Protein Complexes and Interactions
Many researchers are interested in analyzing protein complexes and identifying the interaction between proteins, says Charles Piazza, vp, protein analysis, at Invitrogen (www.invitrogen.com).
“People have been interested in looking at complexes because they represent some of the more interesting phenomenon within cells, either cell receptors or ion channels. Cell signaling is usually mediated by protein complexes on the surface of cells and communicated within the cell also by these complexes,“ according to Piazza.
Invitrogen offers the NativePAGE Novex gels for high-resolution analysis of membrane protein complexes and large proteins that can’t be easily analyzed using other methods, he explains. The newest gel of this line, the NativePAGE Novex Bis-Tris Gel system, enables analysis of membrane-protein complexes in their native conformations and provides better resolution than with traditional Tris-glycine native electrophoresis, he claims, adding that its neutral pH provides maximum stability of the proteins and the gel matrix.
There are two additional products the company recently released for detecting protein-protein interactions. These are the ProQuest Two-Hybrid System with Gateway Technology and the Pro-Quest Reverse Two-Hybrid System. The first is a yeast-based system for detecting protein-protein interactions by using three reporter genes and low-copy number of vectors. This helps to minimize false positives.
The second is designed to eliminate background noise by using the company’s SureFrameAllele Library Construction Kit to facilitate in vitro allele library production and select for full-length proteins in E.coli prior to analysis in yeast.
A new approach to analyze protein-protein interactions is being addressed by Stratagene (www.strategene.com). Although traditionally analyzed by genetic methods, such as two-hybrid screening, this approach is limited to two individual interacting proteins and cannot analyze protein complexes. There are two kits available: the Mammalian InterPlay TAP System and the newer InterPlay Adenoviral TAP System.
The first kit reportedly can be used in any cell line difficult to transfect with traditional methods. There are two purification steps, and each uses a different affinity purification tag fused to at least one known component of a protein complex combined with two purification resins specific for each tag. The initial tag is streptavidin-binding peptide and the second is calmodulin-binding peptide.
“By doing two rounds, you get very pure protein at the end. That’s why the kit is called Tandem Affinity Purification or TAP,“ says Melissa Stolow, Ph.D., director of product marketing, functional biology.
The second part of the process involves taking the protein and performing mass spectroscopy or Western blot or 2-D gels to identify the proteins interacting with the protein of interest.
The Adenoviral TAP System contains the two purification tags, but also includes the AdEasy XL Adenoviral Vector System, a viral-based gene delivery system. This has the ability to transfect additional cell types, such as primary cells, which are known to be difficult to transfect.
“Now customers can get the DNA into cells at a high efficiency rate and into more cell types. It provides more options in terms of looking for interacting proteins in different cell types,“ explains Dr. Stolow. In addition, recombinant adenoviruses can infect both dividing and nondividing cells and can insert up to 6.6 kb of foreign DNA.