Methods and compositions for producing recombinant proteins using a gene for TRNA
Inventors
Blattner, Frederick R. • Campbell, John Walter • Plunkett, Guy
Assignees
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Publication Number
US-7989181-B2
Publication Date
2011-08-02
Expiration Date
Abstract
The invention relates to host cells with improved protein expressed properties. The host cells comprise rare tRNA genes within the one or more rRNA operons.
Core Innovation
The invention relates to an E. coli host cell engineered to include rRNA operons rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH, wherein one or more rRNA operons comprise one or more polynucleotides encoding a tRNA. The tRNA-encoding polynucleotides have a sequence selected from SEQ ID NOS: 1-7.
Expression and processing of rRNA from the operons rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH is maintained, while the tRNA-encoding polynucleotides are embedded within the rRNA operons. The description addresses selecting tRNA genes based on rare codon frequency and supports inclusion using rRNA promoter-driven expression and maintenance of rRNA processing.
The described construction includes insertion positioning within rRNA operons, including insertion within components or at the 3′ end of P1/P2, with optional 5S distal regions to reduce homologous recombination. An example inserts argU and ileX into the 3′ end of rrnG in MDS42 and increases expression of P. falciparum DHPS versus an unmodified control, assessed by SDS-PAGE/Western and enzyme assays.
Claims Coverage
The independent claim covers an E. coli host cell with specific rRNA operons (rrnA–rrnH) that include tRNA-encoding polynucleotides selected from SEQ ID NOS: 1-7, while maintaining rRNA expression and processing for those operons. Dependent coverage refines the selectable tRNA SEQ IDs, narrows the rrn operon context, specifies insertion positioning-related features, and includes an associated method for preparing a recombinant heterologous protein.
tRNA-encoded polynucleotides embedded within rRNA operons
An E. coli host cell comprising rRNA operons rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH, wherein one or more rRNA operons comprise one or more polynucleotides encoding a tRNA.
tRNA polynucleotide sequence selected from SEQ ID NOS: 1-7
The one or more polynucleotides encoding a tRNA comprise a sequence selected from the group consisting of SEQ ID NOS: 1-7.
Maintained rRNA expression and processing from rrnA–rrnH
Expression and processing of rRNA from operons rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH is maintained.
tRNA gene subset specified by SEQ ID NOS: 1 and 7
The host cell is characterized by an rRNA operon comprising SEQ ID NOS: 1 and 7.
Inserting a tRNA-encoding polynucleotide into a selected rrn operon
The host cell is characterized by selecting an rRNA operon used for inserting the tRNA-encoding polynucleotide from rrnA, rrnB, rrnC, rrnD, rrnE, rrnG, or rrnH.
Recombinant heterologous protein prepared by expressing a gene in the host cell
A method prepares a recombinant heterologous protein by providing a host cell and expressing a gene encoding the heterologous protein.
Overall claim coverage centers on an E. coli host cell where tRNA-encoding polynucleotides (SEQ ID NOS: 1-7) are incorporated into rRNA operons rrnA–rrnH while maintaining rRNA expression and processing. Dependent refinements narrow the tRNA SEQ IDs and specify insertion into particular rrn operons, and an associated method covers preparing a recombinant heterologous protein by gene expression in the engineered host cell.
Stated Advantages
Increases expression of P. falciparum DHPS versus an unmodified control.
Documented Applications
Recombinant heterologous protein production, illustrated by increased expression of P. falciparum dihydropteroate synthetase (DHPS) in an engineered E. coli host cell.
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