Proteins are complicated molecules, well beyond conventional chemical synthesis processes. Biology, however, has been manufacturing proteins at scale for billions of years. Recombinant protein expression technology puts those biological tools in human hands and opens new fields of study, including gene regulation, biotherapeutic drug discovery, structural analysis, investigation of protein function in vivo, and more. Whether proteins are needed for research, medicinal, or industrial purposes, they can be expressed with biological systems crafted for that purpose, and there are many available. Here’s a rundown.

Protein Expression Host Systems

There are five primary categories of biological system for protein expression:

• Cell-free protein expression is the in vitro synthesis of protein using translation-competent extracts of whole HeLa, rabbit, or coli cells. In principle, whole-cell extracts contain all the macromolecular components needed for translation and posttranslational modification. When supplemented with RNA polymerase, cofactors, ribonucleotides, and the specific gene template, these extracts can synthesize proteins of interest in a few hours. They provide the simplicity of not needing to introduce anything to a host genome or maintain a cell culture, but without the complex post-translational machinery typically found in intact eukaryotic cells, may not be enough for especially complex proteins.
• Bacterial systems are chosen for generating recombinant proteins because of their ease of use, relatively high yields, and simple scalability. However, their differences from eukaryotic cells mean that they struggle to effect complex post-translational modifications such as glycosylation and may also be unable to engage in complex protein folding.
• Yeast systems have proven to be extremely useful for the expression and analysis of recombinant eukaryotic proteins and are ideally suited for large-scale production. These single-celled eukaryotic organisms are well understood genetically and are known to perform many posttranslational modifications. They grow quickly in defined medium, are easier and less expensive to work with than insect or mammalian cells, and are easily adapted to fermentation.
• Insect systems are, contrary to what the term might imply, not based on working with whole insects. Rather, cell cultures derived from insects serve as the protein-manufacturing machinery instead. These systems offer high levels of protein expression with posttranslational modification approaching that of mammalian cells, ease of scale-up, and simplified cell growth that can be readily adapted to high-density suspension culture for large-scale expression. Most of the posttranslational modification pathways present in mammalian systems also occur in insect cells, allowing the production of recombinant protein that is more antigenically, immunogenically, and functionally similar to the native mammalian protein than if expressed in yeast or other eukaryotes.
• Mammalian systems are the preferred expression platform for producing proteins that have the most native structure and activity. Mammalian expression is the system of choice for studying the function of a particular protein in the most physiologically relevant environment because it allows for the highest level of posttranslational processing and functional activity of the protein. These systems are commonly used to produce antibodies and therapeutic proteins, as well as for proteins that will be used for human use in functional cell-based assays.
• More rarely, transgenic plants or animals might be used as protein expression systems. These dramatically increase the challenge, time commitment, and government regulation involved in producing proteins and tend to be options of last resort.

Building the Protein Factory

The successful expression of a recombinant protein starts with making sure one’s host system, whatever it might be, can produce the desired protein. For nearly all host systems, that means the first step is generating an expression vector that contains the gene sequence of the desired protein. (As mentioned above, cell-free systems synthesize protein directly from provided nucleic acids.) Numerous tools are available for this purpose, including restriction enzyme and ligase systems (REaL cloning), ligation-independent cloning (LIC), recombinatorial cloning, and outsourcing the matter entirely to Invitrogen^TM GeneArt^TM Gene Synthesis and Plasmid Services. Which method makes the most sense depends on the gene sequence, the host system, and other factors.

Expression experiments are performed either transiently or stably with cell lines that express the protein of interest.  Both methods involve the artificial delivery of nucleic acids (the expression vector) into host cells. Transient expression, which typically results in high levels of expression for a few days, is exceptional for rapid protein production and quick data generation. Stable expression requires the generation of cell lines in which the expression construct is integrated into the host genome. These stable cell lines can be used over a long experimental time course or used over many experiments. Because the expression vector has a selection marker (such as an antibiotic resistance gene), cells that have integrated the construct can be selected by the addition of a selection agent (such as the antibiotic) to the medium.

Gibco offers numerous tools for culturing recombinant cells for protein expression, tailored to all available host systems.

Harvesting the Protein

Once the host system is churning out the protein of interest, the next step is collecting the protein. Harvesting and protein purification is the penultimate step in the expression process and a purification method is dictated by the features of the protein itself and the intended downstream applications. Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture.  The most common purification strategies include affinity chromatography, ion-exchange chromatography, and size exclusion chromatography (SEC). Which technologies are best and which additional purification steps may or may not be necessary depends on what else is in the culture media, whether the expressed protein is intracellular, membrane, or secreted, and the chemical properties of the protein itself.

Assessing the quantity, quality, and activity of the protein once it is isolated is a final critical part of the process. Analytical techniques such as SDS-PAGE, Western Blotting, Analytical size exclusion chromatography (AnSEC), and mass spectrometry (ESI-MS) can provide a clear snapshot of the size, shape, purity, aggregation state, and behavior of any purified protein.

Gibco Has You Covered

Gibco and the rest of Thermo Fisher Scientific specialize in high-end equipment, reagents, and consumables for all your protein expression needs. We have the products and technical support to help you implement and maintain an optimized protein expression workflow. Have a look at our catalogues of products, online courses, web resources, and web trools to learn more.

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