Tandem affinity purification is the principal method for purifying and identifying stable protein complexes system-wide in whole cells. Although highly effective, this approach is laborious and impractical in organisms where genetic manipulation is not possible. Here, we propose a novel "tagless" strategy that combines multidimensional separation of endogenous complexes with mass spectrometric ...

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression ...

Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusio...

BACKGROUND Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column sc...

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an e...

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GF...

background: phenylalanine dehydrogenase (phedh ec 1.4.1.20) is a nad+-dependent enzyme that performs the reversible oxidative deamination of l-phenylalanine to phenylpyruvate. it plays an important role in detection and screening of phenylketonuria (pku) diseases and production of chiral intermediates as well. the main goal of this study was to find a simple and rapid alternative method for pur...

Polarity Reversed-phase chromatography and hydrophobic-interaction chromatography Centrifugation: Analytical Centrifugation. Chromatography: Protein Separation. III/Enzymes: Liquid Chromatography. Proteins: Capillary Electrophoresis; Centrifugation; Crystallization; Electrophoresis; Glycoproteins: Liquid Chromatography; High-Speed Countercurrent Chromatography; Ion Exchange; Metalloproteins: Ch...

Small interfering RNA (siRNA)-induced gene silencing shows great promise in genomic research and therapeutic applications. siRNA duplexes are typically assembled from complementary synthetic oligonucleotides. High-purity single-stranded species are required for in vivo applications. Methods for separation, characterization, and purification of short RNA strands have been developed based on reve...