Kinetoplastid protein expression system and methods

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BACKGROUND OF THE INVENTION:Efficient and high-level heterologous expression of proteins is an important alternative to the isolation of protein from native sources and is especially useful when the native protein is normally produced in limited amounts or by sources which are impossible, expensive and/or dangerous to obtain or propagate. Although a number of expression systems have proven useful for production of various heterologous proteins, none of these systems is universally applicable for the production of all proteins. For instance, E. coli lacks the ability to provide many post-translational modifications to heterologous proteins. Yeast can provide some of these post-translational modifications, but rapid degradation of heterologous proteins is common and secretion of heterologous proteins with long, untrimmed oligosaccharide chains sometimes results in biologically inactive or antigenically altered proteins. Moreover, a replacement of the natural mammalian signal peptide with a yeast signal peptide is almost always required for efficient secretion of mammalian proteins by yeast. Expression of heterologous eukaryotic proteins in insect or mammalian cells are good alternatives but both require rather expensive medium for cell propagation. Moreover, yeast, cultured insect cells and mammalian cells all have a long doubling time.

Protozoans represent an alternative for the expression of heterologous proteins, however only pathogenic protozoans have been characterized to the extent necessary for routine heterologous protein expression. Well-characterized pathogenic protozoans include Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp. A number of shuttle vectors designed for episomal replication (i.e., integrated into the chromosome and replicating independently of nuclear replication) and coding region expression in pathogenic protozoans have been developed. An inducible coding region expression system has been established for pathogenic T. brucei (Wirtz, E., et al., Science, 268, 1179-1183 (1995)). Vectors that allow efficient coding region expression in different hosts like E. coli and mammalian cells have also been developed (Al-Qahtani, A., et al., Nucleic Acids Res., 24 1173-1174 (1996)). It was recently determined that mammalian and protozoan signal peptides function in T. cruzi to target proteins to different cellular compartments. Also, bioactive cytokines (IL-2 and IFN-gamma) have been produced in both T. cruzi and Leishmania, suggesting that mammalian signal peptides are recognized and processed by these protozoans. However, pathogenic protozoans have not been exploited as a general purpose protein expression system, presumably because they are difficult or expensive to grow in large numbers and/or are infectious to human beings.

There have been unsuccessful attempts to use the nonpathogenic protozoan Crithidia to express heterologous proteins. In one study, Crithidia was transfected with vectors that contained a putative rRNA promoter and one of three reporter coding regions encoding luciferase, chloramphenicol acetyltransferase or β-galactosidase (Biebinger et al., Exp. Parasitol., 83, 252-258 (1996)). The reporter coding regions were inserted between a 5′-trans splicing signal and a 3′-untranslated region isolated from the Crithidia phosphoglycerate kinase coding region. This 5′-trans splicing signal had previously been shown to function in T. brucei. However, despite using regulatory regions endogenous to Crithidia, no activity of the reporter was detected in transient expression assays. When coding regions encoding resistance to hygromycin or G418 were used instead of the reporter coding regions, drug resistant cells were obtained, but at low efficiency. There was no evidence that integration of any of the vectors into genomic DNA had occurred.

In another study, shuttle vectors designed for episomal replication and coding region expression in Leishmania spp. (Coburn, C. M., et al., Mol. Biochem. Parasitol., 46, 169-179 (1991)) were introduced into Crithidia. The vectors were stably maintained in Crithidia at a copy number higher than occurred in Leishmania. However, in Crithidia the level of the protein encoded by the coding regions present on the vectors were significantly lower than the levels expressed in Leishmania.

A protein expression system that provides for the efficient expression and isolation of correctly post-ranslationally modified heterologous proteins in a nonpathogenic host would constitute a much desired advance in the art.

SUMMARY OF THE INVENTION:The invention provides a method for producing a polypeptide that involves providing a host cell containing a vector that includes a 5” regulatory region, a 3’ regulatory region, and a coding region encoding a polypeptide Blocated therebetween, then culturing the host cell under conditions that allow expression of the coding region such that the polypeptide encoded by the coding region is produced. The coding region is operably linked to the 5’ regulatory region and the 3’ regulatory region, and the host cell is a nonpathogenic protozoan. The nonpathogenic protozoan can be a member of the order Kinetoplastida, including the nonpathogenic protozoan Crithidia. The 5’ regulatory region and the 3’ regulatory region can be derived from a protozoan, including a Leishmania HMTXr 5’ or 3’ regulatory region, a Leishmania DHFR 5’ or 3’ regulatory region, or a Leishmania A2 5’ or 3’ regulatory region. Optionally, the polypeptide can be isolated.