B receptor and its domain-truncated forms
were cloned and expressed in Pichia pastoris. Ligand
binding studies with expressed proteins were carried out using
biotinylated endothelins. Competitive binding and liposome
incorporation studies showed that the extracellular region
is essential for ligand binding and that longer peptides have
higher affinity.
[Back to top]
Conformational Studies of Glutenin Polymers from Different
Wheat Cultivars by Circular Dichroism Spectroscopy
S. Fisichella and A. Savarino
A strong correlation between baking quality and size distribution
of wheat glutenin polymers exists, so we have utilized Circular
Dichroism Spectroscopy in presence of some denaturant agents
to study glutenin polymers. Spectra indicated that all the
changes induced by urea and by sodium dodecyl sulphate followed
a multi-step transition process.
[Back to top]
Expression and Purification of Uniformly 15N-Labeled
Amyloid β
Peptide 1-40 in Escherichia coli
M. Nagata-Uchiyama, M. Yaguchi, Y. Hirano and T. Ueda
For biophysical studies using heteronuclear NMR analysis of
amyloid beta peptide, construction of an efficient and high
yield expression system of uniformly isotopic labeled amyloid
beta peptide is desirable. Here we succeeded in obtaining
15N-labeled amyloid beta
1-40 expressed by attachment to hen egg white lysozyme as
a fusion protein.
[Back to top]
Expression of a Lipid Transfer Protein in Escherichia
coli and Its Phosphorylation by a Membrane-Bound Calcium-Dependent
Protein Kinase
M.L. Martin, E. Espinosa Vidal and L. de la Canal
Ha-AP10 is a basic antifungal peptide from sunflower seeds
(Helianthus annuus antifungal peptide of 10 kDa)
belonging to the family of plant lipid transfer proteins.
We report here its expression in E. coli [Glutathione
S-transferase (GST) system]
and its phosphorylation by endogenous membrane-bound calcium-dependent
protein kinases.
[Back to top]
A Novel Lectin Isolated from the Hemolymph of the
Marine Hair Crab Erimacrus isenbeckii
Y.J. Na, Y.J. Kim, B.T. Park, B.W. Jung, K.W. Hwang and
H. Kim
A lectin that induces hemagglutination activity in mouse and
rabbit erythrocytes has been purified from the hemolymph of
the marine hair crab Erimacrus isenbeckii. The results
of SDS-PAGE, gel-filtration, affinity and anion-exchange chromatography
indicate that this lectin, designated EIL (E. isenbeckii
lectin), was successfully purified as a single protein, and
comprises a mixture of a major (90%) dimeric and a minor (10%)
oligomeric protein with a molecular mass of 116 kDa, with
covalent linking between two subunits of 62 and 54 kDa. The
activity was maximal at pH 5.6–8.0 and at temperatures
below 50°C. The N-terminal amino acid sequences were determined,
and these differed greatly from those of other reported lectins
from invertebrates, vertebrates, or plants. EIL binds with
high specificities to both the O-acetylsialic acid
and mannose that are present in bacterial pathogens, which
suggests that EIL can act as a defense protein against infection
in this crab.
[Back to top]
Overexpression in Escherichia coli, Purification
and Characterization of Thermoanaerobacter tengcongensis
Elongation Factor G with Multiple Rare Codons
L. Zhang, P. Xue and H. Zhang
The fusA gene encoding a thermophilic protein EF-G with multiple
rare condons was cloned from Thermoanaerobacter tengcongensis
(TteEF-G) and overexpressed in Escherichia coli
by cotransfering a RIG plasmid to overcome the potential
codon-bias problem originated from Arg, Ile and Gly. The recombinant
protein was identified by MALDI-TOF-MS for molecular mass
with approximation of 76 kDa and by trypsin digestion coupled
LC-MS/MS for peptide sequence coverage of 61.3%. The in
vivo complementary assay indicates that TteEF-G
could significantly rescue the E. coli LJ14 (frrts)
at the non-permission temperature of 42C in the bi-transformant
of TteRRF and TteEF-G. This study indicated
that coexpression of rare codons’ cognate tRNA is a
useful method for protein overexpression in E. coli.
[Back to top]
Prediction of Protein Structure Classes with Pseudo-Amino
Acid Composition and Fuzzy Support Vector Machine Network
Y.-S. Ding, T.-L. Zhang and K.-C. Chou
It is a critical challenge to develop automated methods for
fast and accurately determining the structures of proteins
because of the increasingly widening gap between the number
of sequence-known proteins and that of structure-known proteins
in the post-genomic age. The knowledge of protein structural
class can provide useful information towards the determination
of protein structure. Thus, it is highly desirable to develop
computational methods for identifying the structural classes
of newly found proteins based on their primary sequence. In
this study, according to the concept of Chou’s pseudo
amino acid composition (PseAA), eight PseAA vectors are used
to represent protein samples. Each of the PseAA vectors is
a 40-D (dimensional) vector, which is constructed by the conventional
amino acid composition (AA) and a series of sequence-order
correlation factors as original introduced by Chou. The difference
among the eight PseAA representations is that different physicochemical
properties are used to incorporate the sequence-order effects
for the protein samples. Based on such a framework, a dual-layer
fuzzy support vector machine (FSVM) network is proposed to
predict protein structural classes. In the first layer of
the FSVM network, eight FSVM classifiers trained by different
PseAA vectors are established. The 2nd
layer FSVM classifier is applied to reclassify the outputs
of the first layer. The results thus obtained are quite promising,
indicating that the new method may become a useful tool for
predicting not only the structural classification of proteins
but also their other attributes.
[Back to top]
Screening of a Phage Display Library of Exendin-4
Mutants with the Extracellular Domain of Rat GLP-1 Receptor
X. Yin, Y. Ma, M. Liu, W. Gao and D. Wei
A phage display library of exendin-4 mutants was screened
with the extracellular domain of rat glucagon-like peptide
1 receptor as the target. A novel variant of exendin-4 with
higher affinity for the receptor fraction than that of the
wild type was identified. The increased affinity was attributed
to the substitution of Glu16
by Val16. Although the substitution
probably caused an increased entropic cost to the helix region,
the linker around Val16 is
more flexible resulting in the increase of affinity for the
receptor.
[Back to top]
Expression, Purification and Characterization of Haemophilus
influenzae 3-Isopropylmalate Dehydrogenase (LeuB)
S. Martignon, F. Rossi and M. Rizzi
3-isopropylmalate dehydrogenase (3IPMDH) is the third enzyme
in leucine biosynthesis and a promising target for the development
of broad-spectrum antibacterial agents. We report here the
expression, purification and biochemical characterisation
of Haemophilus influenzae 3-isopropylmalate dehydrogenase.
The observed enzyme inhibition by the reaction product NADH
could represent a regulatory mechanism for 3IPMDH.
[Back to top]
Chitinase-Like Proteins with Antifungal Activity from
Emperor Banana Fruits
V.S.M. Ho and T.B. Ng
Two 30-kDa proteins with N-terminal sequence homology to chitinases
have been isolated from fruits of the emperor banana by using
a protocol that involved (NH4)2SO4
precipitation, affinity chromatography on Affi-gel blue gel,
ion exchange chromatography by fast protein liquid chromatography
(FPLC) on Mono S and gel filtration by FPLC on Superdex 75.
The proteins were adsorbed on Affi-gel blue gel and Mono S.
They both inhibited mycelial growth in Fusarium oxysporum
but not in Mycosphaerella arachidicola. The chitinase-like
protein more strongly bound on Mono S was obtained with a
slightly lower yield and exhibited a higher antifungal potency
toward F. oxysporum when compared with the less strongly
bound chitinase-like protein.
[Back to top]
Pigment Epithelium-Derived Factor (PEDF) Prevents
Advanced Glycation End Products (AGEs)-Elicited Endothelial
Nitric Oxide Synthase (eNOS) Reduction Through Its Anti-Oxidative
Properties
S.-I. Yamagishi, S. Ueda, T. Matsui, K. Nakamura, T. Imaizumi,
M. Takeuchi and S. Okuda
PEDF is an effective inhibitor of angiogenesis in the eye
with neuronal differentiating activity. Here, we show that
PEDF prevents the AGEs-elicited eNOS reduction through its
anti-oxidative properties. Our present study suggests that
PEDF may also play a protective role against atherosclerosis.
[Back to top]
Preparation, Crystallization and Preliminary X-Ray
Analysis of Threonine Synthase from Streptococcus mutans
D.-W. Tang, L.-F. Li, Y.-M. Yu, X.-Y. Liu, X.-D. Su, X.
Zhao and Y.-H. Liang
The thrC gene of Streptococcus mutans encodes
threonine synthase, which is a potential target for drug design.
To study the structure and function of the enzyme, the thrC
gene was amplified from Streptococcus mutans genomic
DNA and cloned into the expression vector pET28α.
The protein was expressed in Escherichia coli in
soluble form and purified to homogeneity. Crystals suitable
for X-ray diffraction were obtained by hanging-drop vapor
diffusion method. The crystal diffracted to 2.5 Å and
belonged to space group P31
or P32, with unit cell parameters
a=b=60.39 Å, c=118.62 Å.
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