Figure 1 shows two typical protein expression workflows. One workflow leads to the generation of a purified protein. The other leads to the generation of a cell line expressing a recombinant protein. In real life, these two workflows may overlap if, for example, a stable mammalian cell line is to be used as the source material from which to purify a recombinant protein.

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Figure 1. Typical protein expression workflows.

The advantages, disadvantages and potential applications of a number of the commonly used recombinant expression systems are listed in Table 1. A number of publications provide detailed information on these systems: Escherichia coli [1-6] ; Saccharomyces cerevisiae [4, 7-10] ; Pichia pastoris [5, 8, 9, 11, 12] ; baculovirus / insect cells [1, 5, 13, 14] ; mammalian cell lines [5, 15] ; and cell-free / in vitro protein production systems [16]. Table 1 summarizes the fundamental properties of these expression systems. Researchers are actively working to improve these fundamental properties (see [17] for a review). Several E. coli strains are now commercially available that aim to overcome the problems of codon bias (Rossetta 2 [18], CodonPlus ril/rp), inefficient disulfide bond formation (SHuffle, Origami [19, 20] ) and poor expression of membrane proteins (C41 and C43). Similarly, E. coli expression vectors utilizing tags such as SUMO, maltose binding protein [21] and thioredoxin [21] designed to promote soluble expression are commercially available. Pre-expression of sulfhydryl oxidase may markedly promote disulfide bond formation [22]. These approaches work very well for some proteins, but for others, it is still not possible to obtain soluble, functional recombinant expression in E. coli [20, 23]. Proteins expressed in bacteria are contaminated with endotoxins, which can be removed by, for example, Toxineraser endotoxin removal kit from GeneScript [24] before being used in vivo. E. coli strains have even been engineered to perform protein N-glycosylation, though the efficiency is low [17, 25], or optimized for His-tagged protein expression (LOBSTR strain) [26, 27]. Likewise, approaches are being developed to express proteins with more mammalian-like glycosylation in the baculovirus/insect cell system and hence expand its utility [28]. Baculovirus variants that promote greater protein secretion are also being developed [17]. Staus DP et al minimized the number of cysteine residues in a truncated rat beta-arrestin 1 gene to increase its expression and stability in BL21/DE3 bacteria [29].

The critical problem facing the researcher is that it is still not possible to predict which expression system will work best for a particular protein and a specific end-use. A universally applicable expression system does not yet exist [30]. When selecting an expression system, the researcher should bear in mind the fundamental properties of each system, their pros and cons and how any particular limitations of that system can be overcome. Decisions should be informed by knowledge of the protein expression target/family members and the ultimate use of the recombinant protein. If resources permit, it may be prudent to explore two (or more) expression systems in parallel.

Interestingly, one structural genomics effort, the RIKEN Structural Genomics Initiative in Japan, has concentrated on the use of cell-free (in vitro transcription/translation) to generate proteins for its structure determination efforts [3, 36], which demonstrates the synthetic capacity of modern cell-free protein expression systems. However, cell-free systems tend to have low efficiency for proper folding.

Key aspects of protein expression for structural biology have recently been reviewed in a special edition of Current Opinion in Structural Biology in 2013.

This article has focussed on the most commonly used expression vector-host systems. These are the systems that are likely to be the first port of call when planning to express a recombinant protein. However, it should be noted that many other, more esoteric, expression systems are available. These may be of interest to those researchers with experience of protein expression, or in those situations where the more ‘mainstream’ expression systems do not meet the needs of a particular study. By way of example, the following microbial/plant cell host systems have been described: yeast (Hansenula polymorpha, Arxula adeninivorans, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe [8] ); bacteria (Bacillus brevis, Bacillus megaterium, Bacillus subtilis and Caulobacter crescentis [2], Corynebacterium [37], hyperthermophilic sulfolobus islandicus [38] ); and algae [39]. For a recent review of alternative/less common expression systems used for structural biology see [40]. The non-pathogenic Mycobacterium smegmatis was used for the soluble expression of proteins from pathogenic Mycobacterium tuberculosis. This is of note as it is reported that the expression of mycobacterial proteins in E. coli can be problematic [41].

The following viral expression vectors are available for recombinant protein expression in mammalian cells: semliki forest virus [42] ; lentivirus [43] ; adenovirus [44] ; adeno-associated virus. Semliki forest virus has proved popular for the expression of membrane proteins for drug discovery and structural genomics [42]. Lentiviral and adenoviral vectors are currently of great interest in the field of gene therapy [45, 46]. There is also considerable interest in the expression of therapeutic recombinant proteins in the milk of transgenic animals [47]. Transposase-based systems are also gaining popularity for hyperactive constitutive expression, such as the sleeping beauty vector [48].

There has been a substantial recent interest in developing systems for the recombinant expression of multi-protein complexes for both structural biology and drug discovery/development [49-52].

The Worldwide Protein Data Bank (wwPDB: http://www.wwpdb.org/) is an international collaboration of four organisations: RCSB PDB (USA: http://www.rcsb.org/); MSD-EBI (Europe: http://www.ebi.ac.uk/pdbe/); PDBj (Japan: http://www.pdbj.org/); and BMRB (USA). The wwPDB is a repository of macromolecular structural data whose "mission is to maintain a single PDB archive of macromolecular structural data that is freely and publicly available to the global community" [53, 54]. The vast majority of the structures in the wwPDB are of proteins.

To understand our current practice on protein expression, we select the PDB entries with publications within the last 10 years (from 2009 onwards), resulting 27096 articles, which correspond to 64713 records.

The majority of the wwPDB entries cited Escherichia coli as the expression host, with 23041 out of 27096 publications (85%) reporting its use. The common strain is BL21 (11628 articles).

Table 2 lists the top 5 expression hosts and the top 2 or 3 expression vectors for each of these host organisms.

It is noteworthy that Escherichia coli is the most common expression host in the wwPDB dataset by a very substantial margin. Escherichia coli is a Gram-negative, rod-shaped bacterium. It is one of the key model organisms in life science research and has been extensively exploited in both academic and industrial settings. It should be noted that the lipopolysaccharides in the outer membrane are the source of endotoxin, which may elicit severe inflammatory responses in cellular and in vivo experimental models. Spodoptera frugiperda cells used for protein expression are cell lines (Sf 9 and Sf 21) derived from the ovarian tissues of the Fall Armyworm. Novavax expressed modified SARS-CoV-2 spike protein in Sf 9 cells to produce a COVID 19 vaccine [55]. Trichoplusia ni cells (available commercially as High Five) are derived from Cabbage Looper ovary cells. Trichoplusia ni is reported to be better than Sf9 cells for the production of secreted proteins using the baculovirus/insect cell system [56]. Trichoplusia ni cells are also superior to Sf9 cells as a host for the production of virus-like particles for recombinant vaccine production [57]. Pichia pastoris is respiratory, methylotrophic yeast that can utilize methanol as its sole carbon and energy source. For example, Kitchen P et al expressed full-length AQP4 protein in Pichia pastoris [58] and D Wrapp et al expressed bivalent VHHs in the pKai61 vector in Pichia pastoris [59]. Cricetulus griseus cell lines are derived from Chinese hamster ovary cells (CHO). This extensively used cell line can be adapted to suspension growth. Most recombinant antibodies are produced in CHO cell lines. Maun HR et al, for example, expressed human alpha- and beta-tryptase genes in CHO DKO cell line [60].

For each expression host, the top 2 or 3 most frequently cited expression vectors account for only a relatively small proportion of the total publications (Table 2). This reflects the wide range of different expression vectors that have been used for each expression host. Figures 1 and 2 show plasmid maps for pET28 and pcDNA3.3 (the latest version of pcDNA3), respectively. These plasmids are the archetypal expression vectors for E. coli and mammalian cells, respectively.

With pET vectors the T7 RNA polymerase promoter drives expression of the recombinant gene. The pET28 plasmid encodes an N-terminal His-tag/thrombin cleavage site/T7-tag sequence and an optional C-terminal His-Tag sequence. These vectors are used with lambda DE3 lysogen strains of E. coli. In these strains expression of a genomic copy of the T7 RNA polymerase is under control of the lac repressor. Expression of the recombinant protein is induced by the addition of isopropyl-b-D-thio-galactoside (IPTG) to the culture medium. Interestingly, vectors in which expression is induced by other molecules (e.g., arabinose) do not feature prominently in the wwPDB dataset.

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Figure 2. Plasmid map of pET28. pET28 images provided with permission of EMD Millipore Corporation.

With the pcDNA3 series of plasmids, the expression is driven by the immediate early promoter of human cytomegalovirus (CMV). This is a strong promoter, constitutively active in mammalian cells. pcDNA3 is the original version of the pcDNA3 series of vectors and is no longer commercially available. The latest development of this series of vectors is pcDNA3.3. It should be noted that pcDNA3 was also identified as one of the most commonly used mammalian expression vectors in a survey of formal publications (like pcDNA3.1 [61, 62] or pcDNA3.3 from Invitrogen (K8300-01) [63] ).

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Figure 3. Plasmid map of pcDNA3.3. pcDNA3.3 images provided with permission of Life Technologies Corporation.

The most commonly used baculovirus/insect cell vectors all utilize the strong polyhedrin promoter to drive constitutive expression of the recombinant protein. The plasmids (baculovirus transfer vectors) do not themselves directly drive protein expression. They are used to generate recombinant baculovirus containing the gene of interest under the control of the polyhedrin promoter. pFastBac is a newer generation of plasmids that utilize site-specific transposition to generate recombinant baculovirus. This reduces the time taken to generate recombinant baculovirus to around 2 weeks compared to the 4-6 weeks required with older generation plasmids such as pVL1392/3 and pAcGP67.

The Pichia pastoris vectors both utilize the strong AOX1 promoter. The expression is induced by methanol. Despite Pichia pastoris being an excellent system for the production of secreted proteins (see below), the two most commonly used Pichia pastoris expression vectors in the wwPDB dataset are both designed for cytoplasmic expression.

The data in Table 2 correspond to expression hosts and expression vectors that were specifically employed for producing proteins for structural studies. Consequently, this dataset is biased towards those expression vectors/hosts that are capable of generating large amounts of purified proteins that are required for 3-dimensional structure determination studies. The inability of Escherichia coli to glycosylate proteins and the relative ease with which insect cell N-glycosylation can be removed enzymatically (see Table 1) may also contribute to the frequent use of these systems in the wwPDB dataset. Unglycosylated proteins are generally preferred for structural studies, unless the sugar molecules are essential for function.

For other applications (such as enzyme assays, cellular assays, production of antigen for antibody generation, over-expression to study cellular function or localization) it may not be necessary to express and purify such substantial quantities of protein. Indeed, for cell-based studies purification of the recombinantly-expressed protein is unlikely to be a consideration. Nonetheless, the data on expression vectors/hosts obtained from the wwPDB is a precious resource. The data can help guide protein expression projects for many applications.

To avoid the potential bias of the wwPDB dataset, Labome surveyed a randomly selected set of formal publications that cited plasmids. The top 3 most commonly used groups of expression vectors are shown in Table 3. As observed for the wwPDB dataset, the most commonly cited expression vectors account for only a small proportion of the total publications. Again, this reflects the diversity of expression vectors that researchers use for any given expression host. Vector pcDNA3.1 drives expression in mammalian cells via the constitutive CMV promoter (see above). Plasmid pGL3 is a luciferase reporter vector designed for the quantitative study of the regulation of mammalian gene expression. The increasing popularity of this vector presumably reflects a growing desire of researchers to study the function of the human genome at both the transcriptional and proteomic levels.

Similarly, pEGFP is a mammalian expression vector in which expression is driven constitutively by the CMV promoter. Enhanced green fluorescent protein (EGFP) is expressed as either an N-terminal (pEGFP-C1) or a C-terminal (pEGFP-N1) fusion with the protein of interest. These pEGFP vectors may be used to study the subcellular localization or trafficking of proteins by monitoring the EGFP fluorescence [64, 65]. Other vectors such as pRK5 expression vector [66] are also utilized.

It is notable that the most commonly used mammalian expression vectors, both in the publication survey and in the wwPDB dataset, drive constitutive expression. This is despite the commercial availability of inducible mammalian expression vectors such as the T-Rex system, like Thermo Fisher Flp-In T-REx 293 cells used in the production of ApoE3 proteins [70] or other proteins [67], pGEX vectors from GE Healthcare [71], or the pF12 RM Flexi system. Although not featuring highly in academic publications, the BacMam system has proven popular in the pharmaceutical industry for expressing proteins both for cellular studies and for purification [72-74] and is now commercially available. BacMam utilizes a modified baculovirus in which the usual promoter is replaced with the mammalian cell-active CMV promoter. The BacMam virus drives non-replicative, non-lytic expression in a wide range of mammalian cell types.

Unlike in research settings, the production of newly approved protein pharmaceuticals is often in mammalian expression systems. Gary Walsh summarized the expression systems used in the production of protein pharmaceuticals approved by US/EU regulatory authorities from Jan 2014 to Dec 2018 [79]. Fifty-two out of 62 novel recombinant protein pharmaceuticals are expressed in mammalian cell lines, one (Sebelipase alfa) in a mammalian transgenic system, 5 in E. coli, and 4 in S. cerevisiae. CHO cell-based systems are the most common mammalian expression host. Among the 68 monoclonal antibody drugs (novel or biosimilar) approved during the same period, 57 are produced in CHO cell lines, 9 in NS0 cells and 2 in Sp2/0 cells.

A frequently encountered problem is the expression of recombinant proteins that are toxic to the host cells in which they are expressed. Several strategies are available to overcome this issue. The researcher generally has to empirically determine which potential solution works best for their particular protein. Literature precedent, target class knowledge and ‘in-house’ experience of the target protein can all be used to guide the choice of strategy.

The critical issue with E. coli expression is the leaky expression of the toxic protein before induction. This leads to plasmid loss/rearrangement, poor cell growth and reduced protein expression [80]. Several approaches have been developed to address the problem of toxic protein expression in E. coli.

• Tightly regulated (i.e., non-leaky) expression systems such as the pBAD system utilizing the araBAD promoter (Invitrogen/Life Sciences) can be used to minimize basal expression [80].
• With T7 promoter-based plasmids, pre-induction expression can be reduced by the use of pLysS/pLysE/pLysY host cells expressing T7 lysozyme that inhibits T7 RNA polymerase and thus reduces promoter activity prior to IPTG induction. Similarly, glucose can be used to repress promoter activity prior to induction with IPTG [80].
• The pETcocoTM system (EMD Millipore) allows plasmid copy number to be maintained at a very low level during cell growth thus minimizing basal expression and maximizing plasmid stability prior to induction. Plasmid copy number is markedly upregulated and target gene expression induced by IPTG [80].
• Another approach is to use host strains, such as C41(DE3) and C43(DE3), empirically selected for their ability to express toxic proteins more effectively than the parental BL21(DE3) strain (Avidis, Lucigen) [80].
• Empirically screening fusion tags such as maltose binding protein, GST, thioredoxin or SUMO may identify a fusion partner that overcomes the toxicity of the target protein (Invitrogen/Life Sciences, New England Biolabs, LifeSensors) [80].
• Directing expression to the periplasm can potentially overcome toxicity associated with cytosolic accumulation [80].
• Batch-fed culture may also be a useful approach to the expression of toxic proteins [81].

Many mammalian expression systems use a constitutively active CMV promoter. This is problematic for the expression of proteins that are toxic to the host cells. However, researchers frequently wish to study the cellular function of such proteins or wish to express and purify such proteins bearing full mammalian cell post-translational modifications. Multiple inducible mammalian expression systems, utilizing the strong CMV promoter, are now commercially available in which expression is induced by tetracycline (T-RexTM Invitrogen/Life Sciences, Tet-On 3G Clontech), ecdysone (Agilent Technologies/Stratagene) and IPTG (pTUNE Origene). These systems facilitate the growth of sufficient cell numbers prior to the induction of the target protein. Nakagawa T expressed a DualTetONGluA2-FLAG/CNIH3-1D4 plasmid in HEKTetON cell (CLONTECH) to alleviate the toxicity by the activation of the ion channel GluA2 [82].

If one particular expression system fails, it may be advantageous to switch to a different system (e.g., yeast, insect, bacterial, mammalian) if other considerations allow (e.g., end use, post-translational modifications). A protein that is toxic in one system may not be toxic in another [80].

If relatively small quantities of (purified) protein are required then cell-free protein production is an attractive option to circumvent issues of cellular toxicity [80].