Your Protein Research Handbook

Cloning and protein expression Recombinant protein expression technology enables analysis of gene regulation and protein structure and function. Utilization of recombinant protein expression varies widely—from investigation of function in vivo to large-scale production for structural studies and biotherapeutic drug discovery. Protein sample preparation Protein purification Protein biology workflow Crude protein Immunoassays Immunoassays Protein purification Protein biology workflow Cloning and protein expression Cloning and protein expression Protein gel electrophoresis Protein gel electrophoresis Crude protein Mass spectrometry Mass spectrometry Protein bioconjugation, crosslinking, and modification Protein bioconjugation, crosslinking, and modification Cryo-EM sample preparation Cryo-EM sample preparation Protein sample preparation Western blotting Western blotting Protein sample preparation Protein purification Protein biology workflow Crude protein Protein purification Protein biology workflow Cloning and protein expression Cloning and protein expression Crude protein Protein sample preparation Tools to optimize your cloning step 7 Recombinant protein expression systems 8 Ordering information 11 Cloning and protein expression thermofisher.com/proteinbiology 5Introduction To investigate how particular proteins regulate biology, researchers usually require a means of producing (manufacturing) functional proteins of interest. Given the size and complexity of proteins, de novo synthesis is not a viable option for this endeavor. Instead, living cells or their cellular machinery can be harnessed as factories to build and construct proteins based on supplied genetic templates (Figure 1). Most vectors contain a promoter for expression by a specific host system; however, some offer the option to add your own promoter. Once cloning is completed, plasmids are taken up into Using the right expression system for your specific application is the key to success. Protein solubility, functionality, purification speed, and yield are often crucial factors to consider when choosing an expression system. Additionally, each system has its own strengths and challenges, which are important when choosing an expression system. For example, bacterial host cells are low-cost, easy to culture, and easily scalable but are limited to the expression of bacterial proteins or simple eukaryotic proteins with limited posttranslational modifications. Since most proteins undergo some degree of posttranslational modification, bacterial host cells limit the range of the proteins expressed. Insect and mammalian host systems support the most complex proteins and maximum protein quality. Insect host systems support multi-protein complexes with posttranslational modifications similar to mammalian cells and are a good option for proteins that are toxic to mammalian cells. If the protein studied must be identical to its in vivo mammalian counterpart, then a mammalian host system is ideal. Mammalian host systems retain the most posttranslational modifications and most closely resemble functional human proteins. However, these systems are also the most expensive. Both insect and mammalian cell lines have more demanding culture conditions, and therefore are not suited to every protein expression case. The most common mammalian cell lines are human embryonic kidney (HEK293) cells and Chinese hamster ovary (CHO) cells. Choice of cell line may also depend on whether the protein of interest expresses better in one cell line or the other, or whether the researcher wants to take a transient or stable expression strategy. Transient expression usually implies a short-term, small-scale production, while stable expression involves the long-term integration of genes into the genome for large-scale production. However, new technologies have transformed transient expression to have robust yields. In prokaryotes, the processes of transcription and translation occur simultaneously. In eukaryotes, the processes are spatially separated and occur sequentially—with transcription happening in the nucleus and translation occurring in the cytoplasm. After translation, polypeptides are modified in various ways to complete their structure, designate their location, or regulate their activity within the cell. Posttranslational modifications (PTMs) are various additions or alterations to the chemical structure of the newly synthesized protein and are critical features of overall cell biology. Cloning refers to the propagation of DNA of interest from an existing organism. This can be accomplished by cloning a gene of interest into an expression vector (Figure 2). DNA AAAAA mRNA Protein Transcription Translation Figure 1. Transcription and translation. Information is flowed from DNA base-pair sequence (gene) to amino acid polypeptide sequence (protein). Figure 2. Example of an expression vector. Choice of promoter: • CMV • EF-1α • UbC • T7 or SP6 • Other Marker for propagation in E. coli Optional epitope tags: • V5 • Myc • His • Xpress • Other Choice of cloning method: • Restriction cloning • Gibson Assembly cloning Origin of replication examples: • pUC ori • pBR322 ori • Gateway technology • TOPO cloning Additional antibiotic selection marker: • Neomycin (Geneticin antibiotic) • Blasticidin S • Hygromycin B • Zeocin antibiotic • Other Expression vector map GOI Ampicillin ri Promoter Selection marker O Table 1. Overview of epitope tags. Purpose Description Examples of tags Detect Well-characterized antibody available against the tag; easily visualized V5, Xpress, Myc, 6xHis, GST, BioEase, capTEV, GFP, Lumio, HA, FLAG tag Purify Resins available to facilitate purification 6xHis, GST, BioEase, capTEV tag Cleave Protease recognition site (TEV, EK, HRV 3C, factor Xa) to remove tag after expression to obtain native protein Any tag with a protease recognition site following the tag (only on N terminus) competent cells (e.g., chemically competent or electrocompetent E. coli) for propagation and storage, by a process called transformation. Epitope tags can be used to allow for easy detection or rapid purification of your protein of interest by fusing a sequence coding for the tag to your gene (Table 1). 6 Cloning and protein expression thermofisher.com/thermofisher.com/proteinbiologyTools to optimize your cloning step GeneArt Gene Synthesis and GeneOptimizer software The power of custom gene synthesis is the ability to design your DNA without the constraints of traditional cloning. Equally important to most researchers, however, is obtaining high yields of mRNA and, ultimately, protein from synthetic genes. We developed Invitrogen™ GeneArt™ GeneOptimizer™ software to maximize the expression of synthetic genes in all commonly used expression systems. Tuning the expression level by choosing the optimal promoter and terminator combination can be an essential part of your expression project. The origin of replication also has a significant influence on the expression level of the foreign protein. We offer a broad range of commercially available, predesigned vectors optimized for various expression systems. Learn more at thermofisher.com/genesynthesis Gibson Assembly kits and GeneArt Strings DNA Fragments Invitrogen™ GeneArt™ Gibson Assembly® cloning kits provide highly efficient, seamless cloning, enabling the assembly of multiple DNA fragments of varying length into any vector. When combined with Invitrogen™ GeneArt™ Strings DNA Fragments or Invitrogen™ GeneArt™ Gene Synthesis, these cloning kits can be used to build simple constructs as well as large and demanding constructs from multiple fragments. Learn more about GeneArt Gibson Assembly cloning kits for protein expression at thermofisher.com/gibsonassembly Gateway cloning Invitrogen™ Gateway™ cloning technology is rapid, robust, and suited for high-throughput clone generation for protein production. It allows shuffling between different expression systems in just a few simple steps. Choose from a diverse selection of host systems, including E. coli, yeasts, and insect or mammalian cells, each of which utilizes unique destination vectors for all your expression applications. Additionally, the Invitrogen™ Gateway™ Vector Conversion System can convert specialized or customized vectors into an Invitrogen™ Gateway™ destination vector to suit your expression workflow. Select the right Gateway vectors for your workflow and review our cloning protocols at thermofisher.com/gateway One Shot chemically competent E. coli E. coli cells are widely used for production of recombinant proteins quickly, economically, and on a large scale. The most popular strain for recombinant protein expression is BL21 and its derivatives. Invitrogen™ One Shot™ chemically competent cells, such as BL21 Star™ (DE3) and BL21-AI™ cells, are optimized for high-level protein or toxic protein expression from T7 promoters. All are induced with IPTG or L-arabinose and come in the convenient Invitrogen™ One Shot™ format, allowing for transformation and recovery in a single tube. Choose chemically competent cells optimized for protein expression at thermofisher.com/compcells Cloning and protein expression thermofisher.com/proteinbiology 7Recombinant protein expression systems Achieving optimal and reliable amounts of recombinant protein is easier because of our wide selection of trusted mammalian, insect, yeast, bacterial, and cell-free protein expression systems (Table 2). Backed by a team of experienced professionals to help you quickly optimize your protein expression experiments, we can accelerate your research and development by offering: • Higher protein yields (3–20x higher than other systems) • Faster protein production (days vs. weeks or months) • High cell density • Lower cost per mg of protein • Complete, optimized systems of cells, media, transfection reagents, enhancers, feeds, and vectors Table 2. Expression system selection guide. Expression system Human (HEK293) Hamster (CHO) Featured application Membrane proteins (GPCRs, ion channels, other membrane proteins) Ig-related proteins Other applications • Difficult-to-express proteins • Secreted proteins • Membrane proteins • Secreted proteins Posttranslational modifications Full Nearly full Protein yield Up to 1 g/L Up to 3 g/L Recommended host system kit Expi293 expression system kits ExpiCHO Expression System Kit Recommended cells Expi293F Cells Expi293F GnTI– Cells Expi293F Inducible Cells Expi293F Inducible GnTI– Cells ExpiCHO-S Cells (cGMP Banked) ExpiCHO-S Cells Recommended media Expi293 Expression Medium ExpiCHO Expression Medium Recommended transfection and delivery ExpiFectamine 293 Transfection Kit ExpiFectamine CHO Transfection Kit Recommended expression vectors pcDNA3.4 TOPO vector pcDNA3.4 TOPO vector Recommended cultureware Nalgene shake flasks Nunc bioreactor tubes Nunc plates Nalgene shake flasks Nunc bioreactor tubes Nunc plates Recommended extraction reagent kit M-PER Mammalian Protein Extraction Reagent M-PER Mammalian Protein Extraction Reagent Recommended protein labeling Expi293 Met (–) Protein Labeling Kit L-Methionine (Methyl-13C) L-Selenomethionine Scale-up Expi293 Expression Medium Single-use bioreactor vessels ExpiCHO Stable Production Medium EfficientFeed C+ 2X Supplement Efficient-Pro Feed 2 Single-use bioreactor vessels Services option Gene-to-protein services Gene-to-protein services The Gibco™ Expi™ platforms are examples of expression systems that are fast and cost-effective with high yield. These systems combine the advantages of both transient and stable strategies for high expression of diverse proteins (Figure 3). For example, the Gibco™ Expi293™ Expression System uses HEK293 cells and can yield up to 1 g/L of protein in just 5–7 days. Figure 3. External collaborator results. In collaboration with 22 labs, expression levels of 98 different proteins were tested using the Expi293 Expression System. The following results were obtained: • 87% of proteins demonstrated increased expression in the Expi293 system compared with the user’s current system • 4.6x average increase for all proteins (n = 98) • 4.0x average increase for mAbs (n = 54); highest level was 826 mg/L • 5.3x average increase for non-mAbs (n = 44); highest level was 790 mg/L 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Fold yield increase 8 Cloning and protein expression thermofisher.com/thermofisher.com/proteinbiologyInsect (Sf9, Sf21) Yeast B