Biomedical subjects

Igor A Kozlov

Publications and source records attributed to Igor A Kozlov.

4 recordsLinked to original sources

PubMed2007

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Binding Sites↗

PubMed2006

A method for rapid protease substrate evaluation and optimization.

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.

Biological Assay↗

PubMed2005

Significant improvement of quality for long oligonucleotides by using controlled pore glass with large pores.

A key factor influencing the quality of long oligonucleotides is the choice of controlled pore glass (CPG) which is used as a solid support during oligonucleotide synthesis. We studied the influence of CPG pore size on the quality of 75-mer oligonucleotides. Using electrophoresis and HPLC, we demonstrated failure modes that can occur at certain oligo lengths with 1000A pore size, and compared yield and purity of 75-mer oligos using 1000A and larger pore size CPG. We showed that oligonucleotides with much better quality are obtained using CPG with pore sizes of 1400A and larger. We also identified the key characteristics for CPG selection that lead to the best CPG performance.

Chromatography, High Pressure Liquid↗

PubMed2004

Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection.

Three methods for the conjugation of oligonucleotides to antibodies and the subsequent application of these conjugates to protein detection at attomole levels in immunoassays are described. The methods are based on chemical modification of both antibody and oligonucleotide. Aldehydes were introduced onto antibodies by modification of primary amines or oxidation of carbohydrate residues. Aldehyde- or hydrazine-modified oligonucleotides were prepared either during phosphoramidite synthesis or by post-synthesis derivatization. Conjugation between the modified oligonucleotide and antibody resulted in the formation of a hydrazone bond that proved to be stable over long periods of time under physiological conditions. The binding activity of each antibody-oligonucleotide conjugate was determined to be comparable to the corresponding unmodified antibody using a standard sandwich ELISA. Each oligonucleotide contained a unique DNA sequence flanked by universal primers at both ends and was assigned to a specific antibody. Highly sensitive immunoassays were performed by immobilizing analyte for each conjugate onto a solid support with cognate capture antibodies. Binding of the antibody-oligonucleotide conjugate to the immobilized analyte allowed for amplification of the attached DNA. Products of amplification were visualized using gel electrophoresis, thus denoting the presence of bound analyte. The preferred conjugation method was used to generate a set of antibody-oligonucleotide conjugates suitable for high-sensitivity protein detection.

Antibodies↗