Production of recombinant proteins from protozoan parasites

Abstract

Although the past decade has witnessed sequencing from an increasing number of parasites, modern high-throughput DNA sequencing technologies have the potential to generate complete genome sequences at even higher rates. Along with the discovery of genes that might constitute potential targets for chemotherapy or vaccination, the need for novel protein expression platforms has become a pressing matter. In addition to reviewing the advantages and limitations of the currently available and emerging expression systems, we discuss novel approaches that could overcome current limitations, including the 'pseudoparasite' concept, an expression platform in which the choice of the surrogate organism is based on its phylogenetic affinity to the target parasite, while taking advantage of the whole engineered organism as a vaccination adjuvant.

Figure 1

(a) Resolved crystal structures of Plasmodium genes. PDB database (www.pdb.org, May 2009) was searched for Plasmodium protein resolved structures and plotted against the year deposited (bars: number of structures deposited in a given year; line: cumulative structure deposited numbers). After the sequence of the genome, there were two high-throughput efforts that resulted in a limited increase in the number of structures resolved. Overall, recombinant protein production remains a bottleneck for many scientific endeavors. TBR: to be released. (b) Heterologous protein expression systems for parasites. A PubMed search of recombinant proteins from parasites in the past seven years reveals that prokaryotic systems are still the workhorse, followed by insect cells/baculovirus. Interestingly, protozoan organisms are used more often than yeast. Cell-free systems are just starting to take off. (c) Solubility as a function of construct length; Ref. [32] is the original source. The overall success rate for soluble recombinant proteins remains very low, especially for protein targets with lengths > 800 amino acids [32]. Dotted line, fraction of cloned targets resulting in successful large-scale purifications. Dashed line, fraction of soluble clones at a 1 ml scale resulted in pure protein at large scale. Solid line, fraction of purified proteins resulting in successful crystal structure determinations.

Figure 2

Most frequently used systems for the production of recombinant protein from parasites. After the selection of the gene of interest, an extensive in silico analysis should corroborate the presence of protein domains, secretion signals, organellar targeting, and post-translational modifications. These characteristics and the use of the recombinant protein will help guide the selection of the cloning vector and expression system. The gene of interest can be directly cloned, often under a strong promoter, into a suitable vector to express in a homologous system or in a surrogate system, ideally, the closest related species that can be grown in large quantities and is amenable for genetic manipulation. When no in vitro culture is available or the scale needed precludes the use of the homologous system, the bacterial system is often the first choice. General guidelines include removing predicted membrane-spanning regions, avoiding disrupting predicted secondary structural elements, respecting the boundaries of globular domains, if known; and avoiding the inclusion of low-complexity regions or hydrophobic residues at the N- and/or C-termini. When post-translational modifications in the recombinant protein are needed for function or antigenicity or the bacterial systems are not performing as expected, eukaryotic systems are available either using a surrogate organism or cell-free system. In either case, and especially for systems that have been in the market for long time, there is a suite of clones, strains, culture conditions, and tags that can be tested during the optimization process.

Figure 3

The ‘pseudoparasite’ concept. The evolutionary relationships between organisms provide us with a ‘magnifying glass’ to select those with shared structures, mechanisms, and perhaps shared processes required for protein function. Therefore, ‘phylogenetically tailored’ heterologous expression systems might lead to the development of novel alternative platforms for expressing recombinant proteins from parasites. The first step would involve the selection of the closest relative to our organism of interest that shares the targeted structure/pathway/gene. Ideally, this organism would be amenable for genetic manipulation and for large-scale culture in axenic conditions and in a cell-free defined medium. In the second step, the cloning vector (cytosolic, organelle targeting, surface, secretion) will be selected depending on the predicted native targeting, and the potential applications of the recombinant protein, ranging from diagnostic tool development to screening and drug profiling. Taking this phylogenetic relationship further, the engineering of a pseudoparasite might be carried out in an organism that expresses genes that are homologous to those in the parasite of interest. These will enable the correct folding and post-translational modifications that take place in the target organism, and that are necessary to elicit a specific, effective immune response, thus, providing a multi-antigen, up-scalable vaccine delivery platform. This approach would enable the identification and selection of the best combination of multiple immunogens to be expressed in their immunologically relevant conformation by the pseudoparasite, while taking advantage of the whole engineered organism as an adjuvant. Hence, the pseudoparasite displaying the selected parasite antigens can be directly tested for the ability to induce an effective immune response in the absence of adjuvants. Gateway systems and alike can also be adopted to incorporate any gene of the organism of interest to the pseudoparasite platform. The example depicted focuses on multiple Plasmodium stages (intra-erythrocytic stage omitted from the diagram) from which candidate genes selected for a malaria vaccine are integrated into the pseudoparasite. Other genes of interest identified in future studies could be incorporated to develop a platform expressing as many genes as necessary to provide an effective vaccine.