What is it about?
Tag-assisted protein purification is a method of choice for both academic researches and large-scale industrial demands. Application of the purification tags in the protein production process can help to save time and cost, but the design and application of tagged fusion proteins are challenging. An appropriate tagging strategy must provide sufficient expression yield and high purity for the final protein products while preserving their native structure and function. Thanks to the recent advances in the bioinformatics and emergence of high-throughput techniques (e.g. SEREX), many new tags are introduced to the market. A variety of interfering and non-interfering tags have currently broadened their application scope beyond the traditional use as a simple purification tool. They can take part in many biochemical and analytical features and act as solubility and protein expression enhancers, probe tracker for online visualization, detectors of post-translational modifications, and carrier-driven tags. Given the variability and growing number of the purification tags, here we reviewed the protein- and peptide-structured purification tags used in the affinity, ion-exchange, reverse phase, and immobilized metal ion affinity chromatographies. We highlighted the demand for purification tags in the pharmaceutical industry and discussed the impact of self-cleavable tags, aggregating tags, and nanotechnology on both the column-based and column-free purification techniques.
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Why is it important?
• The application of tags for protein purification will reduce the cost of preparing the chromatographic columns. • A suitable tagging system may provide a sufficient expression yield and high purity of the protein product. • The introduction of the self-cleavable tags and aggregating tags have been influential in the protein purification industry. • The application of the aggregating tags in a column-free manner can facilitate higher protein expression yield. • Emerging technologies, including bioinformatics, SELEX, and nanotechnology, have helped to improve technical limitations.
Perspectives
Even with many advances that have been made to simplify the protein production process, there are still many considerations which must be taken into account to design and/or choose an appropriate tag for a particular application and much progress has yet to be made toward the maturity of the tagging technology. Despite a large number of tags introduced for laboratory scale applications, it seems that their use in pharmaceutical production is still not well-established and straightforward. Hence, we hope that we could help scientists to deal with the existing challenges by providing an integrated view of the production and purification processes that include tag designing approaches, choosing a suitable tag for a particular purpose, and also using a proper tag removal strategy. The information presented in this article finely describes that apart from the tagging systems, the quality of the expression hosts and the features of the columns and chromatographic resins are essential for a successful and affordable manufacturing process. We also showed that many other technologies, including bioinformatics, SELEX, and nanotechnology, can help to improve many technical limitations that are associated with large-scale protein production. It seems that the protein production techniques are to continue their way to become feasible and more affordable. Although chromatographic systems are now the method of choice for protein purification, no one knows whether the new techniques such as column-free purification can continue their dominance in academic research and industry or not.
Dr. Neda Saraygord-Afshari
Iran University of Medical Sciences
Read the Original
This page is a summary of: Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry, Biotechnology Advances, December 2020, Elsevier,
DOI: 10.1016/j.biotechadv.2020.107653.
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Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry
Today, commercial applications for the large-scale production of recombinant and native proteins have gained widespread attention. Meanwhile, the production of a variety of hormones (Rezaei and Zarkesh-Esfahani, 2012), growth factors (Devesa et al., 2016), and antibodies (Castelli et al., 2019) for theranostic applications using the recombinant DNA technology is also of great historical value (Khan et al., 2016). It is deemed that the turnover of the market for recombinant proteins will reach over $460 billion by 2022 (Van den Hauwe et al., 2018; Yang et al., 2018). Hence, it is not surprising that many researchers from different fields of science are looking for the most efficient and productive approaches to facilitate the production and purification of the engineered proteins (Mishra et al., 2017). However, although various techniques have been developed to enrich the proteins of interest from crude biological extracts, the most effective method is still affinity purification, whereby the proteins are enriched due to their specific binding properties to an immobilized ligand (Guan and Chen, 2014a). An efficient and demanding strategy to enhance the efficacy of a protein purification process is to take control of the interactions between proteins and immobilized ligands using specific fusion peptide or protein sequences known as tags (Yang et al., 2018). Tags may include a whole enzyme, a protein domain, or a small polypeptide chain. They can conjoin a range of substrates such as carbohydrates, small biomolecules, metal chelators, and high-specificity antibodies and help to enhance the recovery rate and yield of their associated counterparts (Beloborodov et al., 2018). Initially, tag-based systems were used only for purification purposes (Sambrook et al., 1989; Zhao et al., 2013), but now they are used in various techniques and considered an essential tool in western blotting (Horinouchi et al., 2016), flow cytometry (Horinouchi et al., 2016), microarray (Damin et al., 2018), mass spectrometry (Zhang and Elias, 2017), lab-on-a-chip (Wu et al., 2018), and protein localization studies (Huang et al., 2019). Together with the purification t
https://www.sciencedirect.com/science/article/abs/pii/S0734975020301555
An efficient and demanding strategy to enhance the efficacy of a protein purification process is to take control of the interactions between proteins and immobilized ligands using specific fusion peptide or protein sequences known as tags (Yang et al., 2018). Tags may include a whole enzyme, a protein domain, or a small polypeptide chain. They can conjoin a range of substrates such as carbohydrates, small biomolecules, metal chelators, and high-specificity antibodies and help to enhance the recovery rate and yield of their associated counterparts (Beloborodov et al., 2018). Initially, tag-based systems were used only for purification purposes (Sambrook et al., 1989; Zhao et al., 2013), but now they are used in various techniques and considered an essential tool in western blotting (Horinouchi et al., 2016), flow cytometry (Horinouchi et al., 2016), microarray (Damin et al., 2018), mass spectrometry (Zhang and Elias, 2017), lab-on-a-chip (Wu et al., 2018), and protein localization studies (Huang et al., 2019).
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