In the post-genomic era, the continuing growth in the need of recombinant proteins leads to the development of many strategies and techniques to improve recombinant protein production yield, purity, and quality. For example, with advanced molecular biology and chemical synthesis of DNA, it is now possible to synthesize almost any gene of interest for the expression of a recombinant protein. Moreover, in order to improve the yield, it is possible to optimize gene codons for the selected expression system and host species. However, the optimal expression conditions differ from one protein to another due to the intrinsic variability in quantity, solubility, stability and functionality. Thus it is crucial for any laboratory of recombinant protein production to use techniques for testing many different conditions of expression as possible in a timely manner. 

The bacterial system is commonly used for recombinant protein expression. In cases where post-translational protein modifications are required for proper activity or folding, researchers prefer to use an eukaryotic protein production system such as mammalian cells. The choice of the system depends ultimately on the nature of the protein to be produced and the application in which the recombinant product will be used. Mammalian cell expression systems provide the most comprehensive post-translational modifications, which are critical to the protein function. For example, therapeutic proteins including antibodies (composed of 82-96% protein and 4-18% carbohydrate) often require glycosylation that can only be achieved in a mammalian expression system.

Protein Fusions and Epitope Tags

When expressing and purifying large quantities of soluble proteins, the major obstacles often include poor yield and solubility due to the formation of aggregates. There are a number of advances in recombinant protein expression, including the optimization of expression vector and host system, improving transcriptional level and stability, increasing translation with host-specific codon optimization, maximizing the use of secretory pathways, co-expression with chaperones, and decreasing proteolytic degradation. In many cases, protein fusions or peptide (epitope) tags can simplify the detection and purification, improve the solubility, and/or promote the proper folding of the protein of interest. 

Protein fusions refer to those with more than a dozen of amino acids, such as GFP, while epitope tags refer to the short peptides, such as HA, Myc, and poly-histidine (His-tag). Among them many are used as affinity tags for protein purification due to their ability of binding to a specific chemical ligand or antibody. Nowadays almost all recombinant proteins are expressed through fusions or tags. The localization and expression of protein of interest, even without a suitable detecting antibody, can be monitored through the tag or fusion. For example, tags or fusions can be used for protein detection by Western blot, ELISA, IP, flow cytometry, immunohistochemistry, and fluorescence microscopy, while some of them can be further utilized for protein purification (see a list of commonly used protein fusions and tags below). 

Proper design and judicious use of the right fusion or tag can enhance the stability and solubility of the protein of interest. There are a few basic considerations during the design of the proper tag or fusion. For example, the sequence of the tag/fusion must be in-frame with that of the protein of interest. Codon usage should be considered when different host species is used. The tag or fusion can be placed on the either end of target protein. Some fusions or tags can be used for easy in situ detection, such as HRP fusion (a mammalian expression optimized version of the enzyme is offered by G&P Biosciences for both intracellular and extracellular expression). Some can be used in tandem to increase desired features or applications (e.g., GFP & His-tag together for detection & purification). Some can be explored to solve significant protein expression problems, e.g., extending half-lives (e.g., Fc fusion) and increasing the protein solubility and folding (e.g., SUMO, MBP, thioredoxin, or GST fusion).

Cleavage Proteases

It is often not necessary to remove the tag/fusion, especially for the epitope tags due to their small size. In addition many Fc-fusion proteins are successfully used in clinic as therapeutic agents. However, a linker can be easily added between the tag/fusion and protein of interest to enable, e.g., easy cleavage of the tag/fusion, without interfering with the structure and activity of target protein. The key criterion for selecting a proper cleavage site and protease is that the protease must be very specific to a tag or a linker, and also the target protein does not have the recognition site.

Thrombin:

Due to its high specificity, Thrombin is one of most commonly used tag/fusion cleavage proteases. It cleaves after the arginine residue in the consensus cleavage site, Leu-Val-Pro-Arg|Gly-Ser, (L-V-P-R|G-S),which is often included in the linker region, and upon cleavage, there are residual amino acids in the fusion partner protein.

Factor Xa:

Factor Xa is also a commonly used protease in removing the tag/fusion. It cleaves after the arginine residue in its preferred cleavage site Ile-(Glu/Asp)-Gly-Arg|, (I-(E/D)-G-R|), and thus can completely remove the tag from the N-terminus of the target protein. However it sometimes cleave at other basic residues, depending on the conformation of the target protein. It does not cleave a site followed by proline or arginine.

TEV:

Tobacco etch virus (TEV) protease has a specific recognition site, and cleaves at very high precision. It cleaves between Gln and (Gly/Ser) in the consensus site, Glu-Asn-Leu-Tyr-Phe-Gln|(Gly/Ser), (E-N-L-Y-F-Q|(G/S)). Its activity is not inhibited by low concentration of urea, which can prevent protein aggregation, and increase protein solubility.

Enterokinase:

Enterokinase recognizes the D-D-D-D-K| and cleaves at the carboxyl site of lysine. FLAG-tag DYKDDDDK contains such a cleavage site. 

In addition to above protease, SUMO can be cleaved by SUMO protease, which recognizes the confirmation of SUMO rather than a specific amino acid sequence. G&P Biosciences offer the ability to cleave tags or fusions used for purification as an integral part of the choice of a fusion or tag. We prefer to utilize the TEV cleavage (the recognition site can easily be integrated during gene synthesis and cloning between the tag/fusion and the protein of interest ) followed by affinity purification and an on-column cleavage. We have a panel of expression vectors with TEV cleavage sites built-in, allowing high throughput cloning and expression of genes of interest.

Overall the choice of tag or fusion depends on the nature and downstream application of expressed protein. Every protein is unique and no single fusion/tag or cleavage method will answer every need. His-tag and Fc fusion are the most commonly used and it is easy to apply others according to the needs. Through genetic engineering, researchers can alter the protein sequence and hence its structure, binding behavior, physical property or even biological function. For example, the technique allows the incorporation of unnatural amino acids into a recombinant protein product using artificial tRNAs and may allow the rational design of new proteins or protein-like biologics with novel affinity moieties for easy detection, modification, and/or purification.

» Go to the next topic: Post Genomic Recombinant Protein Purification

« Return to Technical Support mainpage