b'Introduction competent cells (e.g., chemically competent or electrocompetent To investigate how particular proteins regulate biology,E. coli) for propagation and storage, by a process called researchers usually require a means of producing (manufacturing)transformation. Epitope tags can be used to allow for easy functional proteins of interest. Given the size and complexitydetection or rapid purification of your protein of interest by fusing of proteins, de novo synthesis is not a viable option for thisa sequence coding for the tag to your gene (Table 1).endeavor. Instead, living cells or their cellular machinery can beTable 1. Overview of epitope tags.harnessed as factories to build and construct proteins based on supplied genetic templates (Figure 1). Purpose Description Examples of tagsDetect Well-characterized antibodyV5, Xpress, Myc, 6xHis, available against the tag; easilyGST, BioEase, capTEV, Transcription Translation visualized GFP, Lumio, HA, FLAG tagPurify Resins available to facilitate6xHis, GST, BioEase, purification capTEV tagCleave Protease recognition site (TEV,Any tag with a protease AAAAA EK, HRV 3C, factor Xa) torecognition site following DNA mRNA Protein remove tag after expression tothe tag (only on N obtain native proteinterminus)Figure 1. Transcription and translation. Information is flowed from DNA base-pair sequence (gene) to amino acid polypeptide sequence (protein). Using the right expression system for your specific application In prokaryotes, the processes of transcription and translationis the key to success. Protein solubility, functionality, purification occur simultaneously. In eukaryotes, the processes are spatiallyspeed, and yield are often crucial factors to consider when separated and occur sequentiallywith transcription happeningchoosing an expression system. Additionally, each system has in the nucleus and translation occurring in the cytoplasm.its own strengths and challenges, which are important when After translation, polypeptides are modified in various ways tochoosing an expression system. For example, bacterial host complete their structure, designate their location, or regulate theircells are low-cost, easy to culture, and easily scalable but are activity within the cell. Posttranslational modifications (PTMs) arelimited to the expression of bacterial proteins or simple eukaryotic various additions or alterations to the chemical structure of theproteins with limited posttranslational modifications. Since most newly synthesized protein and are critical features of overall cellproteins undergo some degree of posttranslational modification, biology. bacterial host cells limit the range of the proteins expressed.Cloning refers to the propagation of DNA of interest from anInsect and mammalian host systems support the most existing organism. This can be accomplished by cloning a genecomplex proteins and maximum protein quality. Insect host of interest into an expression vector (Figure 2). systems support multi-protein complexes with posttranslational modifications similar to mammalian cells and are a good option Choice of cloning method:Restriction cloning for proteins that are toxic to mammalian cells. If the proteinGibson Assembly cloning Gateway technology studied must be identical to its in vivo mammalian counterpart,TOPO cloningthen a mammalian host system is ideal. Mammalian host systems Choice of promoter:Optional epitope tags:CMVV5 EF-1Myc retain the most posttranslational modifications and most closely GOI UbC romoterHis T7 or SP6 PXpress resemble functional human proteins. However, these systemsOther Otherare also the most expensive. Both insect and mammalian cell lines have more demanding culture conditions, and therefore are Expression not suited to every protein expression case. The most common Additional antibiotic vector map r selection marker: ke mammalian cell lines are human embryonic kidney (HEK293) cells rAammMarker forpNeomycin noipropagationc(Geneticin antibiotic) and Chinese hamster ovary (CHO) cells.itilli ecin E. coli n SelBlasticidin S Hygromycin B Zeocin antibiotic OtherChoice of cell line may also depend on whether the protein of interest expresses better in one cell line or the other, or whether OriOrigin of replication examples: the researcher wants to take a transient or stable expressionpUC ori pBR322 ori strategy. Transient expression usually implies a short-term, Figure 2. Example of an expression vector. small-scale production, while stable expression involves the Most vectors contain a promoter for expression by a specificlong-term integration of genes into the genome for large-scale host system; however, some offer the option to add your ownproduction. However, new technologies have transformed promoter. Once cloning is completed, plasmids are taken up intotransient expression to have robust yields.6 Cloning and protein expression thermofisher.com/thermofisher.com/proteinbiology'