Lu Yinghua ^{1, 2*} , Wu Xiaoxia ¹, Xu Zhinan ³, Li Qingbiao ^{1, 2}, Deng Xu ¹
(¹ Department of Chemical and Biochemical Engineering, Xiamen University, Xiamen 361005;  ² Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005; ³ Institute of Biochemical Engineering, Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China )

Received Jun. 20, 2004; Supported by the National Natural Science Fund (No. 20306025，30370039)

Advances in genetic engineering have made possible the production of therapeutics and vaccines in the form of recombinant proteins for the treatment of many ailments like cancer, tumors, hypertension and AIDS. Construction of recombinant strains/cells using genetic technology and production of target proteins through cultivation of recombinant strains/cells have become increasing interested in modern biotechnology, which are widely used in production of heterologous proteins and metabolic engineering. At the current time frequently used expression host systems are prokaryotic bacterial system and eukaryotic yeast, insect, mammalian cells. However, these expression systems have different degrees of limitations for overexpression of eukaryotic genes.
    In recent years, the single-celled amoeba of Dictyostelium discoideum(Dd) has emerged as a promising eukaryotic alternative system for the expression of recombinant proteins of high value^[1]. In this article the advantages of Dd in comparison to other expression systems and advances in Dd for expression of heterologous proteins are reviewed.

1. Limitations of the general prokaryotic and eukaryotic systems in the biosynthesis of heterologous proteins
The prokaryotic bacterial system is by far a widely employed host. It has two distinct advantages, that is, it is generally easy to handle and can grow very rapidly to high cell densities in simple and cheap media, which make it an ideal tool for expression of many proteins. However, there are serious limitations in using bacteria for the production of eukaryotic proteins. Common bacterial expression system such as E. coli is not capable of post-translational modifications, such as phosphorylation, acylation, N- and O-linked glycosylation. These modifications affect bioactivity, function, structure, solubility, stability, half-life, protease resistance and compartment of proteins requiring post-translational modifications^[2,6]. In addition, the bacteria have low levels of protein secretion. Although some recombinant proteins can be secreted into the periplasm, in general it is impossible for proteins to be secreted into the extracellular medium. Protein expressed in large amounts often precipitates into insoluble aggregates called inclusion bodies, from which it can only be recovered in an active form by solubilization in denaturing agents followed by careful renaturation. This complicates downstream process, especially by large-scale production of heterologous recombinant proteins. Lysis to recover the cytoplasmic proteins often results in the release of nucleic acids, pyrogens, endotoxins and liposaccharides, which must be removed from the final product. In addition, compared with the extracellular circumstance, the concentration of proteases is higher inside the cells. So the activities and yields of the product may be influenced by the proteases. Therefore, it is not suitable to use the bacterial system for glycoproteins expression.
    Yeast is a favored lower eukaryotic system for the expression of foreign proteins. As a food organism, yeast does not contain viruses and does not produce toxins, which makes it a safe system in genetic engineering. Its gene expression and control mechanism is fully understood and it can be manipulated readily as well. In addition, it can grow rapidly (doubling time: 90 min) on simple media and to high cell densities. Also, yeast can secret the target protein into the media. Although this lower eukaryotic system is able to glycosylate the target proteins, both N- and O-linked oligosaccharide structures are significantly different from their mammalian counterparts. Hypermannosylation (additon of a large number of mannose residues to the core oligosaccharide) is a common feature in yeast, hindering proper folding and therefore the activity of the protein. Hence, the production of glycoproteins by this expression system for use as human therapeutics is unattractive^[2].
    Baculovirus-infected insect cells such as Spodoptera (SF9 or SF21) show some features, which make them become popular eukaryotic expression systems for overproducing recombinant proteins^[3]. Being eukaryotes, they use many of the protein modifications, processing, and transport systems present in higher eukaryotic cells. Baculoviruses can be propagated to high titers in insect cells, making it possible to express large amounts of recombinant proteins with relative ease. Expressed proteins are usually expressed in the proper cellular compartment, i.e. membrane proteins are usually localized to the membrane, nuclear proteins to the nucleus and secreted proteins secreted into the medium. Viral genome is large (130kb) and thus can accommodate large fragments of foreign DNA. However, expression using baculoviral vectors also has some limitations with respect to some post-translational modifications, e.g. internal proteolytic cleavages at arginine- or lysine-rich sequences are highly inefficient. The glycosylation capability of insect cells is generally limited to producing only high mannose type and not processed to complex type oligosaccharides containing fucose, galactose and sialic acid. In addition, insect cells grow slowly and usually require expensive media, which makes cultivation expensive and also limits its commercial use.
    Despite of many shortcomings, mammalian cells are still the ideal expression systems to express some complex-structured large-molecule proteins, especially those pharmaceutical proteins whose conformation and bioactivity depend on mammalian post-translational modifications like glycosylation and phosphorylation, as they offer the greatest degree of product fidelity. However, oligosaccharide processing is species- and cell type-dependent among mammalian cells. Differences in glycosylation pattern are reported in rodent cell lines and human tissues^[3]. Even the use of human cell line is not perfect, since the transformation event required in most cases to produce a stable cell line may itself result in altered glycosylation profiles. Also mammalian expression techniques are time consuming, and mammalian cells grow very slowly and the products are likely to be contaminated by viruses, thus it is much more difficult to perform cell cultivation on a large scale. In addition, complex nutrient requirement and low product concentration make the end product extremely expensive.

3. Advances of the expression of heterologous proteins in Dictyostelium discoideum    
As Dd is an attractive model system for studying cell and development biology, scientific efforts were expended on the techniques for culturing Dd cells on axenic medium in the 1960's and 1970's. A variety of axenic medium were exploited and more than 50 kinds of axenic strains (AX2, AX3, AX4) were obtained, which can grow in axenic (liquid) medium in suspension culture. Since 1970's, many researches have been carried out in the genetics and development biology of Dd. Several families of high copy number nuclear plasmids were found in Dictyostelium species, and two plasmids (Ddp1 and Ddp2) were used to construct expression vectors. In the meantime, shuttle vectors were constructed that allow replication in E. coli as well as expression in Dd at low or high copy number, among which the high copy number vectors replicate in the form of extrachromosomal vectors, while the low copy number vectors are integrated into the Dd chromosomes. At the late eighties and early nineties of last century, the Dd system was validated by expression of homologous genes, which made this system very powerful in studies on the cytoskeleton, signal transduction and multicellular development, etc. The most notable achievement was the expression of a 380 kDa myosin protein, one of the biggest proteins to be genetically engineered hitherto ^[9]. Such research has laid a good foundation for the expression of heterologous proteins in this system.
    Since 1990's, researchers have begun to carry out the expression of heterologous proteins in Dd. Firstly, they found that the Dd genome shows a very AT-biased codon usage, that is, the Dd genome has high AT content. It is reported that minimizing the number of rare codons of the first ten codons at the N-termini of the target gene can remarkably improve expression levels. Secondly, they found that an authentic Dd signal sequence is able to lead to secretion of foreign protein^[6]. Finally, they constructed a series of shuttle vectors that allow replication in E. coli as well as expression in Dd. After almost ten-years' work, about ten kinds of heterologous proteins have been successfully expressed in Dd by the biologists in America, Germany, Britain, Japan, Australia, Holland and China, which are mainly the complex parasitic, viral and human (glyco)proteins. Those proteins include the Plasmodium falciparum circumsporozoite antigen, human antithrombin III, Rotavirus SA11 protein VP7, human muscarinic receptor gene m2, human choriogonadotropin, human gonadrotropin, Schistosoma japonicum glutathione S-transferase, E. coli b -glucuronidase, a soluble form of the D. discoideum surface membrane protein-PsA and human soluble Fas ligand (Table 1).

4. The problems in Dd as an expression system          
Although in the past ten years, about ten kinds of complex glycoproteins in a functional form have been successfully expressed in Dd, there are still some problems such as the mechanism of high level expression and the glycosylation features need to be elucidated, and strategies for the high cell density cultivation of Dd should be developed. Firstly, expression levels of target proteins expressed by Dd are comparable to or even higher than those of mammalian cell lines (such as CHO), but they are much lower than those of other microorganisms like bacteria and yeast. Therefore, regulation and expression units of expression vector still have to be optimized. Secondly, although Dd is capable of performing a whole spectrum of post-translational protein modifications, little is known about the regulation and the machinery of glycosylation, which disfavours the understanding of glycosylation machinery and the realization of artificial control of glycosylation. Thirdly, although Dd cells can grow either on bacteria or axenic medium without any special factors, showing advantages over animal cells, it is reported that the maximal cell densities of Dd growing in suspension culture or feeding on bacteria are only in the range of 1-2×10⁷ mL^-1, which is a low cell density in comparison to other microbial expression systems. Little attention has been paid to improve its cultivation so far. The difficulty in mass cultivation of Dd has become the main obstacle that restricts Dd to become general expression system ^[31]. It is commonly assumed that the cell density of Dd is regulated by some autocrine factors secreted by Dd during the vegetative growth phase, like prestarvation factor (PSF) and the conditioned medium factor (CMF) ^[16].
    Another main factor that disfavours the application of this expression system is slow growth rates in the presence of axenic (liquid) media. Although Dd cells can grow on bacteria with a doubling time of 3-4 h, this would certainly not conform with reglementations in the pharmaceutical industry. Dd cells can grow on complex and semi-synthetic medium with a doubling time of about 8-12 h or on synthetic FM medium with a doubling time of about 12-14 h. Therefore, the application of this system is seriously affected by slow growth rates as well as low maximal cell densities. Recently, some researches have been done in respect of high cell density cultivation of Dd. Stephan et al.^[17] investigated the influence of certain medium components of complex media on cell density and doubling time. Beshay et al.^[18] have reported that Dd might be immobilized by entrapment of cells inside porous carriers. Cell densities in the pores of the supports were reached 1-2×10⁸ mL^-1, which were about 10-15 times higher than those in submerged culture. Also, a synthetic modified FM medium, called SIH medium, yielded much higher cell densities (5×10⁷ mL^-1) than axenic complex media was reported by Han et al^{[19, 20]}. The suspension cell density can be further improved by means of continuous cultivation of Dd in a bioreactor on SIH medium with cell retention through microfiltration^[15]. At low space velocity very high cell densities of up to 2.4×10⁸ mL^-1 were achieved. However, the growth rate of Dd on SIH medium was quite low----a doubling time of 14-16 h was usually observed. In consequence, it is still essential to develop new strategies for the mass cultivation of Dd in order to improve its productivity.

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