Barrie C. Rooney

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs)

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials.

Characterisation And Analysis Of Human Interferon-Y Glycoforms Produced In Baculovirus Infected Spodoptera Frugiperda (SF9) And Estigmena Acrea (EA) Cell Lines

A large percentage (~50%) of the human therapeutic proteins that are of clinical relevance are glycosylated [1] and these carbohydrate moieties can have a great influence on efficacy, antigenicity, and circulatory half life of the protein. There can be considerable diversity in the carbohydrate structure attached at each glycosylation site. Several factors are known to affect the micro-heterogeneity that is observed; the cell line, the availability of sugar residues, the energy state of the cell, or the amount and type of glycosyl transferases present. Of these factors the least understood is the effect of host cell type on the glycosylation. Baculovirus infected insect cells are being used for the production of human therapeutic proteins. In the study presented here we investigate the glycosylation capability of two insect cell lines to produce glycoproteins.

Expression of Recombinant Receptors in Insect Cells

Membrane bound receptors are key molecules in intracellular communication systems but structure function studies are often hampered by low level of native expression of these proteins. An essential feature of any recombinant system used to over express receptors is the authenticity of the recombinant product. The Baculovirus/insect cell expression system BEVS has been widely used to express many mammalian proteins as it has been shown to carry out many eukaryotic post-translational modifications. This paper will describe its use to over express members of the single transmembrane domain, tyrosine kinase receptor family (PDGF-receptors), and members of the G-protein linked, seven transmembrane domain family (Dopamine receptors). In particular production and activity studies on full length and truncated forms of the PDGF-receptors and comparisons made with mammalian expressed constructs will be discussed. A full pharmacological profile of recombinant dopamine receptors will be presented and the significance of variations between sub-types will be discussed in the context of possible physiological significance.

EXPRESSION OF THE D2 AND D3 DOPAMINE RECEPTORS IN INSECT CELLS USING THE BACULOVIRUS SYSTEM

The rat D2 and D3 dopamine receptor isoforms have been successfully expressed in Spodoptera frugiperda cells to a high level using the baculovirus system. Both recombinant receptors have been examined at the ligand binding level to ascertain their biological integrity and thus their suitability for use in structure / function studies. Ligand binding profiles for the D2 and D3 receptors expressed in Sf-21 insect cells have been compared to those generated from recombinant receptors in Chinese Hamster Ovary cells. The D3 receptor appears to conform to the results predicted from these studies. However, the D2 receptor expressed in insect cells deviates from the profile expected.