Polypeptide expression in ciliates

Abstract

This invention is directed to methods for recombinant polypeptide production and, in particular, methods and products for the production of recombinant polypeptides in ciliates.

What is claimed is: 
     
         1 . A nucleic acid construct comprising:
 a) a selection cassette encoding a resistance marker,   b) a transgene cassette encoding a recombinant polypeptide, and   c) a C3 allele rDNA locus comprising a 17S rDNA gene that does not confer paromomycin resistance to a ciliate transformed with the nucleic acid construct.   
     
     
         2 . The nucleic acid construct of  claim 1 , wherein the construct is a linear DNA or RNA 
     
     
         3 . The nucleic construct of  claim 1 , wherein the construct is a circular DNA or RNA. 
     
     
         4 . The nucleic acid construct of  claim 1 , wherein the construct is a vector, a plasmid, a cosmid, a chromosome or minichromosome, a transposon, an rDNA or any combination thereof. 
     
     
         5 . The nucleic acid construct of  claim 4 , wherein the vector is an rDNA vector. 
     
     
         6 . The nucleic acid construct of  claim 1 , wherein the selection cassette does not confer paromomycin resistance to a ciliate transformed with the nucleic acid construct. 
     
     
         7 . The nucleic acid construct of  claim 1 , wherein the 17S rDNA gene is a B allele 17S rRNA. 
     
     
         8 . A method for producing a recombinant polypeptide in a ciliate, the method comprising:
 a) transforming the ciliate with the vector of  claim 1 ,   b) culturing the ciliate and expressing the recombinant polypeptide, and   c) isolating the recombinant polypeptide.   
     
     
         9 . A genetically modified ciliate having a micronuclear genotype having a micronuclear rDNA locus comprising one or more non-functional rDNA genes. 
     
     
         10 . The genetically modified ciliate of  claim 9 , wherein the non-functional micronuclear rDNA locus comprises a modification of micronuclear rDNA locus nucleotide sequence. 
     
     
         11 . The genetically modified ciliate of  claim 9 , wherein the modification of micronuclear rDNA locus results in the formation of a non-functional rDNA chromosome after sexual conjugation of the ciliate. 
     
     
         12 . The genetically modified ciliate of  claim 9 , wherein the ciliate has a conditionally conjugation-lethal phenotype. 
     
     
         13 . The genetically modified ciliate of  claim 9 , wherein the rDNA gene is selected from the list comprising 5.8S, 17S or 26S. 
     
     
         14 . The genetically modified ciliate of  claim 9 , wherein the modification is selected from the list consisting of: a deletion, an insertion, a substitution or an inversion. 
     
     
         15 . A method for producing a recombinant polypeptide a ciliate, the method comprising:
 a) causing the genetically modified ciliate to undergo sexual reproduction by conjugation;   b) transforming a genetically modified ciliate of any of  claim 9  with an rDNA rescue vector comprising a transgene cassette encoding the recombinant polypeptide;   c) culturing the genetically modified ciliate to produce the recombinant polypeptide; and   d) isolating the recombinant polypeptide.