Biomedical subjects

R Assenberg

Publications and source records attributed to R Assenberg.

3 recordsLinked to original sources

PubMed2006

Eukaryotic expression: developments for structural proteomics.

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Animals↗

PubMed2001

Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure.

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2001

The electrophoretic mobility of DNA three-way junctions is affected by the sequence of overhanging single-stranded ends.

The folding of three- and four-way DNA junctions is often assessed by comparing the electrophoretic mobility of restriction enzyme fragments, using the long-short arm assay. We have compared the mobility of synthetic three-way junctions that contain identical branch point sequences, but different restriction sites in the arms. We show that the mobility of fragments is affected by the sequence of the overhanging ends. In general, GC-rich overhangs produce fragments with anomalous mobilities. These anomalies can be prevented by treating the cleaved junctions with mung bean endonuclease, elevating the electrophoresis temperature or using blunt cleaving restriction endonucleases.

DNA, Single-Stranded↗