Production of Therapeutic Proteins in Photosynthetic Organisms
The present disclosure relates to methods of expressing therapeutic proteins in photosynthetic organisms and the therapeutic proteins produced by the methods. The therapeutic proteins include high-mobility group box 1 (HMGB1) protein, fibronectin domain (10) (10FN3), fibronectin domain (14) (14FN3), interferon beta (IFNβ), proinsulin and vascular endothelial growth factor (VEGF). The photosynthetic organisms include prokaryotes such as cyanobacteria and eukaryotes such as alga and plants. Transformation of eukaryotes is preferably the plastid genome, more preferably the chloroplast genome.
1 . An isolated polynucleotide comprising a nucleotide sequence encoding a high-mobility group box 1 (HMGB1) protein that is capable of transforming an alga.
2 . The polynucleotide of claim 1 , wherein the nucleotide sequence is codon optimized.
3 . The polynucleotide of claim 2 , wherein the nucleotide sequence is codon optimized for expression in a chloroplast of the alga.
4 . The polynucleotide of claim 2 , wherein the nucleotide sequence is codon optimized for nuclear expression in the alga.
5 . The polynucleotide of claim 1 , wherein the nucleotide sequence comprises a nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 90.
6 . The polynucleotide of claim 1 , wherein the nucleotide sequence comprises a nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 90 wherein the nucleic acid sequence is modified by deleting at least one nucleic acid, adding at least one nucleic acid, or replacing at least one nucleic acid, and wherein the HMGB1 protein is biologically active.
7 . The polynucleotide of claim 1 , wherein the nucleotide sequence comprises a nucleic acid sequence that has about 80% homology, about 85% homology, about 90% homology, about 95% homology, or about 99% homology to a nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 90, and wherein the HMGB1 protein is biologically active.
8 . The polynucleotide of claim 1 , wherein the protein comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 28.
9 . The polynucleotide of any one of claims 1 to 7 , wherein the polynucleotide further comprises a nucleotide sequence encoding a fusion protein fused to the 5′ end of the nucleotide sequence encoding HMGB1.
10 . The polynucleotide of claim 9 , wherein the fusion protein is mammary-associated serum amyloid (M-SAA).
11 . The polynucleotide of claim 9 , wherein the polynucleotide further comprises a proteolytic cleavage site between the nucleotide sequence encoding the fusion protein and the nucleotide sequence encoding HMGB1.
12 . The polynucleotide of claim 1 , wherein the polynucleotide further comprises a nucleotide sequence (SEQ ID NO: 92) that comprises a nucleic acid sequence coding for a psbA promoter and 5′UTR, an atpA promoter (SEQ ID NO: 63) and 5′ UTR, or a psbD promoter and 5′ UTR (SEQ ID NO: 65) that is upstream of the nucleotide sequence encoding HMGB 1.
13 . The polynucleotide of claim 1 , wherein the polynucleotide further comprises a nucleotide sequence coding for a psbA 3′ UTR (SEQ ID NO: 66) or a rbeL 3′ UTR (SEQ ID NO: 67) that is downstream of the nucleotide sequence encoding HMGB1.
14 . An alga transformed with a polynucleotide comprising a nucleotide sequence encoding a HMGB1 protein.
15 . The alga of claim 14 , wherein the nucleotide sequence is codon optimized.
16 . The alga of claim 15 , wherein the nucleotide sequence is codon optimized for expression in the chloroplast of the alga.
17 . The alga of claim 14 , 15 , or 16 , wherein the nucleotide sequence comprises a nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 90.
18 . The alga of claim 14 , wherein the protein comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 28.
19 . A method of expressing a high-mobility group box 1 (HMGB1) protein in a photosynthetic organism, comprising:
a) transforming the photosynthetic organism with a polynucleotide comprising a nucleotide sequence encoding HMGB1 ; and
b) expressing the HMGB1.
20 . The method of claim 19 , wherein the polynucleotide further comprises a 5′ UTR.
21 - 206 . (canceled)