Protein profiling by microscale solution isoelectrofocusing (MicroSol-IEF).

Sample prefractionation is essential for more comprehensive coverage and reliable detection of low-abundance proteins in complex proteomes. An efficient and reproducible new method for sample prefractionation is microscale solution isoelectrofocusing (MicroSol-IEF), in which samples are separated into chambers defined by membranes of specific pH, yielding well resolved fractions on the basis of isoelectric point (pI). The output seamlessly interfaces with narrow-pH-range 2-D gels, enhancing data obtained from protein profiling studies, including quantitative proteome comparisons.

Overview of proteome analysis.

This unit reviews the new discipline of proteomics, which includes any large-scale protein-based systematic analysis of the proteome or defined sub-proteome from a cell, tissue, or entire organism. Proteomics originated in the mid-1990 s due to two key enabling advances, availability of complete genome sequences, and mass spectrometry advances that allowed high sensitivity identifications of proteins. Proteome analyses can be broadly categorized into three types of studies: quantitative protein profile comparisons, analysis of protein-protein interactions, and compositional analysis of simple proteomes or subproteomes such as organelles or large protein complexes.

Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO.

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed.

Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli.

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein and 5-lipoxygenase-activating protein (FLAP) are among a large number of membrane proteins that are poorly expressed when traditional expression systems and methods are employed. Therefore to efficiently express difficult membrane proteins, molecular biologists will have to develop novel or innovative expression systems. To this end, we have expressed the SARS-CoV M and FLAP proteins in Escherichia coli by utilizing a novel gene fusion expression system that takes advantage of the natural chaperoning properties of the SUMO (small ubiquitin-related modifier) tag.

Expression and purification of SARS coronavirus proteins using SUMO-fusions.

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale.

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical.

Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis.

Comprehensive analysis of complex proteomes requires prefractionation of samples prior to two-dimensional gel electrophoresis (2-DE). This study demonstrates the utility of using a high resolution sample prefractionation method and slightly overlapping narrow pH range two-dimensional gel electrophoresis to enhance quantitative comparisons of complex proteomes. A key feature of this strategy is to prefractionate samples into a few well-defined pools using microscale solution isoelectric focusing (nusol-IEF) prior to 2-DE protein analysis.