Abstract
Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements.
Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.
Key words
1 Protein Folding
1.1 Protein Synthesis and Folding
Protein expression in cells is a highly regulated process that permits to build the whole essential protein apparatus for the cells. Nucleic acid codons, through the ribosomal machinery, lead to the formation of linear amino acid sequences that will result in a 3D polypeptide structure. The formation process of this defined spatial structure is called protein folding. Since 1961, when Anfinsen showed that the DNA sequence owns the information for the final tridimensional structure, a lot has been learnt and discussed about the protein folding phenomena [1]. Nowadays, the folding process can be described as the way by which the proteins reach the most favored status at the bottom of an energetic funnel, rolling down into different energetics status.
The number of theoretical conformations that a relatively small protein can reach is really high. As an example, a 100 amino acid peptide can fold in 1,030 possible conformations. Folding for accidental scanning among all conformation permitted, but not functionally exact, could take up to 1,011 year. Despite of this statistics, inside the cells, the protein emerging from ribosome folds spontaneously and rapidly, under hydrophobic driving forces [2].
One of the major issues in protein folding is that, until the whole protein is synthesized, the N-terminal overhanging polypeptide chains lacks complete information for a correct folding. It is now clear that in vivo, newly growing synthesized proteins must be protected in order to avoid misfolding or aggregation until the whole translation is complete. Moreover, once the synthesis is complete, proteins should be immediately folded in order to avoid nonspecific interaction with other components of the crowded cytoplasmic environment. Otherwise, if proteins are required to be moved to another cellular compartment, they must maintain the unfolded state in order to permit the membrane translocation to the appropriate subcellular target site.
During the evolution, cells have developed a protein quality control system, which control protein synthesis, folding, unfolding, and turnover. This system is constituted by a class of highly conserved proteins called chaperones and also by a clearance mechanism, which act together [3]. Chaperones synthesis can be induced by heat shock, among other factors, and, because of that, they are called heat shock proteins (Hsps) [2]. Most of chaperones interact with other regulatory and cooperating proteins which support their functions and are extremely important for the cells, especially under stress situations. There are two major classes of chaperones, Hsp70s and Hsp60s (or chaperonins). Both are characterized by being ATP hydrolysis-dependent to assist the specific protein folding in eukaryotic and prokaryotic cells [4, 5]. Despite their analogy on ATP hydrolysis and substrate binding, they show a completely different mechanism of action.
1.2 Soluble and Insoluble Proteins
In biotechnology, proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. Expressing and purifying the maximum amount of recombinant active protein as possible are among the main goals in this field. In this context, it is important to point out that the selection of the optimal expression organism, as well as the most appropriate growth conditions, should be done according to the protein characteristics and downstream requirements [2].
Under stress situations, such as thermal or oxidative stress, or under protein overexpression conditions, protein folding machinery is saturated, and this promotes protein misfolding and, consequently, protein aggregation. Other causes of aggregation could be a mutation in the protein primary structure sequence due to a RNA/DNA mutation or to a translational misincorporation, or the high concentration of the newly synthesized protein [6–8]. Thus, aggregation process is a common phenomenon observed during recombinant protein production. These protein-based aggregates are generally present in low copy number in the cell cytoplasm or periplasm, and they are formed by a wide range of different conformational populations, including those polypeptides that are partially folded as well as by proteins that have reached their native form [9]. Protein aggregates are found in both eukaryotic and prokaryotic cells under homologous or heterologous protein overexpression being its formations favored at high growth temperatures. Specifically, protein aggregates formed in bacteria are known as inclusion bodies (IBs) (see Subheading 3.2, Chapters 4, 6, and 16), while in mammalian cells they are named aggresomes (see Subheading 3.3 and Chapter 17).
In contrast to what occurs during IB formation, aggresomes are not aggregates of only single protein species; chaperones, chaperonins residues, and proteasome subunits are also found in aggresome immunohistochemical analysis [10, 11]. It is being thought that concentrating aggregates in a defined area have the function to remove aggregates from cytosol and also promote their disposal by autophagy [12].
Besides aggregation, it is important to emphasize that in many cases, misfolded proteins can be degraded through the proteasome complex.
2 Expression Systems for Recombinant Protein Expression (Fig. 1)
Since the production of soluble and functional proteins through a cost-effective and easily scalable process is one of the main challenges nowadays, most of the efforts in this context are aimed at developing and optimizing gene expression systems to minimize the formation of insoluble proteins.
2.1 Prokaryotic Systems
2.1.1 The Preferred Expression System: Escherichia coli (See Chapter 2)
E. coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. The key of success is related to the easy of handle, reduced cost, high yield, and the possibility to optimize downstream processes by affordable scaling-up processes. In addition, a large amount of protein expression tools are available. In fact, the use of E. coli as the preferred expression system is patent in the amount of released PDB entries from proteins obtained in this host organism, representing more than 88 % of the stored structures while only in 12 % of them an E. coli gene is expressed, demonstrating the great power of this expression system for heterologous protein production.
However, as has been already broadly discussed [13, 14], this expression system poses severe restrictions for heterologous proteins such as full-length mammalian proteins and the difficult-to-express membrane proteins [15–17]. In that sense, the E. coli expression system of biopharmaceutical proteins on the market drops to 30 % [18] when compared to eukaryotic expression systems. The limitation of this prokaryotic expression system relies in the reduced capacity to fulfill certain specific posttranslational modifications of the eukaryotic world which can be related to protein solubility and/or biological activity. In other instances, the protein is not even transcribed or translated, and, in most of the cases, aggregation takes place, making the purification process from the soluble cellular fraction a laborious or impossible task.
In summary two main problems are encountered when producing recombinant proteins in E. coli: reduced or lack of heterologous gene expression and aggregation.
The strategies to improve protein yield mainly relies on the gene design aimed to optimize the rate of transcription, the stability of the mRNA, and the rate of translation [19–24]. On the other hand, improving solubility involves changes in cellular metabolism or/and the protein quality control system.
In the E. coli cell, the protein quality control system is composed by two key elements: chaperones and proteases that control the correct folding of proteins and eliminate reluctant protein species that cannot be properly processed, respectively. There are two types of chaperones depending on their effect on protein folding. On the one hand, holding chaperones detect and bind to unfolded or partially folded protein species to let the quality control system to try to fold the polypeptide. Trigger factor binds to nascent polypeptides, and the small heat shock proteins IbpA and IbpB bind to hydrophobic patches in partially folded proteins. On the other hand, two sets of folding chaperones interact with partially folded polypeptides to assist them in their proper folding (GroEL with the accessory protein GroES and DnaK and co-chaperone DnaJ and GrpE). The GroELS complex has a broad specificity and is essential for cell viability, while DnaKJE complex shows substrate preference for nascent polypeptides and is not essential. Finally, the quality control system removes unfolded or folding reluctant proteins by cellular proteases as Lon, ClpA, and ClpB, releasing small peptides in the cytosol that can be recycled in protein synthesis.
This finely tuned system seems to be overcome when overexpression of a recombinant gene takes place in an E. coli cell as in many other expression systems, and the limiting step in protein production and solubility might be related to the limitation of one or more protein factors involved in protein folding. For that reason, many chaperone cocktails have been co-expressed with the gene of interest as a strategy to compensate for the stress produced to the cell. However, the outcome of the supplementation of chaperones is variable, and not a single, universal cocktail has been described being a matter of trial and error process for each and every protein that has to be attempted to be produced.
In the case of cellular metabolism, one of the most explored variables has been media formulation that has a great impact in protein yield [25] as well as in protein solubility [26]. During gene expression induction, expressing cells suffer metabolic stress derived from the reduced access to oxygen, substrates, and also pH changes among others. In addition, limiting cofactors may have a great impact in the proper protein folding and stabilization even in the presence of optimized media formulations [27]. In that scenario, the establishment of optimal growth conditions in fermentation systems guarantees the reproducibility of the process, although controlled batch experiments give not negligible results [25, 28].
Additionally, reduction of growth temperature has a positive effect over solubility since hydrophobic interactions are promoted at high temperatures and expression of chaperones is induced. In summary, less newly synthesized polypeptides are produced, having less hydrophobic interactions and more access to the folding machinery. Obviously, at low growth temperatures, protein yield is compromised, yet protein solubility has been demonstrated to be favored [29].
As it would be discussed in the following sections, aggregation of proteins in E. coli does not seem to be a dead-end for some recombinant proteins as it was assumed in the near past. On the one hand, the recovery of functional recombinant protein coming from IBs after denaturing-refolding processes [30–32] has been widely documented, while on the other hand, biologically active proteins are detected in the formed IB during recombinant gene overexpression [33–40]. In this latter case, solubilization protocols allow the partial recovery of the entrapped protein [41], and more interestingly, innovative biotechnological applications of intact IB are underway to use them as biocatalysts, nanopills, or cell proliferation factors in regenerative medicine among others [42–46].
In any case, several approximations to improve the solubility/aggregation rate of recombinant proteins in E. coli have been developed as solubility has been linked to conformational quality and biological activity. However, it is important to note that this link might be a simplistic view since the soluble cellular fraction has been demonstrated to contain a wide spectrum of soluble protein species, reaching threshold protein conformations in which proteins have a high tendency to aggregate and therefore accumulating and forming part of IBs [47]. In agreement with this observation, it has been described lost in the specific activity of the produced recombinant protein while gaining solubility in some cases [48, 49]. This phenomenon has been ascribed to the way in which the cellular quality control system copes with overexpression of recombinant proteins imposing solubility over folding efficiency.
2.1.2 Lactic Acid Bacteria
It is important to emphasize that recombinant production of difficult-to-express proteins, including those mostly insoluble and prone to aggregate, is one of the main important challenges in the biotechnology field [50]. In this context, the use of lactic acid bacteria (LAB) as a recombinant cell factory is gaining importance, especially for the recombinant production of membrane proteins, which are known to have a huge tendency to precipitate, being mostly insoluble (see Chapter 8). LAB, being a prokaryotic expression system, not only present the same advantages as E. coli (cheap and easily scalable system) but also an important added value, since they do not contain endotoxins in their membrane, which are pyrogenic in humans and other mammals [50–53]. Thus, since the presence of bacterial endotoxins in proteins is becoming one major concern by regulatory agencies [54], many efforts are being addressed to the development of alternative expression systems, being LAB, classified as Generally Recognized As Safe (GRAS), an excellent candidate.
Additionally, it is worth stressing that the endotoxin removal process has not only important associated costs but also presents a risk to destroy protein folding and protein function, being a step that should be avoided, especially for those proteins that are difficult to isolate such as insoluble proteins [55].
Besides, LAB have in general an efficient protein secretion system, being another important characteristic to be considered in the protein production system when overexpressing proteins difficult to produce and purify.
Thus, as described in Chapter 8, it is important to point out that nowadays it is already possible to find commercial proteins produced in recombinant LAB, being Bacillus subtilis and Lactococcus lactis widely used as host microorganisms.
2.1.3 Pseudoalteromonas haloplanktis
Protein aggregation is mainly driven by stereospecific interactions between solvent-exposed hydrophobic patches [9, 56]. Such interactions are weakened when temperature decreases. Thus, the production of recombinant proteins in psychrophilic bacteria (cultured at 4 °C or below) represents an exciting model to improve the quality/solubility of recombinant proteins. In this context, a few cold-adapted bacterial species are under early but intense exploration as cell factories, with Pseudoalteromonas haloplanktis TAC125 as a representative example. P. haloplanktis TAC125 is a Gram-negative bacterium isolated from an Antarctic coastal seawater sample [57], being able to duplicate in the range of 0–30 °C [58], and even at lower temperatures, making it one of the faster growing psychrophiles so far characterized, and an attractive host as cell factory (see Chapter 13).
P. haloplanktis TAC125 versatility has been improved by the development of genetically engineered strains with improved features as cell factories [59, 60]. P. haloplanktis TAC125 was also the first Antarctic bacterium in which an efficient gene expression technology was set up, by the proper assembly of psychrophilic molecular signals [58, 61] into a modified E. coli cloning vector [62]. Several generations of cold-adapted expression vectors allow the production of recombinant proteins either by constitutive [61] or inducible profiles [63] and address the product toward any cell compartment or to the extracellular medium [64].
Beneficial effects in using this cold-adapted platform with respect of the conventional mesophilic E. coli have been reported during the production of antibody fragments [65, 66] or in the production of some “difficult-to-express” proteins such as the human nerve growth factor, h-NGF [67], or the alpha-glucosidase from Saccharomyces cerevisiae [63]. While when produced in E. coli the h-NGF fails to fold and accumulates into IBs [68], its production in P. haloplanktis TAC125 results in fully soluble and periplasmically translocated protein, accumulating in almost fully dimeric form [67]. In the same line, alpha-glucosidase from S. cerevisiae is largely insoluble when expressed in E. coli, but its recombinant production in P. haloplanktis TAC125 renders a recombinant enzyme totally soluble and highly active [63].
Observation that insoluble aggregates of recombinant proteins have never been observed in P. haloplanktis TAC125 (even at high expression levels [63]) suggests that its cellular physicochemical conditions and/or folding processes are quite different from those observed in mesophilic bacteria [69].
Recently a synthetic medium for P. haloplanktis TAC125 growth was obtained, and the new optimized medium was used for P. haloplanktis TAC125 chemostat cultivation [66]. Moreover, a P. haloplanktis TAC125 fed-batch fermentation strategy could be established, which is feasible to be used in lab-scale or for industrial purposes [70]. The next challenges for the industrial application of P. haloplanktis TAC125 as nonconventional system for protein production include the development of efficient fermentation scheme to upscale the production in automated bioreactors.
2.2 Eukaryotic Systems
2.2.1 Mammalian Cells as Expression System
Prokaryotic protein expression systems, such as E. coli, often fail to produce correctly folded, functional eukaryotic proteins. The expression of these proteins greatly benefits of using a eukaryotic expression system, such as mammalian cells, due to their ability to perform proper posttranslational modifications, usually essential for the functionality of therapeutic proteins.
Recent advances have significantly improved the expression levels in mammalian cell lines, reaching up to a few grams of recombinant antibodies per liter in stably transfected Chinese hamster ovary (CHO) cells [71, 72]. The development of a process for recombinant protein production in mammalian cells usually follows a well-established scheme. Unfortunately, this process can take several months, being the major drawback of the stable CHO cell lines. Thus, faster and cheaper approaches for recombinant protein production are needed when many proteins (or several variants of a single protein) must be rapidly evaluated as potential biotechnological or biopharmaceuticals products. For that purpose, a different strategy (called “transient gene expression” or TGE) is preferred. In TGE, recombinant gene is not incorporated into the host cell genome, and selection and isolation of stable transfectants is bypassed so that protein expression is obtained rapidly but only for a limited period of time. By TGE approach, it is possible to produce milligram quantities of recombinant proteins within days or weeks [73].
CHO cells have become the standard mammalian host cells used for the production of recombinant proteins, since it grows rapidly, offers process versatility, can be cultured as either an adherent or a suspension-adapted culture, and is capable of growing in protein-free medium [74, 75]. Apart from CHO, other cell lines as mouse myeloma (NS0), baby hamster kidney (BHK), human embryonic kidney (HEK-293), or human retina-derived (PERC6) cells have proved to be good alternatives. Volumetric yields of secreted recombinant proteins are usually higher when using HEK-293 cells [76]. Thus, HEK-293 cells have also been adapted to grow in serum-free medium, and it has been demonstrated the feasibility of transfecting these cells in suspension and in large-scale volumes (see Chapters 11 and 12) [77, 78].
As in any other expression system, high expression levels are pursued when using mammalian cells as a “cell factory.” However, if the synthesis rate of the recombinant protein exceeds its combined folding and degradation rates, some of the protein will be unable to reach its native, soluble form and will accumulate into insoluble aggregates, in subcellular structures called “aggresomes,” as described by Johnston in the late 1990s [79]. Cells have special machinery responsible for the transport of such protein aggregates, in a microtubule-dependent manner, to the centrosome, forming there the aggresome [6, 79, 80].
Aggresomes formation is usually related to overexpression of recombinant proteins. For example, overexpression of the cystic fibrosis transmembrane conductance regulator and presenilin-1 [79], mutant forms of superoxide dismutase [79], synphilin 1 [81], or a chimera between green fluorescent protein (GFP) and a fragment of p115, a membrane protein [10] led to their accumulation into aggregates. Therefore, protein misfolding and aggregation into aggresomes are issues that must be considered in the design of biotechnological procedures.
Finally, recent data revealed that aggresomes formed by different mutants of GFP were fluorescent [10, 82, 83], indicating that protein embedded into such aggresomes is not completely inactivated by aggregation. Such observations could have interesting theoretical and practical implications: for example, aggresomes formed by proteins with biomedical or biotechnological interest could be used as nanopills or as immobilized biocatalysts. However, these new, putative applications of aggresomes have not been further investigated.
2.2.2 Insect Cells
Insect cells expression system is an appealing alternative for many biotechnological applications since insect cells perform similar posttranslational modifications present in mammalian proteins. However, in the case of glycosylation, the metabolic pathways diverge, and in biomedical applications, these differences need to be analyzed. For instance, when recombinant proteins obtained from insect cells are intended to be included in vaccine formulations, this difference might represent a positive adjuvant effect [84–86]. In some other applications, mainly when the recombinant protein is intended to be repeatedly administered as a therapeutic component or vehicle, undesired immunostimulation might be triggered [87]. Some efforts have been made to obtain transgenic insect cell lines capable of performing humanized glycosylation patterns (MimicTM Sf9 insect cells from Life Technologies), although resulting proteins show insufficient terminal glycosylation [88].
Two different approaches can be followed with this expression system. On the one hand, stable insect cell lines provide continuous production of the protein of interest in the same way as mammalian cell stable cell lines do, while on the other hand, the insect cell-baculovirus tandem offers an important issue that has not been solved yet by any of the other expression systems which corresponds to the expression of up to four genes at a time in the same infected cell (pABAC) using viral strong promoters (see Chapter 10).
Recombinant protein aggregation in that expression system has been also documented although less studied, and it has been associated with the accumulation of the recombinant protein in cell aggresomes [89, 90]. Several strategies have been tried to improve protein solubility including lowering growth temperature, using softer cell lysis methods and adding high salt concentrations or detergents to the lysis buffer [91]. In addition, co-expression of chaperones offers an alternative as in the E. coli expression system [92]. However, since this expression system is mostly used for the secretion of recombinant proteins, the study of the insoluble cell fraction remains mostly unexplored, and the real impact of protein aggregation needs to be further investigated.
2.2.3 Yeast
Yeast cells combine the eukaryotic ability to perform posttranslational modifications with the bacterial capacity to grow to high cell densities, usually rendering higher yields of recombinant proteins and better scalability than mammalian cells. Moreover, yeasts are able to secrete recombinant proteins to the extracellular medium, which is a major advantage during downstream processes (see Chapter 9).
S. cerevisiae, known for ages as a beer and bread producer, is one of the most common yeast used to produce therapeutic proteins [18]. S. cerevisiae genome was the first from a eukaryotic organism to be sequenced, their genetics and physiology are widely known, and tools for molecular biology are very well established.
Several therapeutic proteins have been produced and commercialized using S. cerevisiae as expression system. As an example, Ardiani and coauthors [93] reviewed the use of recombinant S. cerevisiae cells engineered to express viral or tumoral antigens as therapeutic vaccines. Fusion of carrier proteins to therapeutic proteins or peptides is receiving increasing interest because of its potential advantages over the first generation of therapeutic products. In general, such fusions goal is to increase the circulation half-life of the protein of interest [94, 95].
All the commercialized therapeutic proteins produced in S. cerevisiae to date are non-glycosylated, although yeast glycoprotein expression is potentially envisaged as a main source of human glycoproteins in the future [96, 97]. In that respect, a huge effort is being done to generate a collection of new strains with humanized sugar contents, starting with a S. cerevisiae mutant strain with a deletion in the alpha-1,6-mannosyltransferase OCH1 gene [98].
Despite the successful commercialization of several therapeutic proteins obtained in this system, S. cerevisiae has been reported to show limitations in the soluble production of particular protein species [99–101]. This can be exemplified by the failing expression of virus surface glycoproteins (namely, mumps or measles hemagglutinin [102]) that renders inactive aggregates.
Apart from S. cerevisiae, a number of alternative yeast expression systems have been developed (reviewed in [103]). Among them, the methylotrophic yeast Pichia pastoris as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris was originally developed as a single-cell protein production system by Philips Petroleum (Bartlesville, OK, USA) but was subsequently adapted for heterologous protein expression. More than 120 recombinant proteins have been expressed in this host, many of them being of human or mammalian origin [104].
Recombinant protein production in P. pastoris has several advantages over other eukaryotic and prokaryotic expression systems: rapid growth rate; ease high cell-density fermentation; high levels of productivity; elimination of endotoxin and bacteriophage contamination; ease genetic manipulation; absence of known human pathogenicity in the spectrum of P. pastoris lytic viruses; diverse posttranslational modifications including polypeptide folding, glycosylation, methylation, acylation, or proteolytic adjustment; and the ability to engineer secreted proteins that can be purified from growth medium without harvesting the yeast cells themselves [105].
Several products from P. pastoris like human serum albumin, insulin, interferon-alpha, and hepatitis B vaccine are marketed in India and/or Japan [106]. Several reviews [107–109] have described different recombinant proteins with application in diverse areas expressed in P. pastoris.
As mentioned before, S. cerevisiae is by far the best studied yeast with respect to molecular and cell biology, including protein folding and secretion. However, some evidence shows that its secretion pathway differs more from higher eukaryotes than that of P. pastoris. The regulation pattern of unfolded protein response, the major regulon controlling folding limitations, shows significant differences between these two yeasts [110]. With the advent of humanized yeast strains and their ability to control glycosylation, development of a significant number of biopharmaceuticals produced in yeast-based expression systems can be easily envisaged.
2.2.4 Trichoderma reesei
Trichoderma reesei is an efficient secretory filamentous fungus with reported production yields in excess of 100 g/L [111], of industrially applicable native enzymes. This fungus is a soil-based microorganism able to utilize cellulose as its source of nutrition, allowing for both low-cost fermentation media and also strong induction when using the cellobiohydrolase Ι (cbh1) promoter [112].
Therapeutic protein production in T. reesei is an emerging but promising field, particularly considering that the major N-glycan form synthesized by T. reesei GlcMac2 MAN5 [113, 114] is a suitable precursor for mammalian glycosylation. Thus, the possibilities for humanization of the T. reesei glycosylation pathway are better than, for example, in yeast systems. In that respect, human N-acetylglucosaminyltransferase I has already been expressed in T. reesei to transfer a GLcNAc residue to the GlcNac2Man5 fungal glycans [115]. In terms of potential for pharmaceutical protein production, T. reesei is not only well established in large-scale fermentation but is also already approved as a GRAS organism for food applications, thereby presenting a platform for progression toward regulatory approval for therapeutic uses.
Since a wide number of proteins have an important tendency to aggregate, an interesting approach under current study is promoting or favoring the accumulation of the recombinant protein into intracellular insoluble aggregates by the fusion of specific signals, such as the ZERA peptide [116] or the endogenous hydrophobin [117] fusion partners. Such systems allow for accumulation of the fusion protein within a protein body structure (similar to IBs or aggresomes). Then, purification can be achieved by utilizing the highly hydrophobic properties of the fusion, by mechanical gravity separation, or by two-phase extraction for ZERA peptide or hydrophobin fusions, respectively, with the need for additional downstream purification.
Development of improved T. reesei strains for production of therapeutic proteins must concentrate on both overcoming the bottlenecks of not only expression and purification but also refining the molecular mechanisms involved in determining tertiary structural characteristics, in order to yield molecules of high efficacy and immunogenic compatibility to humans.
2.2.5 Microalgae
The term “microalgae” includes a diverse photosynthetic group of both prokaryotic (cyanobacteria) and eukaryotic organisms. Historically, microalgae have been used in applications ranging from enhancing the nutritional value of animal feed to as producers of highly valuable molecules, like polyunsaturated fatty acid oils, pigments, or human nutritional supplements [118, 119]. Apart from these traditional uses, during the last years, microalgae have received the attention of researchers as an alternative to current recombinant protein expression systems [120–122], due to the feasibility of microalgae to be genetically modified and express heterologous genes. In this context, microalgae show the benefits of plants (they share the same basic photosynthetic mechanism), together with the high productivities of microbial systems. Being most microalgae photoautotrophs, they require only light, water, and basic nutrients for their culture. Some microalgae can also be grown as heterotrophs in fermenters without light as energy source, thus requiring a supply of sugars for energy and as a carbon source. At the same time, microalgae can be grown in large-scale liquid cultures (either in controlled, closed bioreactors or in open ponds). The potential for large-scale culture (on scales ranging from a few milliliters to 500,000 liters in a cost-effective manner) makes microalgae a desirable target as cell factories for the synthesis of high-value therapeutic proteins. More advantages making microalgae ideal candidates for recombinant protein production include the fact that (1) transgenic algae can be generated quickly, requiring only a few weeks between the generation of transformants and their scale up to production volumes, (2) both chloroplast and nuclear genomes of microalgae can be genetically transformed, and (3) green algae fall into the GRAS category. Since there is no gene flow by pollen or other vehicles of gene escaping, transgenic microalgae are harmless to the environment [123].
Regarding economic issues, and according to a recombinant antibody production study, the cost of production per gram of functional antibody was $150, $0.05, and $0.002 (USD) in mammalian, plant, and microalgae expression systems, respectively, data that makes microalgae-based expression systems very appealing for biotechnological industries [124].
Despite the increasing examples of successful transformation of different microalgae species, Chlamydomonas, Chlorella, Volvox, Haematococcus, and Dunaliella remain the most widely used [125, 126]. However, current work is mainly performed with Chlamydomonas reinhardtii, as it is the best characterized microalgae specie, and for which stable genetic transformation at both chloroplast [127] and nuclear [128, 129] level was first reported. In order to achieve high expression levels of protein in C. reinhardtii chloroplast, codon-optimized reporter genes has been developed [130, 131] and used to examine a variety of promoter and translational elements [132]. Using this strategy, GFP accumulation up to 0.5 % of total soluble protein (TSP) was achieved in transgenic chloroplasts [131, 132].
Considerable progress has been made in metabolic engineering toward increasing the expression of naturally produced compounds, with varying levels of success [126, 133]. The expanding genetic engineering toolbox for microalgae has allowed the expression of fully functional antibodies [134, 135], therapeutics [136, 137], and bactericides [138]. However, many obstacles still remain to be solved before microalgae can be seen as standard expression systems. So far, success essentially remains anecdotal, and no wide-ranging system or protocol leading to high-level expression has been fully established.
2.2.6 Transgenic Animals and Plants
The expanding recombinant protein market seems to be limited by the achieved yield of the conventional expression systems described above. Therefore, production systems derived from transgenic animals and plants have been developed with the aim to increase the production potential. In the case of animals, the most promising systems are proteins secreted in the milk (first approved biopharmaceutical of that type is the anticoagulant human antithrombin, from goat) and semen or accumulated in white yolk of hen eggs. Unfortunately, aggregation of the recombinant protein in animals is not determined. Due to the fact that the protein of interest is recovered in a secreted form, protein accumulation in producing cells is not analized. Therefore, information related to the amount of protein retained in the insoluble fraction of the producing cell is not considered in the production studies.
In the case of plants, the recombinant protein is usually produced at low levels, and the purification process tends to be relatively expensive and complicated [139]. Interestingly, plants have specialized tissues (seeds) that are able to store recombinant proteins at a high purity [140]. In addition, unlike for most of the expression systems, delivery of recombinant protein to aggregated structures in plants offers many advantages. Plants make use of protein aggregation to accumulate proteins for storage purposes in specific cell compartments. For instance, proteins of interest can be sent to protein bodies derived from endoplasmic reticulum by including a proline-rich domain in the recombinant gene [141]. The recovery from this specialized membranous structure simplifies downstream processing by increasing capture of the recombinant protein. In addition, this technology can be transferred to non-seed tissues in plants and also to other eukaryotic expression systems [142, 143].
2.2.7 Cell-Free System
Synthesis of proteins without the entire machinery of a living cell, better known as cell-free protein system (CFPS), is an emerging technology for simple and effective protein productions (see Chapter 7) [144, 145].
This platform takes advantage of catalytic components and the necessary elements for transcription, translation, and protein folding that are extracted from crude lysates of E. coli (ECE), rabbit reticulocytes (RRL), wheat germ (WGE), or insect cells (ICE) principally. Production in CFPS is quick and simple and not restricted by the eventually toxic effects of the final product.
The appropriate cell-free system should be chosen depending on protein complexity, posttranslational modifications, downstream process, and yield required [146, 147].
Proteins’ tendency to aggregate when overexpressed in prokaryote’s expression system can be often overcome changing to eukaryote CFPS organism. This technology also permits to perform denaturing and refolding process as well as permits to add components that assist in protein folding, avoiding aggregation [148, 149].
The biggest problem noticed by using those extracts is that cell lysate contains cellular proteins as proteases or nucleases, and nucleic acids not required for protein of interest expression. These components can act in an unpredictable and often unknown way, causing problems to the reaction.
The “PURE” in vitro system, which consists in purified translation factor components of E. coli, has been shown as an efficient alternative to the crude lysates [150, 151].
Recently, the CFPS evolution has permitted protein yield at milligram levels thanks also to the development of various reaction schemes as the continuous exchange, continuous flow, hollow fiber systems, or batch-type improvements [152–157].
Another interesting aspect is that the final product purification is simplified [158].
Despite these improvements, costs, lack of wide experience of use, and the problem in reproducing the folding environment, resulting in a non-correct protein folding, are still limiting factors of this technique.
3 Purification and Characterization
3.1 Protein Purification
As already mentioned, in Subheadings 1 and 2, many insoluble proteins aggregate during the expression process, being necessary to optimize different parameters. However, in other cases, protein aggregation occurs during downstream purification processes. In these situations, it is crucial to develop a suitable and optimized purification protocol for each protein (see Chapter 14). In fact, it is important not only to carefully evaluate the best purification protocol but also the appropriate characterization (see Chapter 21) and dialysis and storage conditions (see Chapter 18).
3.2 Inclusion Bodies Purification
IBs are protein aggregates formed in both bacterial cell cytoplasm and periplasm when overexpressing insoluble proteins, which have a huge tendency to aggregate. IBs show, in general, a sphere-like shape varying between 0.1 to 0.8 μm in diameter, depending on the cell host dimension, growth conditions, and protein sequence (see Chapter 22). Interestingly, they show higher stability than that of their soluble counterparts and are essentially formed by the protein of interest. In this regard, it is important to stress that it was found that IBs show a spongelike organization, which combine both active and inactive protein forms. Inactive forms correspond to proteins which adopt an amyloid-like organization forming a protease K-resistant fibrillar scaffold that is fully embedded by functional proteins [159–161] (see Chapters 19 and 20).
Thus, since these insoluble aggregates are mainly composed by the target protein, they are an important source of the protein of interest. In this regard, several protocols that aim obtaining protein from IBs have been developed in the last decades. These protocols include denaturation and refolding processes, which allow the purification of variable amounts of the recombinant protein of interest (see Chapter 15).
Given that in recent years several groups have shown the huge potential of IBs for diverse applications such as catalysis and tissue engineering, some researchers have focused their efforts on the development of protocols for obtaining highly pure IBs to be used in the applications mentioned above (see Chapters 16 and 24). In this regard, this book reviews the newest and most widely used for both isolation of soluble proteins from IBs and protocols for IB purification.
3.3 Aggresomes Purification
Many studies have attempted to identify proteins associated with aggresomes (see Chapter 17). For that, the simplest approach would be to isolate the aggresome and determine its composition, since factors involved in the formation of such aggregates may be physically associated with them. However, a detailed analysis of the components associated with aggresomes is hampered by the difficulties found for their isolation. Aggresome heterogeneity in size and charge precludes application of conventional biochemical methods (e.g., gel filtration, or ion exchange or affinity chromatography) for their isolation.
Protein aggregate isolation has been attempted by using the ionic detergent insolubility of amyloids [162] or density gradient fractionation [163]. Such methods may be useful to address certain questions, but they are inadequate to identify aggregate-associated proteins. SDS treatment dissociates most of the associated proteins, and the use of density gradients (apart of being a tedious procedure) renders a high number of nonspecifically associated polypeptides. Due to the abovementioned difficulties, most of the published studies regarding aggresome composition rely on immunocytochemistry of cells overexpressing certain recombinant proteins and that, consequently, produce cytoplasmic aggregates [164–166]. In such studies, aggresomes isolation is bypassed, since detection of their components is performed directly on cells producing such structures.
As an alternative, other approaches have been proposed to obtain cellular insoluble fractions containing aggresomes. For example, a protocol for the isolation of aggresomes formed by a GFP fusion protein has been proposed by García-Mata and colleagues [10]. In this protocol, pellets of cells producing aggresomes were washed with phosphate buffered saline (PBS) and lysed for 30 min on ice with different detergent-containing buffers. Lysates were then passed through a 27 gauge needle, and finally the insoluble material was recovered by centrifugation. Such isolated insoluble material allowed to gain insight into formation and composition of aggresomes, but the presence of some ionic detergents like SDS in some of the buffers used for cell lysis could render misleading results.
Finally, Wang and colleagues have developed a protocol for the isolation of aggregates based on affinity purification without involvement of a solid phase [167, 168]. This highly reproducible procedure yielded a fraction of polyQ aggregates of diverse size and charge, which could be separated by 2D gel and analyzed by mass spectrometry to identify aggregate-associated proteins. Also, the method allows for semiquantitative comparison of the identified proteins. Another advantage of this protocol is that it avoids exposure of the aggregates to extreme pH or ionic strength as well as to ionic detergents, thus preserving putative weak protein interactions.
The apparition of future new applications for protein aggresomes will surely result in the development of faster and better purification protocols for such aggregates, for example, those based on magnetic micro- and nanoparticles.
4 General Overview
The development of recombinant DNA technology represented a breakthrough in the treatment of some human diseases, increasing life expectancy and life quality of patients. The first therapeutic recombinant protein product, human insulin, was approved in 1982, opening up a new pharmaceutical market with an unceasing demand and steady global sale increase [169, 170].
Industrial market also benefits from the recombinant DNA technology in the enzyme and agricultural industry [171].
In this arena, different expression systems have been established to fulfil the production needs [172]. It is widely accepted that prokaryotic expression systems represent a cost-effective alternative when comparing with eukaryotic expression systems [173]. However, at least in the use of therapeutic proteins, regulatory and functional constraints impose the use of the eukaryotic expression systems [174, 175].
In any case, the recombinant protein production process copes with similar limitations in any of the available expression systems [171]. In the extremely crowded cell cytosol, the appearance of a great amount of newly synthesized polypeptide chains challenges the folding machinery, and, consequently, protein aggregation is detected [172].
In some instances, modifications in the growth parameters can modulate the ratio of the amount of protein in the soluble/insoluble cell fraction, but in some cases, the valuable recombinant protein is reluctant to solubilize [176] (see Chapter 23).
In this book, the reviewed strategies to improve protein solubility are disclosed in addition to established approaches to obtain soluble protein from protein aggregates. In addition, novel applications for the use of protein aggregates in nanomedicine are also shown.
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Acknowledgments
The authors acknowledge the financial support granted to E.G.F. from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria—MINECO (RTA2012-00028-C02-02) and Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina. Z.X. acknowledges financial support from China Scholarship Council. PS has received a predoctoral fellowship from Instituto de Salud Carlos III. The authors are also indebted to the Protein Production Platform (CIBER-BBN—UAB) for helpful technical assistance (http://www.bbn.ciber-bbn.es/programas/plataformas/equipamiento).
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Ferrer-Miralles, N., Saccardo, P., Corchero, J.L., Xu, Z., García-Fruitós, E. (2015). General Introduction: Recombinant Protein Production and Purification of Insoluble Proteins. In: García-Fruitós, E. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 1258. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2205-5_1
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