21 research outputs found
Understanding heterologous protein overproduction under the T7 promoter - A practical exercise
Because various genome projects have been advanced many genes are known,
and large amounts of proteins are required to elucidate their function.
Most biomolecular research laboratories have a need to overexpress a
certain gene, or a part of it, in eukaryotic or prokaryotic expression
systems. It is therefore important for young students to become familiar
with the technology of heterologous gene expression systems. Gene
expression in eukaryotic cells is rather complicated and costly and is
therefore not ideally suited to exercises for students. The goal of this
paper is to describe an experimental example of a well known and broadly
used prokaryotic system, the pET system, that works under the strong T7
promoter. The clones described in this paper are suitable for the
practical exercise and are available upon request
Cloning, overproduction, purification and crystallization of the DNA binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima
The humar gene encoding for the histone-like DNA-binding protein HU from
the hyperthermophilic eubacterium Thermotoga maritima was efficiently
overexpressed in Escherichia coli under the T7 promoter. The HU protein
was purified using SP-Sepharose ion-exchange and heparin-affinity
chromatography and was successfully crystallized in ammonium sulfate.
The crystals were grown in the tetragonal form in space group P4(3) or
P4(1) and have unit-cell dimensions a = b = 46.12, c = 77.56 Angstrom, a
= beta = gamma = 90 degrees. The crystals diffract X-rays to 1.6
Angstrom resolution using synchrotron radiation and are suitable for
determination of the HU structure at high resolution
Isolation, cloning, and overexpression of a chitinase gene fragment from the hyperthermophilic archaeon Thermococcus chitonophagus: semi-denaturing purification of the recombinant peptide and investigation of its relation with other chitinases
A 189-bp sequence was isolated from the hyperthermophilic archaeon
Thermococcus chitonophagus and was found to present strong homology with
a large number of chitinase genes from a variety of organisms and
particularly with the chitinaseA gene from Pyrococcus kodakaraensis
(Pk-chiA). This fragment was subcloned to an expression vector and
overexpressed in Escherichia coli. The E coli BLR21(DE3)pLysS
transformant, harbouring the gene on the pET-31b plasmid vector, was
found to overproduce the target protein at high levels. The 63
aminoacid-long peptide was efficiently purified to homogeneity, with a
one-step, semi-denaturing affinity chromatography, on a metal chelation
resin and was used for the production of a specific, polyclonal antibody
from rabbits. The produced antibody was demonstrated to display strong
and specific affinity for the chitinase A from Serratia marcescens
(Sm-chiA), as well as the membrane-bound chitinase70 from Thermococcus
chitonophagus (Tc-Chi70). The strong sequence homology, in combination
with the demonstrated specific immunochemical affinity, indicates that
the isolated peptide is part of a chitinolytic enzyme of T
chitonophagus. In particular, it could belong to the membrane-bound
chi70, or to a distinct chitinase, coded by a different gene, or even by
the same gene, following post-transcriptional or post-translational
modifications. (C) 2004 Elsevier Inc. All rights reserved
Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis
A gene encoding a highly thermostable extracellular alpha-amylase from
the hyperthermophilic archaeon Pyrococcus furiosus was identified, The
gene was cloned, sequenced, and expressed in Escherichia coli and
Bacillus subtilis. The gene is 1383 base pairs long and encodes a
protein of 461 amino acids, The open reading frame of the gene was
Verified by microsequencing of the recombinant purified enzyme. The
deduced amino acid sequence is 25 amino acids longer at the N terminus
than that determined by sequencing of the purified protein, suggesting
that a leader sequence is removed during transport of the enzyme across
the membrane. The recombinant alpha-amylase was biochemically
characterized and shows an activity optimum at pH 4.5, whereas the
optimun temperature for enzymatic activity is close to 100 degrees C.
alpha-Amylase shows sequence homology to the other known alpha-amylases
and belongs to family 13 of glycosyl hydrolases. This extracellular
alpha-amylase is not homologous to the subcellular alpha-amylase
previously isolated from the same organism
Structure and dynamics of the DNA-binding protein HU of B-stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy
The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a
90-residue homodimer that binds nonspecifically to B DNA. Although the
structure of the HUBst:DNA complex is not known, the proposed
DNA-binding surface consists of extended arms that project from an
alpha-helical platform. Here, we report Raman and ultraviolet-resonance
Raman (UVRR) spectra diagnostic of subunit secondary structures and
indicative of key side-chains lining the proposed DNA-binding surface.
Raman conformation markers show that the DNA-binding arms of the dimer
contain beta-stranded structure in excess (eight +/- two residues per
subunit) of that reported previously. Important among side-chain markers
are Met (701 cm(-1)), Ala (908 cm(-1)), Arg (1082 cm(-1)), and Pro (1457
cm(-1)). The Ala marker undergoes a substantial shift (908 –> 893
cm(-1)) on deuteration of alanyl peptide sites, indicating a coupled
side-chain/main-chain mode of diagnostic value in the identification of
exchange-protected alanines. A large subset of alanines (67%) in the
a-helical core exhibits robust resistance to exchange. A quantitative
study of NH –> ND exchange exploiting newly identified amide II’
markers of helical (1440 cm(-1)) and nonhelical (1472 cm(-1))
conformations of HUBst indicates unexpected flexibility at the dimer
interface, which is manifested in rapid exchange of 80% of peptide
sites. The results establish a basis for subsequent Raman and UVRR
investigations of HUBst:DNA complexes and provide a framework for
applications to other DNA-binding architectural proteins
De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin
The purification scheme of chitinase A (ChiA) from S. marcescens has
been extensively revised. The pure enzyme crystallizes readily under new
crystallization conditions. The ChiA crystal structure has been refined
to 1.55 Angstrom resolution and the crystal structure of ChiA
co-crystallized with the inhibitor allosamidin has been refined to 1.9
Angstrom resolution. Allosamidin is located in the deep active-site
tunnel of ChiA and interacts with three important residues: Glu315, the
proton donor of the catalysis, Asp313, which adopts two conformations in
the native structure but is oriented towards Glu315 in the inhibitor
complex, and Tyr390, which lies opposite Glu315 in the active-site
tunnel
Inhibition of two family 18 chitinases by various allosamidin derivatives
The inhibitory activities of several allosamidin derivatives on two
family 18 chitinases, an insect enzyme from the epithelial cell line
from Chironomus tentans, and a bacterial enzyme, chitinase A from
Serratia marcescens, were evaluated. The following structural
requirements are necessary for inhibition of the Chironomus enzyme:
1. One N-acetylallosamine residue can be omitted without impairment of
enzyme inhibition.
2. At least one N-acetylallosamine sugar must be present.
3. Glucosamine can replace the allosamine moiety without a negative
effect on the inhibitory activity.
4. The spatial arrangement of the allosamizoline moiety is important for
inhibition.
5. If one sugar is omitted and the arrangement of the cyclitol residue
is changed, the inhibitory effect is diminished further.
For purified chitinase A from Serratia marcescens the arrangement of the
aglycone moiety is equally important, but recognition of the sugar is
different:
1. Omission of one allosamine residue decreases the inhibitory activity
considerably.
2. Inhibition is improved if the remaining N-acetylallosamine is
replaced by the epimer N-acetylglucosamine.
Only endochitinase activity is affected, since chitin formation (up to
10(-4) M) and N-acetylglucosaminidase activity (up to 10(-3) M) are not
impaired, at least in Chironomus cells. (C) 1998 SCI
Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation
The stereochemistry of the reaction catalysed by Serratia marcescens
chitobiase was determined by HPLC separation of the anomers of
N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl
N-acetyl-beta-D-glucosaminide (PNP-GlcNAc). In the early stages of the
reaction, the beta-anomer was found to prevail, whereas the alpha-anomer
dominated at mutarotation equilibrium. This established that chitobiase
hydrolyses glycosidic bonds with overall retention of the anomeric
configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was
competitively inhibited by a series of chito-oligosaccharides (degree of
polymerization 2-5) that were selectively de-N-acetylated at their
non-reducing end. The results are in accord with the participation of
the acetamido group at C-2 of the substrate in the catalytic mechanism
of chitobiase and related enzymes
High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis
Chitinase A (ChiA) from the bacterium Serratia marcescens is a
hydrolytic enzyme, which cleaves beta -1,4-glycosidic bonds of the
natural biopolymer chitin to generate di-N-acetyl-chitobiose. The
refined structure of ChiA at 1.55 Angstrom shows that residue Asp313,
which is located near the catalytic proton donor residue Glu315, is
found in two alternative conformations of equal occupancy. In addition,
the structures of the cocrystallized mutant proteins D313A, E315Q,
Y390F, and D391A with octa- or hexa- N-acetyl-glucosamine have been
refined at high resolution and the interactions with the substrate have
been characterized. The obtained results clearly show that the active
site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates
the substrate in the vicinity of the scissile bond. Furthermore, the
enzyme imposes a critical “chair” to “boat” conformational
change on the sugar residue bound to the - 1 subsite. According to our
results, we suggest that residues Asp313 and Tyr390 along with Glu315
play a central role in the catalysis. We propose that after the
protonation of the substrate glycosidic bond, Asp313 that interacts with
Asp311 flips to its alternative position where it interacts with Glu315
thus forcing the substrate acetamido group of - 1 sugar to rotate around
the C2-N2 bond. As a result of these structural changes, the water
molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido
group is displaced to a position that allows the completion of
hydrolysis. The presented results suggest a mechanism for ChiA that
modifies the earlier proposed “substrate assisted” catalysis
Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant
We have studied the stability of the histone-like, DNA-binding protein
HU from the hyperthermophilic eubacterium Thermotoga maritima and its
E34D mutant by differential scanning microcalorimetry and CD under
acidic conditions at various concentrations within the range of 2-225
mum of monomer. The thermal unfolding of both proteins is highly
reversible and clearly follows a two-state dissociation/unfolding model
from the folded, dimeric state to the unfolded, monomeric one. The
unfolding enthalpy is very low even when taking into account that the
two disordered DNA-binding arms probably do not contribute to the
cooperative unfolding, whereas the quite small value for the unfolding
heat capacity change (3.7 kJ.K-1.mol(-1)) stabilizes the protein within
a broad temperature range, as shown by the stability curves (Gibbs
energy functions vs. temperature), even though the Gibbs energy of
unfolding is not very high either. The protein is stable at pH 4.00 and
3.75, but becomes considerably less so at pH 3.50 and below, to the
point that a simple decrease in concentration will lead to unfolding of
both the wild-type and the mutant protein at pH 3.50 and low
temperatures. This indicates that various acid residues lose their
charges leaving uncompensated positively charged clusters. The wild-type
protein is more stable than its E34D mutant, particularly at pH 4.00 and
3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25
degreesC for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which
seems to be related to the effect of a salt bridge between E34 and K13