Search results for "ERAS"

High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization.

2015

We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, b…

Chemical conjugation of dexamethasone to a polyaspartamide and in vitro evaluation studies

2004

Two macromolecular conjugates of dexamethasone containing different drug amounts were synthesized using PHEA as the polymeric carrier and a succinic group as spacer. The content of linked drug was equal to 25.3% w/w (conjugate A) and 12.7% w/w (conjugate B). Both polymeric conjugates, unlike the free drug, were water-soluble and the amount of unlinked drug was evaluated to be approximately about 0.01% w/w. Both conjugates were relatively stable in vitro at pH 7.4 whereas in the presence of esterase only the conjugate B was able to release drug under the used experimental conditions. This dissimilar behavior has been attributed to the distinct macromolecular conformations assumed in aqueous …

Estimation of the density of the protocatechuate-degrading bacterial community in soil by real-time PCR

2008

Summary The β-ketoadipate pathway is the major route for degradation of aromatic compounds by various soil microorganisms. Protocatechuate 3,4-dioxygenase, a key enzyme of this pathway and which is encoded by pcaGH genes, catalyses the ring cleavage of protocatechuate. Microorganisms harbouring pcaGH genes are widely distributed in the environment but little is known about their relative abundance within the total microflora. Hence, this paper reports the development of a real-time PCR assay to quantify the bacterial pcaH sequence that encodes the β sub-unit of the protocatechuate 3,4-dioxygenase. This real-time PCR assay was linear over seven orders of magnitude with a calculated efficienc…

The Catalytic Mechanism of Carboxylesterases: A Computational Study

2014

The catalytic mechanism of carboxylesterases (CEs, EC 3.1.1.1) is explored by computational means. CEs hydrolyze ester, amide, and carbamate bonds found in xenobiotics and endobiotics. They can also perform transesterification, a reaction important, for instance, in cholesterol homeostasis. The catalytic mechanisms with three different substrates (ester, thioester, and amide) have been established at the M06-2X/6-311++G**//B3LYP/6-31G* level of theory. It was found that the reactions proceed through a mechanism involving four steps instead of two as is generally proposed: (i) nucleophilic attack of serine to the substrate, forming the first tetrahedral intermediate, (ii) formation of the ac…

RAS proteins and control of the cell cycle inSaccharomyces cerevisiae

1995

Genes related to the mammalian H-, K-, and N-ras oncogenes were identified in S. cerevisiae by DNA hybridization techniques (for reviews, see Tamanoi, 1988; Gibbs and Marshall, 1989; Broach and Deschenes, 1990). According to the rules of yeast genetics (dominant genes are indicated by three capital letters followed by a number), the yeast genes were denominated RAS1 and RAS2 (collectively referred to as RAS). The corresponding RAS1 and RAS2 proteins were 309 and 322 amino acids long, respectively. The sequence similarity between the human and yeast proteins was very high, reaching 90% identity at the level of the N-terminal 80 amino acids. As a consequence, perfect sequence conservation was…

2000

In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome. We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 27…

N-glycosylation efficiency is determined by the distance to the C-terminus and the amino acid preceding an Asn-Ser-Thr sequon

2010

N-glycosylation is the most common and versatile protein modification. In eukaryotic cells, this modification is catalyzed cotranslationally by the enzyme oligosaccharyltransferase, which targets the β-amide of the asparagine in an Asn-Xaa-Ser/Thr consensus sequon (where Xaa is any amino acid but proline) in nascent proteins as they enter the endoplasmic reticulum. Because modification of the glycosylation acceptor site on membrane proteins occurs in a compartment-specific manner, the presence of glycosylation is used to indicate membrane protein topology. Moreover, glycosylation sites can be added to gain topological information. In this study, we explored the determinants of N-glycosylati…

Biochemical and Kinetic Analyses of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus

1998

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA…

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1981

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for hal…

Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed m…

2002

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the α/β hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model…