Fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. Many different affinity tags have been developed (Terpe, 2002). One method for isolating or immobilizing a specific protein is the use of affinity tags. Other downstream applications such as mass spectrometry do not require protein immobilization to identify protein partners and individual components of protein complexes. Immobilized proteins also can be used in protein pull-down assays to isolate protein binding partners in vivo (mammalian cells) or in vitro. Protein microarrays provide a method for high-throughput identification of protein:DNA interactions. Fluorescently labeled DNA is used to probe the array and identify proteins that bind to the specific probe. Functional protein microarrays normally contain full-length functional proteins or protein domains bound to a solid surface. Immobilization of proteins on chips is a popular approach to analyze protein:DNA and protein:protein interactions and identify components of protein complexes (Hall et al. This immobilization must not interfere with the binding capacity and can be achieved through the use of affinity tags.
1998).Īnalysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. This regulation is required for cell viability, differentiation and growth (Mankan et al. For example, transcription factors play an important role in regulating transcription by binding to specific recognition sites on the chromosome, often at a gene’s promoter, and interacting with other proteins in the nucleus. Analysis of protein:protein interactions can provide valuable insight into the cell signaling cascades involved in these processes, and analysis of protein:nucleic acid interactions often reveals important information about biological processes such as mRNA regulation, chromosomal remodeling and transcription. Isolation of Protein ComplexesĪ major objective in proteomics is the elucidation of protein function and organization of the complex networks that are responsible for key cellular processes. Using expression vectors that include a fusion tag facilitates recombinant protein purification. The biochemical features of different tags influence the stability, solubility and expression of proteins to which they are attached (Stevens et al. Common fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. To simplify purification, affinity purification tags can be fused to a recombinant protein of interest (Nilsson et al. As a result, attaining satisfactory yield and purity depends on highly selective and efficient capture of these proteins from the crude cell lysates. However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge for their purification. Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications (Geisse et al. coli can be purified in relatively high quantities, but these proteins, especially eukaryotic proteins, may not exhibit proper protein activity or folding.
Escherichia coli remains the first choice of many researchers for producing recombinant proteins due to ease of use, rapid cell growth and low cost of culturing. The best protein purification protocol depends not only on the protein being purified but also on many other factors such as the cell used to express the recombinant protein (e.g., prokaryotic versus eukaryotic cells). The optimal approach often must be determined empirically. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction.