Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast
AssigneeThe Scripps Research Institute
InventorStephen P. Mayfield
Life of this patent
Estimating the term…
Abstract
Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.
Claims51
A method of expressing a gene comprising:
- transforming an algal cell by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence, a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site;
- and
- cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
A method of expressing a gene comprising:
- transforming an algal cell by replacing psbA via integration of a chimeric construct comprising a nucleic acid sequence encoding SEQ ID NO:1, a promoter sequence cognate or non-cognate to psbA, and at least one UTR, wherein psbA is re-inserted at a silent distal site;
- and
- cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
An algal extract obtained from an algal cell transformed by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence, a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site.
A method of expressing a gene comprising:
- transforming an algal cell by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence encoding mammary associated serum amyloid A3 (M-SAA3), a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site;
- and
- cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
A method of expressing a gene comprising:
- transforming an algal cell by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence, a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site and driven by a non-cognate promoter;
- and
- cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
Dependencies are inferred from the claim text.