Kinetoplastid protein expression system and methods

A nonpathogenic protozoan comprising a vector comprising a first coding region that encodes a polypeptide, wherein the first coding region is flanked by a first 5′ regulatory region and a first 3′ regulatory region, and a second coding region that encodes a detectable marker, wherein the second coding region is flanked by a second 5′ regulatory region and a second 3′ regulatory region.

A method for producing a polypeptide comprising:

• (a) providing a nonpathogenic protozoan host cell comprising a vector comprising a 5′ regulatory region, a 3′ regulatory region, and a coding region located therebetween, wherein the coding region encodes a polypeptide comprising a signal peptide and is operably linked to the 5′ regulatory region and the 3′ regulatory region;
• and
• (b) culturing the host cell under conditions that allow expression of the coding region to produce the polypeptide.

A method for producing a polypeptide comprising:

• (a) providing a nonpathogenic protozoan host cell comprising a vector comprising a 5′ regulatory region, a 3′ regulatory region, and a coding region located therebetween, wherein the coding region encodes a polypeptide and is operably linked to the 5′ regulatory region and the 3′ regulatory region, and wherein the polypeptide comprises a GPI cleavage/attachment site comprising amino acids 632-679 of amastigote surface protein I (SEQ ID NO:14);
• and
• (b) culturing the host cell under conditions that allow expression of the coding region to produce the polypeptide.

A vector comprising a 5′ regulatory region, a 3′ regulatory region, and a coding region located therebetween, wherein the coding region encodes a polypeptide comprising a signal peptide and is operably linked to the 5′ regulatory region and the 3′ regulatory region, and wherein the 5′ regulatory region and the 3′ regulatory regions are each derived from a protozoan.

A vector comprising a 5′ regulatory region, a 3′ regulatory region, and a coding region located therebetween, wherein the coding region encodes a polypeptide comprising a signal peptide and is operably linked to the 5′ regulatory region and the 3′ regulatory region, wherein the signal peptide is an amino terminal signal peptide selected from the group consisting of amino acids 1-47 of the T. cruzi glycoprotein gp-72 (SEQ ID NO:11) and amino acids 1-18 of influenza hemagglutinin (SEQ ID NO:12).

A vector comprising a 5′ regulatory region, a 3′ regulatory region, and a coding region located therebetween, wherein the coding region encodes a polypeptide comprising a GPI cleavage/attachment site comprising amino acids 632-679 of amastigote surface protein I (SEQ ID NO:14), and the coding region is operably linked to the 5′ regulatory region and the 3′ regulatory region.

A vector comprising a first 5′ regulatory region, a first 3′ regulatory region, and a first coding region located therebetween, wherein the coding region encodes a polypeptide and is operably linked to the first 5′ regulatory region and the first 3′ regulatory region, and a second 5′ regulatory region, a second 3′ regulatory region, and a second coding region located therebetween, wherein the second coding region encodes a detectable marker and is operably linked to the second 5′ regulatory region and the second 3′ regulatory region, wherein the first and second 5′ regulatory regions and the first and second 3′ regulatory regions are each derived from a protozoan.

A vector comprising a first 5′ regulatory region, a first 3′ regulatory region, and a first coding region located therebetween, wherein the coding region encodes a polypeptide and is operably linked to the first 5′ regulatory region and the first 3′ regulatory region, and a second 5′ regulatory region, a second 3′ regulatory region, and a second coding region located therebetween, wherein the second coding region encodes a detectable marker and is operably linked to the second 5′ regulatory region and the second 3′ regulatory region, wherein the second 5′ regulatory region and the second 3′ regulatory region are each derived from a protozoan.

A vector comprising a first 5′ regulatory region, a first 3′ regulatory region, and a first coding region located therebetween, wherein the coding region encodes a polypeptide and is operably linked to the first 5′ regulatory region and the first 3′ regulatory region, and a second 5′ regulatory region, a second 3′ regulatory region, and a second coding region located therebetween, wherein the second coding region encodes a detectable marker and is operably linked to the second 5′ regulatory region and the second 3′ regulatory region, wherein the polypeptide comprises a signal peptide.

A vector comprising a first 5′ regulatory region, a first 3′ regulatory region, and a first coding region located therebetween, wherein the coding region encodes a polypeptide and is operably linked to the first 5′ regulatory region and the first 3′ regulatory region, and a second 5′ regulatory region, a second 3′ regulatory region, and a second coding region located therebetween, wherein the second coding region encodes a detectable marker and is operably linked to the second 5′ regulatory region and the second 3′ regulatory region, wherein the polypeptide comprises a GPI cleavage/attachment site.

A vector comprising a first 5′ regulatory region, a first 3′ regulatory region, and a first coding region located therebetween, wherein the coding region encodes a polypeptide and is operably linked to the first 5′ regulatory region and the first 3′ regulatory region, and a second 5′ regulatory region, a second 3′ regulatory region, and a second coding region located therebetween, wherein the second coding region encodes a detectable marker and is operably linked to the second 5′ regulatory region and the second 3′ regulatory region, wherein the first and second 5′ regulatory regions each comprise a Leishmania DHFR 5′ regulatory region and the first and second 3′ regulatory regions each comprise a Leishmania DHFR 3′ regulatory region.

A nonpathogenic protozoan comprising a vector comprising a coding region that encodes a polypeptide, wherein the coding region is flanked by a 5′ regulatory region and a 3′ regulatory region, wherein the 5′ regulatory region is selected from the group consisting of a Leishmania HMTXr 5′ regulatory region, a Leishmania DHFR 5′ regulatory region, and a Leishmania A2 5′ regulatory region, and wherein the 3′ regulatory region is selected from the group consisting of a Leishmania HMTXr 3′ regulatory region, a Leishmania DHFR 3′ regulatory region, and a Leishmania A2 3′ regulatory region, and wherein the nonpathogenic protozoan is selected from the group consisting of nonpathogenic members of the genus Trypanosoma, members of the genus Blastocrithidia, members of the genus Herpetomonas, members of the genus Leptomonas, members of the genus Phytomonas, members of the genus Crithidia, and Leishmania chamaelonis.