Kinetoplastid protein expression system and methods

Details Kinetoplastid protein expression system and methods Kinetoplastid protein expression system and methods

1999-12-02

2002-04-09

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S258100, C435S320100, C536S023100, C536S024100

Reexamination Certificate

active

06368827

ABSTRACT:

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 (Garg, N. et al.,
J. Immunol
., 158, 3293-3302 (1997)). Also, bioactive cytokines (IL-2 and IFN-gamma) have been produced in both
T. cruzi
and Leishmania (La Flamme, A. C., et al.,
Mol. Biochem. Parasitol
., 75, 25-31 (1995), and Tobin, J. F., et al.,
J. Immunol
., 150 5059-5069 (1993)), 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 &bgr;-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 HMTX
r
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.
The vector used in the method can further include a second 5′ regulatory region, a second 3′ regulatory region, and a second coding region located therebetween. The second coding region encodes a detectable marker and is operably linked to the second 5′ regulatory region and the second 3′ regulatory region. The second 5′ regulatory region and the second 3′ regulatory region can be derived from a protozoan, including a Leishmania HMTX
r
5′ or 3′ regulatory region, a Leishmania DHFR 5′ or 3′ regulatory region, or a Leishmania A2 5′ or 3′ regulatory region. The detectable marker can be a selectable marker that encodes resistance to a drug.
The polypeptide encoded by the first coding region can include an amino terminal signal peptide, for instance amino acids 1-47 (SEQ ID NO:11) of the
T. cruzi
glycoprotein gp-72, amino acids 1-18 (SEQ ID NO:12) of influenza hemagglutinin, or amino acids 1-22 (SEQ ID NO:13) of murine interleukin-2. The polypeptide encoded by the first coding region can include a GPI cleavage/attachment site, for instance amino acids 632-679 (SEQ ID NO:14) of amastigote surface protein I.
The vector used in the methods can be a plasmid that is maintained either extrachromosomally in the nonpathogenic protozoan host cell or integrated into the genomic DNA of the nonpathogenic protozoan host cell. Further, the vector useful in the method of the invention is also encompassed within the scope of the invention.
The invention also provides a nonpathogenic protozoan that contains a vector of the invention. The nonpathogenic protozoan can be a member of the order Kinetoplastida, including the nonpathogenic protozoan Crithidia.


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Hughes et al., “Introduction of Plasmid DNA Into the Trypanosomatid ProtozoanCrithidia fasciculata,”Proc. Natl. Acad. Sci. USA, 83(16):6058-6062 (1986).
Tobin et al., “Mutational Analysis of a Signal Sequence Required for Protein Secretion inLeishmania major,”Mol. Bioc

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