Methods and DNA constructs for high yield production of polypeptides
OwnerFred Wagner, Peng Luan +2inventors
InventorsFred Wagner, Peng Luan, Yuannan Xia, Barton Holmquist
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Abstract
The invention provides an inclusion body fusion partner to increase peptide and polypeptide production in a cell.
Claims83
An expression cassette comprising the following operably linked nucleic acid sequence:
- 5′ Pr-(TIS)D-(IBFP1)E-(CL1)G-ORF-[CL2-ORF]L-(CL3)M-(IBFP2)Q-(SSC)R-(CL4)T-(Ft)W-(Tr)X-3′
- wherein
- Pr is a promoter sequence,
- TIS encodes a translation initiation sequence,
- IBFP1 encodes a first inclusion body fusion partner comprising an amino acid sequence corresponding to SEQ ID NO:
- 1, or a variant thereof,
- CL1 encodes a first cleavable peptide linker,
- ORF encodes a preselected polypeptide,
- CL2 encodes a second cleavable peptide linker,
- CL3 encodes a third cleavable peptide linker,
- IBFP2 encodes a second inclusion body fusion partner comprising an amino acid sequence corresponding to SEQ ID NO:
- 1, or a variant thereof,
- SSC is a suppressable stop codon,
- CL4 encodes a fourth cleavable peptide linker,
- Ft encodes a fusion tag, and
- Tr is a transcription terminator sequence,
- wherein each of D or X is independently 0 or an integer of 1 to 4,
- wherein R is 0 or an integer of 1 to 2,
- wherein each of E, G, L, M, Q, T or W is independently 0 or an integer of 1 to 20,
- wherein either one or both of IFP1 or IFP2 is present, and
- wherein expression of the expression cassette produces a tandem polypeptide that forms an inclusion body when expressed in a cell.
The expression cassette of claim 6, wherein the isolation enhancement of the inclusion body is self-adhesion, solubility, purification stability, resistance to proteolysis, or altered isoelectric point.
A tandem polypeptide comprising:
- a) a first region comprising an inclusion body fusion partner having an amino acid sequence corresponding to SEQ ID NO:
- 1, or a variant thereof;
- and
- b) a second region not naturally associated with the first region comprising a preselected amino acid sequence.
A method to select an amino acid sequence of an inclusion body fusion partner that confers isolation enhancement to an inclusion body comprising:
- a) altering the amino acid sequence of an inclusion body fusion partner comprising SEQ ID NO:
- 1 that is operably linked to an amino acid sequence not naturally associated with the fusion partner to form a tandem polypeptide that forms the inclusion body, and
- b) determining if the inclusion body exhibits enhanced self-adhesion, controllable solubility, purification stability, or resistance to proteolysis.
An isolated inclusion body fusion partner having an amino acid sequence corresponding to SEQ ID. NO:
- 1, and variants thereof.
A method to produce a tandem polypeptide comprising:
- a) expressing a tandem polypeptide comprising an inclusion body fusion partner that is operably linked to a preselected polypeptide in a cell, and
- b) isolating the tandem polypeptide.
A nucleic acid sequence that encodes an amino acid sequence comprising SEQ ID NOs:
- 1 and 2.
An amino acid sequence comprising SEQ ID NOs:
- 1 and 2.
A nucleic acid sequence corresponding to SEQ ID NO:
- 1.
A nucleic acid sequence having at least 98% sequence identity to a nucleic acid sequence that encodes an amino acid sequence corresponding to SEQ ID NO:
- 1.
A nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence that encodes an amino acid sequence corresponding to SEQ ID NO:
- 1.
A nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence that encodes an amino acid sequence corresponding to SEQ ID NO:
- 1.
A nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence that encodes an amino acid sequence corresponding to SEQ ID NO:
- 1.
An amino acid sequence corresponding to SEQ ID NO:
- 1.
An amino acid sequence corresponding to SEQ ID NO:
- 1.
An amino acid sequence having at least 98% sequence identity to SEQ ID NO:
- 1.
An amino acid sequence having at least 90% sequence identity to SEQ ID NO:
- 1.
An amino acid sequence having at least 80% sequence identity to SEQ ID NO:
- 1.
An amino acid sequence having at least 70% sequence identity to SEQ ID NO:
- 1.
A tandem polypeptide comprising an inclusion body fusion partner having an amino acid sequence corresponding to SEQ ID NO:
- 1 operably linked to a preselected polypeptide.
Dependencies are inferred from the claim text.