Polypeptide expression in ciliates
Inventors
Colussi, Paul • Cassidy-Hanley, Donna • Clark, Theodore G.
Assignees
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Publication Number
US-8664374-B2
Publication Date
2014-03-04
Expiration Date
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.
Core Innovation
The invention relates to nucleic acid constructs and genetically modified ciliates for expressing a recombinant polypeptide with improved genetic stability and yield. The constructs include a selection cassette encoding a resistance marker, a transgene cassette encoding a recombinant polypeptide, and 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.
The disclosed C3 allele rDNA locus is described with rDNA locus biology in ciliates, including CBS and NTS within rDNA loci and the involvement of palindromic minichromosomes and amplification. The document describes embodiments using linear and circular forms and carrier formats, including vectors and rDNA-based loci. It further describes specific rDNA allelic design using a 17S rDNA gene altered such that it does not provide paromomycin resistance.
In addition, the document describes genetically modified ciliates in which micronuclear rDNA is altered to create non-functional micronuclear rDNA that yields conditionally conjugation-lethal phenotypes. It further describes “universal recipient” lines that can be rescued by functional rDNA, optionally carrying transgenes, to enable stable production.
Claims Coverage
The independent claims cover a nucleic acid construct architecture with three coordinated parts (selection cassette, transgene cassette, and a C3 allele rDNA locus with a 17S rDNA gene that does not confer paromomycin resistance), plus genetically modified ciliates carrying the construct and a conjugation-based method for producing the recombinant polypeptide. The main inventive features across the independent claims are centered on the C3 allele rDNA locus with the paromomycin-nonresistant 17S rDNA gene and the coordinated inclusion of selection and transgene cassettes.
Nucleic acid construct with selection, transgene, and C3 allele rDNA locus lacking paromomycin resistance
A nucleic acid construct comprising a selection cassette encoding a resistance marker, a transgene cassette encoding a recombinant polypeptide, and 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.
Genetically modified ciliate containing selection, transgene, and C3 allele rDNA locus lacking paromomycin resistance
A genetically modified ciliate comprising a nucleic acid construct comprising a selection cassette encoding a resistance marker, a transgene cassette encoding a recombinant polypeptide, and 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.
Conjugation-based method using nucleic acid construct with selection, transgene, and C3 allele rDNA locus lacking paromomycin resistance
A method for producing a recombinant polypeptide in a ciliate comprising causing the ciliate to undergo sexual reproduction by conjugation, transforming a genetically modified ciliate with a nucleic acid construct comprising a selection cassette encoding a resistance marker, a transgene cassette encoding a recombinant polypeptide, and 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, culturing the genetically modified ciliate to produce the recombinant polypeptide, and isolating the recombinant polypeptide.
Overall, the independent claims define coordinated construct elements (selection cassette and transgene cassette) with a specific rDNA locus feature: a C3 allele rDNA locus comprising a 17S rDNA gene that does not confer paromomycin resistance, carried by a genetically modified ciliate and used in a conjugation-based production method.
Stated Advantages
Improved genetic stability and yield for recombinant polypeptide production in ciliates.
Documented Applications
Producing a recombinant polypeptide in a ciliate using a nucleic acid construct that includes a C3 allele rDNA locus with a 17S rDNA gene that does not confer paromomycin resistance.
Creating genetically modified ciliate lines with conditionally conjugation-lethal phenotypes and rescuing them using functional rDNA, optionally carrying transgenes, to enable stable production.
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