Overview of Protein Expression Systems

After translation, polypeptides are modified in various ways to complete their structure, designate their location or regulate their activity within the cell. Post-translational modifications (PTMs) are various additions or alterations to the chemical structure and are critical features of the overall cell biology.

Types of post-translational modifications include:

• Polypeptide folding into a globular protein with the help of chaperone proteins to arrive at the lowest energy state
• Modifications of the amino acids present, such as removal of the first methionine residue
• Disulfide bridge formation or reduction
• Protein modifications that facilitate binding functions:
  • Glycosylation
  • Prenylation of proteins for membrane localization
  • Acetylation of histones to modify DNA–histone interactions
• Addition of functional groups that regulate protein activity:
  • Phosphorylation
  • Nitrosylation
  • GTP binding

In general, proteomics research involves investigating any aspect of a protein such as structure, function, modifications, localization or protein interactions. 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, chemical synthesis is not a viable option for this endeavor. Instead, living cells and their cellular machinery are usually harnessed as factories to build and construct proteins based on supplied genetic templates.

Unlike proteins, DNA is simple to construct synthetically or in vitro using well established recombinant DNA techniques. Therefore, DNA templates of specific genes, with or without add-on reporter or affinity tag sequences, can be constructed as templates for protein expression. Proteins produced from such DNA templates are called recombinant proteins.

Traditional strategies for recombinant protein expression involve transfecting cells with a DNA vector that contains the template and then culturing the cells so that they transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic in vivo protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems are summarized in the table below and include mammalian, insect, yeast, bacterial, algal and cell-free. Each system has advantages and challenges, and choosing the right system for the specific application is important for successful recombinant protein expression. The following table provides an overview of recombinant protein expression systems.

A number of recombinant protein expression host options exist for prokaryotic and eukaryotic proteins, including mammalian, insect, yeast, bacterial, algal, and cell-free systems.

The choice of an optimal expression system for a specific protein primarily depends on:

The origin of the target protein—Bacterial proteins are better expressed in bacterial systems, while mammalian proteins are better expressed in mammalian systems. For example, mammalian cell lines such as Chinese Hamster Ovary (CHO) and Human Embryonic Kidney (HEK) are the systems of choice for human protein production.

Required post-translational modifications—Proteins devoid of complex modifications (e.g., glycosylation, alkylation, phosphorylation, or specific proteolytic processing) are more easily produced in simpler systems with short turnaround times such as bacteria and yeast cells. In contrast, complex proteins with significant modifications should be produced in either insect or mammalian cells.

Solubility of the recombinant protein—Some proteins are not properly folded in bacterial systems, as a result, they tend to form insoluble aggregates (or inclusion bodies) that are difficult to extract. In these circumstances, higher eukaryotic systems should be preferred for the recombinant production of the target protein.

The intended application of the recombinant protein—Some downstream applications require large amounts of target protein. In these instances, simpler systems with short growth cycles (e.g., bacterial or yeast) might be the better solution. However, if the downstream application requires a functional protein that is reliant on the final protein conformation, then the choice might be insect or mammalian hosts.

Mammalian expression systems can be used to produce mammalian proteins that have the most native structure and activity due to its physiologically relevant environment. This results in high levels of post-translational processing and functional activity. Mammalian expression systems are the preferred system for the expression of mammalian proteins and can be used for the production of antibodies, complex proteins and proteins for use in functional cell-based assays. However, these benefits are coupled with more demanding culture conditions.

Insect cells can be used for high level protein expression with modifications similar to mammalian systems. There are several systems that can be used to produce recombinant baculovirus, which can then be utilized to express the protein of interest in insect cells. These systems can be easily scaled up and adapted to high-density suspension culture for large-scale expression of protein that is more functionally similar to native mammalian protein. Though yields can be up to 500 mg/L, recombinant baculovirus production can be time consuming and culture conditions more challenging than prokaryotic systems.

Bacterial protein expression systems are popular because bacteria are easy to culture, grow fast and produce high yields of recombinant protein. However, multi-domain eukaryotic proteins expressed in bacteria often are non-functional because the cells are not equipped to accomplish the required post-translational modifications or molecular folding. Also, many proteins become insoluble as inclusion bodies that are very difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding procedures. In the example that follows, a bacterial cell-based system was used to express 8 different recombinant proteins. 

Cell-free protein expression is the in vitro synthesis of a protein using translation-compatible extracts of whole cells. In principle, whole cell extracts contain all the macromolecules and components needed for transcription, translation and even post-translational modification. These components include RNA polymerase, regulatory protein factors, transcription factors, ribosomes and tRNA. When supplemented with cofactors, nucleotides and the specific gene template, these extracts can synthesize proteins of interest in a few hours.

Although not sustainable for large scale production, cell-free, or in vitro translation (IVT) protein expression systems, have several advantages over traditional in vivo systems. Cell-free expression allows for fast synthesis of recombinant proteins without the hassle of cell culture. Cell-free systems enable protein labeling with modified amino acids, as well as expression of proteins that undergo rapid proteolytic degradation by intracellular proteases. Also, with the cell-free method, it is simpler to express many different proteins simultaneously (e.g., testing protein mutations by expression on a small scale from many different recombinant DNA templates). In this representative experiment, an IVT system was used to express human caspase 3 protein.