StudiesProtein Purification Protein purification is at the core of many of the technologies that are transforming the way we learn about cellular processes and the mechanisms of disease. However, as knowledge of protein functionality continues to increase, significant improvements to the field of protein purification appear to have slowed down in recent years. The Science Advisory Board therefore decided to assess the tools and techniques involved in protein purification. Members were invited to comment on the protein expression systems that they prefer to use and to describe the applications for which they use the proteins obtained from these systems. Almost 600 scientists participated in this study. These individuals also provided their views on the factors they felt were important for establishing quality in purification reagents and techniques. Purified proteins are a vital feature in many research applications, however study respondents primarily use them for in vitro activity assays. Other popular applications were antibody development and production and cell-based activity assays. Despite the importance of having access to purified proteins for conducting their experiments, 84% of researchers typically perform less than fifteen purification preps per month, regardless of the required final protein yield. Although this is a relatively low number, these preps do consume valuable time and resources from the lab, “I believe we would prefer to use a core facility and have the proteins of interest prepared for us by them. Alternatively, we could establish an in-house core facility, which would be very well equipped with automated systems for protein purification.” -Post Doctoral Fellow, Europe By far, tagging proteins is the most common method for purifying proteins. The majority of participants in the study indicate that they use tagged proteins primarily because of the ease by which the purification procedure can be accomplished. The polyhistidine/metal affinity system (i.e., His-tag) is, by far, the most popular tag. About one-half of scientists surveyed use it as their primary system to purify tagged proteins. Tagged proteins are the top choice of protein type for applications such as antibody development, assessing protein:protein interactions and in vitro and cell-based activity assays. Of the purification methods available for tagged proteins, scientists most often employ gravity flow systems followed by HPLC/FPLC and spin columns. The scientists who participated in the study are generally satisfied with the performance of commercial purification columns and resins that they use for purifying their tagged proteins. They report being most satisfied with features such as the ease in which the purification products can be used, binding capacity and flow rate. However, these scientists are disappointed in both the protein yield and purity of the protein obtained. To overcome some of these difficulties, some researchers are turning towards automation for help. Automated, high-throughput protein purification systems have been developed to decrease hands-on time and eliminate tedious steps such as manual cell lysis and multiple centrifugations. “While automation increases the speed by which protein samples can be isolated and analyzed, I know researchers continue to struggle with basic issues such as sample purity, sample degradation and low yields,” claims Dr. Tamara Zemlo, Director of Scientific & Medical Communications for The Science Advisory Board. However, automation is not for everybody. Some people would prefer to invest in expensive, high quality reagents instead. “I would probably not invest in a high-throughput system since we are studying a small number of proteins (receptors). If money were no object I would probably use expression in a mammalian cell (native source of protein, for proper folding, etc.) using a highly controllable promoter system with no tag. I would then purify the protein of interest using custom polyclonal antibodies (which I produce myself) to various domains. I could then verify that I have the functional receptor protein of interest by performing ligand blotting assays with native ligands.”-Principal Investigator, North America Others believe it would be better to ensure their lab’s long-term adaptability by employing a qualified technician to perform protein purification preps instead, “I wouldn't invest in a high-throughput platform because our needs for purified proteins have varied quite widely over the years. Sometimes the whole lab will be purifying proteins every day. A year later, the projects have moved on and nobody cracks bugs and runs a column for several months. The large, expensive machine would end up gathering dust and occupying a bench that could be otherwise occupied. If money were no object, my ideal solution would be to have a dedicated technician to produce the proteins. A decent tech will always have the flexibility to contribute to the lab even if nobody wants protein expression for a while.” -Principal Investigator, Europe ### Would you like to find out how your colleagues would purify their proteins if money were NO object? Please click here to check-out their wish lists. Please click here to read additional insights into protein purification by members of The Science Advisory Board. [ View Current & Future Studies ] [ View Past Studies ] |
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