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Published as doi: 10.1096/fj.08-110544.
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(The FASEB Journal. 2008;22:4055-4066.)
© 2008 FASEB

Dictyostelium discoideum—a promising expression system for the production of eukaryotic proteins

Ranjana Arya*,1,2, Alok Bhattacharya{dagger} and Kulvinder Singh Saini*

* Department of Biotechnology and Bioinformatics, Ranbaxy Laboratories Limited, Gurgaon, Haryana, India; and

{dagger} School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

2 Correspondence: Department of Biotechnology and Bioinformatics, Ranbaxy Laboratories Limited, R&D III, Sector-18, Udyog Vihar, Gurgaon-122015, Haryana, India. E-mail: ranjanaa{at}mail.jnu.ac.in


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuverability with respect to large-scale production of recombinant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advantages, mammalian cells remain the preferred host for many eukaryotic proteins of pharmaceutical importance, particularly, those requiring post-translational modifications. Recently, the single-celled slime mold, Dictyostelium discoideum (Dd), has emerged as a promising eukaryotic host for the expression of a variety of heterologous recombinant eukaryotic proteins. This organism possesses the complex cellular machinery required for orchestrating post-translational modifications similar to the one observed in higher eukaryotes. This review summarizes the advantages and disadvantages of Dictyostelium as an alternate system compared to other well-established expression systems. The key lessons learned from the expression of human recombinant proteins in this system are reviewed. Also, the strengths, weaknesses, and challenges associated with industrial-scale production of proteins in Dd expression system are discussed.—Arya, R., Bhattacharya, A., Saini, K. S. Dictyostelium discoideum—a promising expression system for the production of eukaryotic proteins.


Key Words: high-throughput system • recombinant • therapeutic • yield • phosphodiesterase 4D3


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
THE ADVENT OF RECOMBINANT DNA technology has revolutionized the strategies for production of therapeutics and vaccines required for the treatment of many diseases, such as cancer, diabetes, Alzheimer’s, and asthma. The ability to clone and express a foreign gene into a heterologous host has provided scientists in the biopharmaceutical field with proteins in quantities that were unimaginable earlier. Since the approval of insulin in 1982, more than 120 recombinant therapeutics and vaccines have been approved by the U.S. Food and Drug Administration (FDA), and a large number are currently in various phases of clinical development. However, biopharmaceuticals, which constitute a market of $100 billion sales annually, are governed by three major factors: product quality and efficacy, safety, and economy of the manufacturing process. For the production of recombinant enzymes and protein therapeutics, the technology platforms should employ inexpensive media components in their fermentation processes to be economically feasible (1) . With the increase in the number of gene targets for pharmaceuticals in the postgenomic era, the demand for suitable expression hosts has tremendously increased. A variety of these are currently available, ranging from bacteria, yeast, and fungi to mammalian cells, insect cells, and plant cells. All of these expression systems have some advantages, as well as a few limitations, which were outlined by Rai and Padh (2) . As there is no universal expression system that is optimal for all possible proteins, several hosts are normally tested in parallel for their ability to produce a particular protein in desired amounts and quality (1) . Thus, choosing the best expression system for a protein requires evaluation with respect to its structure (i.e., post-translational modifications), the final use of the expressed protein (i.e., structural analysis, in vitro, in vivo functional assays), the amount of protein required (screening applications vs. therapeutic applications), the acceptable cost and quality specification (as outlined in U. S. Pharmacopoeia).

Until the mid-1990s, Escherichia coli was the preferred host for the production of pharmaceutical proteins; however, in recent years, the mammalian cell system has been extensively utilized for the production of biochemically complex proteins requiring post-translational modifications. In addition, baculovirus and yeast expression systems have been used extensively for the expression of recombinant proteins in a number of laboratories for drug discovery research, target validation, or large-scale expression of these proteins for high-throughput screening of drugs. Few comprehensive reviews have clearly outlined the pros and cons of the above-mentioned expression systems (2 3 4) . Another organism that has emerged recently as a potential eukaryotic host for heterologous protein expression is the cellular slime mold, Dictyostelium discoideum (Dd). This review focuses on summarizing the key characteristics of the Dd expression system in comparison to other available expression hosts and also outlines recent technological advances in the overproduction of proteins using this organism. On the basis of experience in an Indian pharmaceutical setting, a comparison of various expression systems has been summarized in Table 1 .


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  		Table 1. Comparison of different expression systems


   EXPRESSION SYSTEMS
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
E. coli as an expression host
E. coli is one of the most widely exploited hosts for the production of heterologous proteins, which is well characterized genetically, simple to maneuver, inexpensive to culture, and requires shorter lead time. E. coli has been successfully used for the cost-efficient production of single subunit proteins, and its various strains have been genetically modified to facilitate the bioprocessing and large-scale fermentation (6 7 8) . The development of highly efficient Gateway cloning system and TOPO directional cloning (Invitrogen) systems have greatly expedited the cloning of genes for multiparallel expression profiling of proteins (9) . Various promoter systems commonly used for E. coli and their gene regulatory functions are now well documented (10) . The generation of recombinant fusion proteins using affinity tags, such as glutathione-S-transferase (GST), maltose binding protein (MBP), or polyhistidine (His) tags, has certainly simplified the downstream purification process (11) . However, despite these advantages of E. coli, not every gene can be expressed efficiently in this organism. The major drawbacks of E. coli as an expression system include its inability to perform post-translational modifications found in eukaryotic proteins, which could be glycosylation, phosphorylation, acetylation, disulfide linkages for proper refolding of protein and/or lack of a secretion mechanism for the efficient release of protein into the culture medium (12) . The production of insulin and human growth hormone was not a problem in E. coli, as these proteins are not glycosylated; however, a large number of therapeutic human proteins are modified by the addition of sugar chains. It is well documented that E. coli lacks the machinery involving hundreds of genes/proteins present in endoplasmic reticulum and Golgi apparatus to perform complex glycosylation. This indicates that the common bacterial expression systems have no capacity to perform N- or O-linked glycosylations as observed in eukaryotes (13) . In addition, one of the major challenges is to obtain a soluble and bioactive recombinant protein, as a large amount of expressed proteins often aggregate into inclusion bodies. This requires extensive solubilization with denaturing agents followed by costly and sophisticated renaturation of the inactive product (14) . There is a further possibility of protein degradation due to host cell proteases and also contamination of recombinant protein preparation with undesirable endotoxins and pyrogens found in E. coli. Furthermore, the codon usage in E. coli is quite different from those in many eukaryotic genes leading to inefficient expression of heterologous genes in E. coli (8) . Nevertheless, many improvements have been made to overcome these above-mentioned shortcomings of protein expression in E. coli (6 , 15 16 17 18) , and a range of pharmaceutical products has successfully entered the market using this expression host.

Yeast as an expression host
Yeasts such as Saccharomyces cerevisiae, Hansenulla polymorpha, and Pichia pastoris are among the simplest eukaryotes that have been used for the production of FDA-approved therapeutic proteins such as insulin, Hepatitis B antigen, desirudin, lepidurin, glucagon, and many more (19) . They can grow quickly to high cell densities on simple chemically defined media and are highly compatible to the fermentation processes. Being a food organism, yeasts do not contain viruses and do not produce endotoxins, which makes them safe for genetic engineering (1) . These yeast strains have been genetically well characterized, and a multitude of differentially regulated and constitutive vector constructs are commercially available. An added advantage is their ability to secrete the target protein, leading to higher recovery of purified protein from broth at significantly lower costs (20) . P. pastoris has given fairly high yields of gelatin, even at an industrial scale (21 , 22) . However, there are also severe drawbacks that need to be addressed; e.g., N-and O-linked glycosylation patterns in yeasts may prove to be different from those in other hosts. S. cerevisiae adds mannose sugars to the structure of basic glycan exported from endoplasmic reticulum, while higher eukaryotes prefer sialic acid O-linked side chains. This leads to hypermannosylation of the glycoprotein that may affect folding, stability, activity, and immunogenicity of the recombinant proteins (23) . By contrast, H. polymorpha and P. pastoris harbor N-linked carbohydrate chains that closely resemble higher eukaryotes, and the extent of hyperglycosylation is lower as compared to the S. cerevisiae. Furthermore, heterologous proteins produced in S. cerevisiae may undergo proteolytic degradation in vacuoles and are specifically coupled to ubiquitin-proteosome system (23) . Nevertheless, several groups have investigated the possibility of reprogramming glycosylation pathways in yeast, by substituting yeast enzymes with human ones, thereby offering an opportunity for the production of complex glycoproteins in yeast (24 , 25) .

Insect cells as an expression host
The baculovirus-mediated insect cell expression is universally recognized as a convenient and versatile tool for heterologous gene expression. Insect cells provide correct folding of the recombinant protein, as well as disulfide bond formation, oligomerization, and other important post-translational modifications as compared to yeast (26) . Proteins may be secreted from cells or targeted to different subcellular locations for appropriate processing. Because the gene of interest is integrated into a nonessential region of viral genome by homologous recombination, large cDNA inserts (up to 15 kb) can be accommodated. Also, recombinant virus can be amplified to better titers, thereby leading to high-level protein production on infection of insect cells (26) . Several modified Autographa californica nuclear polyhedrosis virus (AcNPV) -based transfer vectors combined with Spodoptera frugiperda Sf9 and Sf21 cell lines are commercially available for the expression of functional eukaryotic genes (27) . Baculoviruses are noninfectious and considered safe for humans. Autoantigens, cytochromes, G-protein coupled receptors, and herpes simplex virus capsid proteins have been successfully produced for diagnostic purposes in baculovirus expression system (28) . In addition, a number of products produced in this host have completed late-stage clinical trials and are likely to gain U.S. FDA approval (29) . Despite these advantages, there are a number of major challenges of baculovirus expression system with respect to the composition of carbohydrate modifications of recombinant protein, as compared to the mammalian cells (30) . Essentially, insect cells are unable to produce complex binary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues (9) . Also, the internal proteolytic cleavages at arginine- or lysine-rich sequences appears to be highly inefficient (2) . In addition, the process for simultaneous protein expression and propagation in baculovirus is time consuming and laborious with high operational costs. The growth and culture mediums are very complex, which makes them more expensive as compared to the yeasts or E. coli (1) . Quantitative real-time PCR strategies to determine viral titer can lead to overestimation of active virus due to contamination with cellular material (27) . Late expression of protein in the infection cycle may cause improper folding, thereby leading to aggregates of the recombinant protein in the cell. The stability of recombinant virus has also been a concern as the recombinant expression cassette constructed using bacmid may get lost on subsequent passages in insect cells (31) or on storage (32) . Despite these drawbacks, the insect cell system is the closest to the mammalian cells, and in a serum-free medium, the recombinant protein produced in this host may still pass through clinical development with ease, on its way to meeting the strict FDA standards, and ultimately becoming a therapeutic drug.

Mammalian cells as expression host
Most of the biological products for therapeutic use are expressed in mammalian cells, in particular, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells, as they are capable of performing all the necessary post-translational modifications to produce a functional metabolite or an enzyme (33) . CHO cells have the ability to mimic human glycosylation and produce complex glycoproteins. A further advantage here is that they secrete the recombinant protein into the media in a natural form (1) . These cells are preferred hosts for the production of recombinant proteins for cases in which safety and product authenticity is a major concern, particularly during human therapeutic use. Some of the advancements in expression technologies for therapeutic protein production have been reviewed earlier (16 , 33) . Obviously, there are certain drawbacks with the mammalian cell culture systems. They grow slowly and are resource intensive. The process of generating stable mammalian cell lines is often time consuming and laborious (34) . They are quite expensive to maintain because of their requirement for a complex medium. The yields of recombinant protein are also much lower than those reported for various microbial systems (2 , 22) . The growth conditions are highly stringent, and even slight changes in system parameters can drastically decrease the level of output or lead to other inhibitory metabolites, requiring additional steps in downstream processing (22) . The secreted products may not always be homogenous, like that of yeast, but may contain modified proteins with different glycan moieties and pharmaco-kinetic properties. Thus, it is a major bioprocess challenge to maintain a reproducible composition and ratio of glycoforms in the final product (19) . In fact, differences in glycosylation pattern have been reported in rodent cell lines vs. human tissues (2) . The scaleup from small cell cultures to big fermentors is often critical and remains a challenge for most biopharmaceuticals. Nevertheless, a number of products, including antibodies have been produced from various mammalian cell lines, and various biological entities have been successfully produced and marketed using this expression host (1) .


   WHY CHOOSE Dd?
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
Dd offers unique and attractive features as an expression system for basic biomedical research. It is recommended as a nonmammalian model organism for functional analysis of sequenced genes by the National Institutes of Health, (Bethesda, MD, USA). In addition to its powerful well-established molecular genetics architecture, Dd provides a level of complexity that is greater than yeasts but much simpler than plants or animals (35) . Many of the cellular processes, such as chemotaxis, phagocytosis, cytokinesis, and signal transduction, are observed in Dd but were found to be either absent or have lower activity in other model organisms (36) . In addition, basic development pathways that regulate cell sorting, pattern formation, regulation of gene expression, and cell-type regulation are common between Dictyostelium and metazoans.

Short and simple life cycle
The soil amoeba Dd was first described by Oskar Brefeld in 1869 and was further characterized by Raper in 1935. Dd is a simple eukaryotic microorganism with a 34 Mb genome that contains many conserved genes homologous to higher eukaryotes than to fungi (37) . It is nonpathogenic and the life cycle of Dd is divided into two different phases i.e., vegetative growth phase and development phase. During growth phase, Dd grows as a unicellular amoeba and divides mitotically by phagocytosis of soil bacteria or pinocytosis of growth media. On starvation, amoebae aggregate by chemotaxis to form a mound (a discrete multicelluar assembly surrounded by extracellular matrix) and enter development phase by forming a multicellular organism that differentiates into a fruiting body composed of a cellular stalk supporting environmentally resistant spores (38 , 39) . The spores germinate into amoebas to begin a new life cycle (40) . Under laboratory conditions, the developmental cycle from starving amoebas to the formation of mature fruiting bodies is completed within 24 h. In fact, the generation time in liquid culture is ~10 h, and on bacterial lawns, it is 4 h, suggesting much faster growth rate compared to the mammalian cells. Large quantities of cells can be produced easily in shaken cultures or fermentors in a very short time.

Easy to culture
Dd can be cultured xenically with a bacterial food source or axenically on simple salt medium, with a doubling time of 4–12 h. The axenic medium is a chemically defined medium, which is quite inexpensive and does not require serum or other growth factors (41) . Like E. coli, the aeration conditions required for its growth are simple. The cell lines do not need to be maintained in culture for long, as they can be preserved in liquid nitrogen and recovered by scraping frozen cells directly onto a lawn of bacteria or into axenic medium. Moreover, desiccated spores remain viable on silica gel for years and can be used to revive fresh cultures that attain high cell density within a few hours. As desired, the cells can be harvested from growth or any developmental stage. These cells are easy to lyse as they do not have a cell wall and can be subjected to various biochemical and subcellular fractionation protocols. A schematic overview of protein production in Dd is shown in Fig. 1 .


Figure 1
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  		Figure 1. A protocol for the production of recombinant protein in Dictyostelium discoideum.

Easy mutant selection
The simplicity of the Dd life cycle and haploid genome allows easy mutant selection. With the development of reliable transformation mechanisms, any in vitro modified gene can be reintroduced in Dd (42 , 43) . High-efficiency transformation is generally achieved by electroporation (44) . The transformants are plated on bacterial lawns, and the plaques containing starved Dd cells are picked for mutant propagation. Random mutagenesis protocol allows generation of thousands of individual clones that can be maintained easily just like a microbial fashion (45 , 46) . The secreted form of mutated human choriogonadotropin (hCG) and follicle stimulating hormone (FSH) displayed bioactivities of the same order of magnitude, as the corresponding CHO-generated gonadotropins (46) . In contrast to CHO cells, the generation of a large number of mutants in Dd allows high-throughput screening of mutants for proteins of therapeutic interest. Restriction enzyme-mediated integration (REMI) has been successfully used for isolating genes responsible for cytokinesis, development, and membrane glycosylation (47) . Thus, rapid screening of mutants can be performed for studying the role of exogenous genes and identification of new variants in Dd.

Post-translational modifications
Dd is capable of incorporating both N- as well as O-linked glycosylation to the recombinant protein (48 , 49) . Dd possesses an N-glycosylation pathway and several Golgi-associated O-linked pathways that can mediate phosphoglycosylation (50 , 51) . The existence of a complex and functional cytoplasmic glycosylation system in Dd has emerged from the analysis of cytoplasmic/nuclear protein Skp1, which exhibited the presence of GlcNAc, Fuc, Gal, and possibly Xy1 in the purified protein (52 , 53) . Heterologous protein can also be myristylated or attached to the cell membrane via GPI anchor. The basic structure of N-linked glycosylation in Dd is similar to the mammalian high mannose structure, Man9GlcNAc2. However, Dd lacks the ability to add galactose, N-acetyl galactosamine, or sialic acids often observed on complex N-linked glycans of the mammalian system (54) . The study of O-linked glycosylations of Muc1 and Muc2 showed that N-acetyl galactosamine residues were replaced by the N-acetyl glucosamine in Dd (48) . Serine- and threonine-containing peptides are glycosylated in Dd at the same residues as in higher organisms (including humans), but the sugars added may not always be the same (55) . Biologically active hIGFBP6 (human insulin growth factor binding protein 6) was also produced in Dd, but the glycosylation was either absent or was found to be incorrect (56) . Several therapeutic glycoproteins have been successfully expressed in Dd, including rotavirus VP7, muscarinic receptor m2 and m3, antithrombin III, gonadotropins, erythropoietin, and many others, as discussed elsewhere in this review.

Codon usage
The 34 Mb genome of Dd has an AT content of >75%, with codon usage favoring adenine or thymidine in the third position. The genome contains relatively rare introns and promoters that are more than 90% AT rich, distinguishing them from GC-rich coding region of genes. In contrast, the human genome has an AT content of 60% and may contain genes with codons that are suboptimal for expression in Dd. Under certain circumstances, optimization of preferential codon usage and Kozak adaptation can lead to 6- to 8-fold increase in recombinant protein production in Dd (57) . Minimizing the number of rare codons and using Dd authentic signal sequence and stop codon were also found to be helpful for enhanced expression of certain proteins (46 , 58 , 59) . Thus, the proteins with similar codon preference, which are difficult to express in other systems, can be easily produced in Dd.

Economics
For any recombinant protein expression at large scale, a major concern is the cost of production. Preferred expression hosts must make desirable levels of proteins in a cost-effective fashion. Dd offers great economic advantage over other eukaryotic expression systems (55) . As mentioned above, Dd culture requires simple medium with minimum additional expense. Dd cells can grow to very high cell density (1011 clonal cells) in a few days without any sophisticated equipment. This allows production of milligram quantities of recombinant protein in much shorter time frame as compared to the mammalian cells. As the culture maintenance is not required for long, the cost of FTE (full time employee) requirement per protein production is heavily reduced compared to other expression systems (Table 1) . Most of the recombinant proteins are secreted into the medium, making the downstream process much simpler and easy to maneuver.

Extensively studied model organism
Dd has been extensively used as a model system to study basic cellular and developmental processes such as chemotaxis, phagocytosis, differentiation, and patterning (36 , 60 , 61) . Being a cooperative and genetically tractable phagocyte, Dd has been widely used for analyzing bacterial and fungal virulence (62 63 64 65 66) . The vacuolar cell death phenomenon (vegetative cells differentiating into dead vacuolated stalk cells) in Dd is caspase independent and, therefore, extensively explored to study apoptosis in this model organism (67) . The identification of at least 33 genes orthologous to human disease genes in Dd genome has provided an opportunity to investigate molecular mechanisms underlying human diseases (68) . In fact, the attractive biology and availability of powerful molecular techniques have enabled us to exploit Dd for drug discovery research and also for determining the mechanism of action of new molecules (69 70 71) .


   EXPRESSION VECTORS AVAILABLE IN Dd
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
Dd is one of the rare eukaryotes that expresses extrachromosomal circular nuclear plasmids. Unlike yeast, these plasmids are packaged in a nucleosomal structure similar to the chromatin organization of higher eukaryotes (2) . The recombinant proteins are expressed extrachromosomally with high copy number plasmids, and two plasmids, Ddp1 and Ddp2, are well characterized (72 , 73) . Various expression vectors have been constructed using either Ddp1- (72) or Ddp2-based extrachromosomal replicon (73) . In addition to these two categories, integrating plasmids have been engineered that integrate into Dd chromosomes (74) . Ddp2-based plasmids require an ORF product in trans for plasmid maintenance and ~600 bp origin of replication in cis. Therefore, these plasmids are either transformed in AX3 cells containing integrated copies of ORF gene or cotransformed with pREP plasmid expressing ORF gene (75) . A wide selection of stable cell lines, plasmid, and expression vectors are available to facilitate analysis of protein function (www.dictybase. org), and some of these have now become available from commercial sources as well.

Constitutive expression vectors
For enhanced protein expression, a typical Dd cloning vector carries an expression cassette consisting of strong constitutive actin-15 promoter, translational start site, multiple cloning site, affinity purification or visualization tag, Dd polyadenylation, and termination signals. Dittrich et al. (76) described a constitutive actin-15 promoter-based expression vector that contains pspA signal sequence for efficient secretion of recombinant protein. Furthermore, vectors containing N-or C-terminal polyhis, Glu-Glu-Phe, or c-myc tags were also designed (72 , 73) . This is now followed by construction of vectors with Flag-, Strep- or GST- for affinity purification and GFP-, YFP-, CFP- tags for visualization in living cells (77) . Therefore, it was possible to monitor the dynamics of cytoskeletal proteins using dual-wavelength fluorescence microscope with RFP and GFP tags (78) . Moreover, myosin motor domain and MBP tags have been utilized for purification of recombinant proteins expressed in Dd (79 , 80) . This led to the development of new expression vectors containing octahistidine followed by myosin motor domain tag for large-scale protein production (75) . Recently, a series of expression vectors containing tandem affinity purification (TAP) tags have been described that contains a protein A tag and a calmodulin-binding peptide for purification of native protein and protein complexes (81) .

Regulatable/inducible expression vectors
Inducible expression systems are valuable tools for the study of function of cytotoxic proteins. However, very few regulatable gene expression vectors are available in Dd. The most widely used regulated gene expression system is based on repression of discoidin I{gamma} promoter in the presence of folate (82) . Discoidin-tag expression vectors resulted in high yields of recombinant proteins (83) . For the expression of cell surface protein, the Dd leader peptide sequence is linked to a desired protein sequence under the control of discoidin I promoter, and successful membrane expression of circumsporozoite protein and muscarinic receptor m2 protein has been observed by two different laboratories (59 , 84) . Another conditional expression system based on bacterial tetracyclin repressor/operator system that controls expression of Amber suppressor tRNA gene has been described (85 , 86) . Inducible transcriptional regulation has also been studied using tetracycline-controlled actin promoter. Removal of tetracycline led to induction of gene expression by severalfold, and the presence of even low levels of tetracycline inhibited the gene expression efficiently (87) . A UV-inducible ribonucleotide reductase (rnrB) promoter-based regulated gene expression system can also be used for studying the expression of specific genes at all stages of Dd life cycle (88) .


   THERAPEUTICALLY IMPORTANT PROTEINS PRODUCED IN Dd: KEY LESSONS LEARNED
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
As discussed above, Dd offers many advantages as an expression system. This has encouraged scientists to express therapeutically important recombinant proteins in this host. Its ability to perform post-translational modifications and secrete proteins at high levels has given opportunity to explore recombinant glycoprotein expression in Dd. In fact, some of the complex recombinant human proteins of therapeutic importance have been obtained from Dd in large quantities at much lower cost as compared to the mammalian cells (89) . The recombinant proteins expressed in Dd are listed in Table 2 . Description of some of the studies of human proteins expressed in Dd and the process of their customization for developing standard operating procedures are given below.


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  		Table 2. List of recombinant proteins expressed in Dd

1. Human antithrombin III. Antithrombin III is a 58-kDa glycoprotein that inactivates several enzymes of coagulation system pathway, including thrombin, Factor IXa, and Factor Xa. This protease inhibitor is produced by the liver and secreted into the plasma. It contains three disulfide bonds and four N-linked carbohydrate moieties. A biologically active form of recombinant antithrombin III was isolated from CHO cells that displayed biological activity similar to the human plasma protein. However, antithrombin III expressed in yeast showed reduced heparin cofactor activity. As early as in 1991, human antithrombin III was expressed in glycosylated form in Dd (90) . This protein was secreted into the culture broth, suggesting recognition of authentic human preantithrombin signal peptide sequence in Dd. More important, the stationary cultures produced up to 1 mg/l antithrombin protein within 24 h and were able to efficiently secrete protein for as long as 2 wk, suggesting the possibility of a continuous production system in this organism. However, antithrombin III showed aberrant pattern of glycosylation from that of plasma antithrombin III and reduced heparin cofactor activity, which led to its limited use subsequently.

2. Human muscarinic receptor m2. Human muscarinic receptor m2 belongs to the family of G protein-coupled receptors and transduces cell signaling cascade on activation via adenyl cyclase inhibition. A number of m2 agonist/antagonists are under development as drug targets for Alzheimer’s, Parkinson’s, and chronic obstructive pulmonary diseases. For high-throughput screening of muscarinic agonists/antagonists, Dd was exploited as biological model by expressing functional m2 receptor on the plasma membrane with binding characteristics similar to authentic receptors (84) . This system is highly economical, reproducible, and subject to easy automation as compared to the mammalian cells. Although the number of human m2 receptors expressed per Dd cell (3000 receptors/cell) were less compared to homogenous cAMP receptor (500,000 receptors/cell), the number could be enhanced by using preferential codon bias for human m2cDNA. Thus, this system offers a great screening tool for m2 receptor agonist/antagonists.

3. Human gonadotropins. Human gonadotropins are widely used clinically for the treatment of infertility. hCG is an important therapeutic protein involved in the maintenance of early pregnancy, and along with FSH stimulates the maturation of germ cells. Both of these proteins are highly complex glycoproteins with N-linked glycosylations on two noncovalently associated {alpha}-subunits and β-subunits. A single-chain hCG was produced in Dd as an immunologically active protein that can bind to the human LH/CG receptor and elicit biological response (46 , 91) . The receptor binding affinity of hCG expressed in Dd was found to be comparable to that observed in mammalian cells. These two studies suggest that Dd possesses a large variety of chaperones and folding enzymes necessary to perform all the post-translational modifications for biological activity of glycoprotein hormones. However, hCG derived from Dd has lower biologically activity compared to that produced in CHO cells. This could be due to small differences in the glycosylation patterns, as seen earlier for human antithrombin III. FSH was also successfully synthesized and secreted by Dd. The product binds its human receptors and elicits biological response, suggesting that FSH can be folded and properly assembled in Dd (46) . In addition, gonadotropin mutants were generated by random mutagenesis in a much easier and cost-effective manner (91) .

4. Growth factors. Growth factors and their binding proteins play a major role in regulating normal physiology, as well as pathological states. Their production in E. coli or other prokaryotes is usually cumbersome due to the requirement of post-translational modifications. Among the various growth factors, hIGFBP6 and bovine beta cellulin (bBTC) have been produced in stably transformed Dd (56) . As discussed earlier, hIGFBP6 is modified by O-linked glycosylations, and the protein expressed in Dd was also glycosylated, suggesting that O-linked glycosylation takes place in Dd system. However, the molecular weight of Dd-expressed protein was found to be 3 kDa less than the protein produced in CHO cells, suggesting a different glycosylation pattern. However, this did not seem to affect the protein activity, as both of the proteins, hIGFBP6 and bBTC (requires N- and O-linked glycosylation), were biologically active.

5. Human Fas ligand. Human Fas Ligand (hFasL) is a 37-kDa glycoprotein that belongs to the tumor necrosis factor family and plays important roles in cancer and autoimmune diseases. The hFasL was successfully produced as a bioactive form in Dd (92 , 93) and purified using single-step Ni-affinity chromatography with a recovery of 92% and purity of 91%, respectively (94) .

6. Human erythropoietin. Erythropoietin (EPO) is a heavily glycosylated hormone that is used for the treatment of anemia during chronic renal disease or cancer (95) . The commercial recombinant EPO is produced from CHO cells that show correct post-translational modifications with one O-linked and three N-linked glycosylated residues. The glycosylated form of EPO was also successfully expressed in Dd (96) . However, the glycoforms appear to be homogenous in nature as in trypanosomes or yeasts, and unlike CHO cells, where the N-glycan chains are heterogenous. Although the protein is properly glycosylated, the pattern may be different as compared to the protein produced in mammalian cells. Whether this difference in glycosylation will affect the bioactivity of EPO remains to be deciphered.

7. Human phosphodiesterase. Phosphodiesterases (PDEs), in particular, cAMP-specific PDEs have been viewed as an effective therapeutic target in number of inflammatory diseases, including asthma and chronic obstructive pulmonary disease (COPD). Several PDE4 and PDE7 dual inhibitors are being investigated as potential drugs for the treatment of anti-inflammatory diseases. Although the catalytic domains of PDE4 and PDE7 have been expressed in E. coli and baculovirus, there are technical hurdles in expressing full-length proteins. Expression of full-length PDE isoforms is essential for HTS assays for the identification of PDE subtype selective inhibitors that would offer better efficacy profile over existing molecules. We have successfully expressed and purified active recombinant human PDE4B2 in Dd that showed similar kinetic profile, as compared to PDE4B2 expressed in mammalian cells (97) . Recombinant human PDE7A1 was also expressed in a functionally active form in Dd, and up to 2 mg/l of full-length protein was purified from cell lysates (98) . Production of both full-length proteins on a large scale has greatly expedited the screening of their inhibitors in HTS. Similarly, we have been successful in the expression of the full-length recombinant hPDE4D3 in Dd as GST fusion protein of 102 kDa (Fig. 2A ) (unpublished observations). The expression was further confirmed by immunofluorescence observed in the cells expressing PDE4D3 using specific antibodies. Untransformed cells did not show any specific fluorescence (Fig. 2B ). The expressed protein displayed kinetics and behavior toward inhibitors similar to the protein expressed in mammalian (HEK 293) cells. The Km of the enzyme was found to be 1 ± 0.02 µM. A comparison of protein yields that are expressed in different expression systems from our laboratory is shown in Table 3 . We observed higher yields of PDEs full-length protein in Dd compared to mammalian cells. Another recombinant human protein spleen tyrosine kinase (Syk), anticancer target, was successfully expressed in Dd, as well as baculovirus with comparable yields. However, the cost per mg of Syk produced in Dd was 4 times lower compared to baculovirus (unpublished observations). Thus, Dd offers an excellent expression host for bulk production of full-length recombinant PDEs and related proteins.


Figure 2
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  		Figure 2. Expression of recombinant human phosphodiesterase 4D3 in Dictyostelium discoideum (Dd). The PCR product of full-length human PDE4D3 (accession number L20970) was cloned in pDXA-GST vector at XhoI and XbaI sites. The recombinant construct was used for transforming Dictyostelium wild-type cells and the transformants were selected using G418 selection marker. A) The cell lysates of clone numbers 4–10 were screened for recombinant hPDE4D3 expression by immunoblotting with anti-PDE4D3 antibody, Un: untransformed cell lysate. B) Expression of recombinant hPDE4D3 was confirmed by immunofluorescence i) phase contrast, wild type; ii) green fluorescence, wild type; iii) phase contrast, transformants stained without anti-PDE4D3; iv) green fluorescence, transformants stained without anti-PDE4D3; v) phase contrast, transformants stained with anti-PDE4D3; and vi) green fluorescence, transformants stained with anti-PDE4D3.


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  		Table 3. Comparison of protein yields expressed in Dd vs. other expression system

Besides these human proteins of therapeutic importance, several proteins from genetically diverse organisms have been successfully expressed in Dd as recombinant proteins; for example, S. japonicum GST, E. coli β-glucuronidase (GUS) (76 , 99) ; P. falciparum circumsporozoite protein (59 , 100) ; rotavirus SA11 protein VP7 (101) ; A Victoria GFP (102) ; Arabidopsis aquaporin (103) ; avian Na-K-ATPase (104) ; rat Glut 1 (105) and P. vivax ortholog of pfcrt (106) . In addition, milligram quantities of isotopically labeled recombinant glycoproteins were produced from Dd, which were further subjected to the NMR studies (107) . This clearly indicates that Dd has the potential to overexpress a wide variety of recombinant proteins from different organisms in their functional and bioactive forms and deserved further exploitation at industrial levels.


   CHALLENGES AND PITFALLS
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 
Although Dd offers many advantages as an alternative eukaryotic host, there are a few limitations to this system. Dd has the potential to perform N- and O-linked glycosylations similar to higher eukaryotes, but the glycosylation pattern tends to vary. Poor understanding of the glycosylation regulatory machinery hinders the development of mutants or transformants that can artificially substitute the glycosylating enzymes, as done successfully in yeasts. Industrial-scale protein production has not yet been achieved in Dd, whereas a number of biological entities are in market or under clinical development, in which the expression host was either E. coli or yeast or mammalian cells. For industrial-scale production of recombinant proteins in Dd, further optimizations in bioprocess and fermentation technology are required. It will be interesting to exploit this organism for the production of recombinant or humanized antibodies. However, FDA approval of recombinant protein may be a challenge, as there is no protein to date under consideration by any regulatory body worldwide or even in clinical development produced in this host. Whether the recombinant protein expressed in Dd will raise any immunogenic response in humans remains to be seen. Therefore, manufacturing, regulatory, clinical development, and related issues need to be sorted out before any of the therapeutic protein or biological entities produced in this expression host actually gets the FDA nod for its marketing.


   INDUSTRIAL-SCALE ADVANCEMENTS TO OVERCOME BOTTLENECKS
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
CONCLUSIONS
 REFERENCES
 
To exploit Dd at the industrial level, efforts are under way to maximize cell densities in suspension culture by varying media composition and optimizing culture parameters. The influence of medium components on cell density and doubling time of Dd has been investigated to identify critical nutritional factors limiting its growth kinetics (108 , 109) . It has been suggested that the quality of peptone greatly influences the growth of Dd (110) . More than 50 kinds of mutant strains (AX2, AX3, AX4, etc.) have been generated that can feed on soluble axenic media, as opposed to the wild-type Dd that feeds on bacteria, so as to meet the regulatory requirements of pharmaceutical industry. A synthetically modified FM medium was developed that could lead to cell densities in excess of 5 x 107 ml–1 compared to the standard axenic complex media (111) . Furthermore, mass cultivation of Dd was achieved by batch and fed batch operation in conventional stirred-tank type bioreactors for industrial purposes (112) . This led to a shift in strategy to immobilize Dd on porous carrier particles developed to cultivate Dd more efficiently (113) . Batch and continuous cultivation of Dd in the immobilized state resulted in much higher cell densities (10–15 times higher) than those observed in submerged culture conditions (114) . The immobilized cell densities could be kept constant for a longer duration by repeated renewal or continuous feeding of synthetic media (92) . In addition, the density of cells in suspension culture was improved by means of continuous cultivation of Dd in a bioreactor with cell retention through microfiltration and low space velocity (92) . Clearly, the exploitation of Dd expression system for the overproduction of therapeutically important proteins for the new drug discovery research has achieved quite a few milestones. We need to further characterize this host for the production of new biological entities (NBEs) and biological products that could one day be approved by the FDA for clinical use.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
REFERENCES
 
In the past 10 years or so, considerable advances have been made in developing Dd as a model system for pathogenesis, biomedical research, and new drug discovery research. Dd has also been extensively utilized as an expression host for more than 10 complex glycoproteins in their functional form. The expression of a eukaryotic protein in Dd is reliable, reproducible, and amenable to automation as compared to the mammalian cells. Dd cell culture conditions are simple, inexpensive, less time consuming, and not as labor intensive as observed in the mammalian cells, thereby making it an attractive host for eukaryotic protein expression. Furthermore, successful expression of rotavirus vaccine candidate, SA11 protein VP7, and malarial subunit vaccine circumsporozoite protein in Dd makes it an ideal production system for recombinant subunit vaccines. Our own experience and that of others clearly points to a need of a lot of further developmental work, both preclinical and clinical, on the biological entities expressed in this host, before any of them makes it to the clinic.


   ACKNOWLEDGMENTS
 
We thank Dr. Pradip Bhatnagar for financial support and encouragement on the Dictyostelium expression system project at Ranbaxy. We thank Ms. Sudha Naithani for technical assistance on PDE4D3 work. We thank Ms. Saima Aslam, Ms. Chetali Sachdeva, and Ms. Sarada Lekshmi for literature assistance.


   FOOTNOTES
 
1 Current address: School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India.

Received for publication May 22, 2008. Accepted for publication July 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPRESSION SYSTEMS
 WHY CHOOSE Dd?
 EXPRESSION VECTORS AVAILABLE IN...
 THERAPEUTICALLY IMPORTANT...
 CHALLENGES AND PITFALLS
 INDUSTRIAL-SCALE ADVANCEMENTS TO...
 CONCLUSIONS
 REFERENCES
 

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