Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by enzyme-linked immunosorbent assay.

Capsular polysaccharides of Streptococcus pneumoniae are used in pneumococcal polysaccharide and protein-conjugate vaccines. Cell-wall polysaccharide (C-Ps) is a critical impurity that must be kept at low levels in purified polysaccharide preparations. Hence, accurate and precise methods for determining C-Ps are needed.

O-phthalaldehyde based quantification of polysaccharide modification in conjugate vaccines.

Polysaccharide-based vaccines cannot stimulate long-lasting immune response in infants due to their inability to elicit a T-cell-dependent immune response. This has been addressed using conjugation technology, where conjugates were produced by coupling a carrier protein to polysaccharides using different conjugation chemistries, such as cyanylation, reductive amination, ethylene diamine reaction, and others. Many glycoconjugate vaccines that are manufactured using different conjugation technologies are already in the market for neonates, infants and young children (e.

Individual polysaccharide quantification in polyvalent pneumococcal conjugate vaccine using rate nephelometry.

The multivalent pneumococcal conjugate vaccine (PCV) contains purified polysaccharides of different serotypes conjugated to a carrier protein. Testing the final formulated product for individual serotype polysaccharide content is critical in vaccine quality control which requires an assay specific to each serotype polysaccharide present in the formulated product. Antibodies specific to the serotypes specific polysaccharide were used in rate nephelometry assay for quantifying individual serotype polysaccharides in the formulated vaccine.

High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides.

Capsular polysaccharides of Streptococcus pneumoniae contain a characteristic mix of monosaccharides in their structure resulting in immunologically distinct serotypes. Pneumococcal capsular polysaccharides include sugars such as hexoses, uronic acids, hexosamines, methyl pentoses, other functional groups are attached to the sugars are N and O-acetyl groups, nitrogen and phosphorus. Most of these components can be quantified using different colorimetric methods.

A reversed phase HPLC-UV method for the simultaneous determination of residual formaldehyde and Triton X-100 in vaccine products.

Many of the inactivated viral vaccines for human and animal use are manufactured using formaldehyde as an inactivating agent. Apart from formaldehyde, Triton X-100 is also one of the chemicals commonly used in viral vaccine manufacturing. Triton X-100 is typically used to extract the cell-associated viruses and / or components during manufacturing process.

2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification.

Anthrone is a routinely used reagent for estimating carbohydrates (Polysaccharides) in research, development and pharmaceutical applications. In presence of sulphuric acid, the polysaccharide gets hydrolyzed to monosaccharides in the form of hydroxymethyl furfural or furfural. The furfural then reacts with anthrone to form a green color complex with a maximum absorbance at 625 nm.

E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53.

Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E.

A simple and rapid method to monitor the disassembly and reassembly of virus-like particles.

Protein fluorescence spectra (~300-440 nm) could be used as a simple and sensitive method to monitor the disassembly and reassembly of virus-like particles (VLPs). Insect cell expressed and purified HPV-16 L1 VLPs show significantly high fluorescence intensity, whereas the fluorescence is almost quenched after disassembly by adding the reducing agent. By removing the reducing agent, the fluorescence was restored to its original intensity, indicating the reassembly of VLPs.

Activity of E. coli ClpA bound by nucleoside diphosphates and triphosphates.

The Escherichia coli ClpA protein is a molecular chaperone that binds and translocates protein substrates into the proteolytic cavity of the tetradecameric serine protease ClpP. In the absence of ClpP, ClpA can remodel protein complexes. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleoside triphosphate binding to first assemble into a hexamer.

Application of the sequential n-step kinetic mechanism to polypeptide translocases.

Clp/Hsp100 proteins are essential motor proteins in protein quality control pathways in all organisms. Such enzymes couple the energy derived from ATP binding and hydrolysis to translocate and unfold polypeptide substrates. Often they perform this role in collaboration with proteases for protein removal or with other chaperones for protein disaggregation.

Effect of substituents of alloxazine derivatives on the selectivity and affinity for adenine in AP-site-containing DNA duplexes.

Using the DNA duplex containing an AP site (5'-TCC AGX GCA AC-3'/3'-AGG TCN CGT TG-5', X = AP site, N = A, T, C, or G), we have found that 2-amino-4-hydroxypteridine (pterin) selectively binds to guanine (G), and that the enhanced binding affinity for G is obtained by its methylated derivative 2-amino-6,7-dimethyl-4-hydroxypteridine (diMe pteridine). Similarly, among the cytosine (C)-selective ligands, i.e.

Molecular mechanism of polypeptide translocation catalyzed by the Escherichia coli ClpA protein translocase.

The removal of damaged or unneeded proteins by ATP-dependent proteases is crucial for cell survival in all organisms. Integral components of ATP-dependent proteases are motor proteins that unfold stably folded proteins that have been targeted for removal. These protein unfoldases/polypeptide translocases use ATP to unfold the target proteins and translocate them into a proteolytic component.

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride.

Background: We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA.

Methods: The combination of experimental and theoretical methods was used.

Results: The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases.

Small-molecule binding at an abasic site of DNA: strong binding of lumiflavin for improved recognition of thymine-related single nucleotide polymorphisms.

The binding behavior of lumiflavin, a biologically vital ligand, with DNA duplexes containing an abasic (AP) site and various target nucleobases opposite the AP site is studied. Lumiflavin binds selectively to thymine (T) opposite the AP site in a DNA duplex over other nucleobases. Using 1H NMR spectroscopy and fluorescence measurements, we show that ligand-DNA complexation takes place by hydrogen-bond formation between the ligand and the target nucleobases and by stacking interactions between the ligand and the nucleobases flanking the AP site.

6,7-Dimethyllumazine as a potential ligand for selective recognition of adenine opposite an abasic site in DNA duplexes.

6,7-Dimethyllumazine more selectively binds to adenine (A) base opposite the abasic site in DNA duplexes (5'-TCC AGX[combining low line] GCA AC-3'/3'-AGG TCN CGT TG-5', X = AP site (Spacer C3), N= A, T, C and G) than the other three nucleobases with a dissociation constant K(d) of ca. 1.0 microM; substituted methyl groups enhance the binding affinity to A and the selectivity for A over T, compared to the parent molecule, lumazine.

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: a case study with isoxanthopterin and its derivatives.

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX.

Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity.

Background: The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements.

Methods: Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.

Alloxazine as a ligand for selective binding to adenine opposite AP sites in DNA duplexes and analysis of single-nucleotide polymorphisms.

Alloxazine can bind to adenine selectively over other nucleobases opposite an abasic site in DNA duplexes (5'-TCC AGX GCA AC-3'/3'-AGG TCN CGT TG-5', X=AP site, N=A, T, C, G) with a dissociation constant of 0.82 microM (pH 7.0, I=0.

Improvement of base selectivity and binding affinity by controlling hydrogen bonding motifs between nucleobases and isoxanthopterin: application to the detection of T/C mutation.

At an abasic site in an oligo-DNA duplex, isoxanthopterin (IX)(dagger) can bind to thymine (T) and cytosine (C) with strong affinity compared to adenine and guanine, but the base selectivity for T against C is moderate. In order to improve both binding affinity and base selectivity for T against C, a methyl group is introduced to IX, which is known as 3-methyl isoxanthopterin (3-MIX),(dagger) by which binding affinity for C is expected to decrease. Indeed, 3-MIX specifically binds to T more strongly than IX and loses its binding affinity for C.