Production of seleno-biologics in genomically recoded organisms
Polypeptides that fold into biologies are stabilized by diselenide bonds between selenocysteine amino acids. Methods to produce such polypeptides in genomically recoded organisms (GRO) can be scaled up for industrial production. Since diselenides have the same geometric bond angles and torsions as disulfides, as well as very similar bond lengths, they can be substituted into polypeptides without disrupting the three dimensional structure of the polypeptides. Diselenides render the polypeptides resistant to reduction when they are exposed to blood serum or to reducing components of blood serum or to reducing components components within cells.
1. A protein comprising at least two selenocysteine residues with at least one diselenide bond between the at least two selenocysteine residues, wherein the protein comprises a sequence selected from the group consisting of: SEQ ID NO: 1142, 1144, 1146, 1148, 1150, 1152, 1154 and residues 90-110 of SEQ ID NO: 1630 and sequences that are at least 80% identical to said sequence.
2. The protein of claim 1 , wherein the protein is a modified insulin protein that further comprises a B chain having a sequence of residues 25-54 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence; wherein the protein comprises at least one diselenide bond within the A chain and at least one diselenide bond between the A chain and the B chain.
3. A bacterial host that has been genomically recoded so that an unassigned codon is recognized by a selenocysteine tRNA with a corresponding anticodon, wherein the bacterial host contains a constructed DNA sequence encoding a variant form of a protein in which a pair of cysteine codons has been replaced with the unassigned codon wherein the variant form comprises a modified insulin protein having at least two selenocysteine residues replacing two cysteine residues that form a disulfide bond in a native human insulin protein, with at least one diselenide bond between the at least two selenocysteine residues.
4. A method of producing a modified insulin protein having at least two selenocysteine residues replacing two cysteine residues that form a disulfide bond in a native human insulin protein, with at least one diselenide bond between the at least two selenocysteine residues, the method comprising:
culturing a bacterial host that has been genomically recoded so that an unassigned codon is recognized by a selenocysteine tRNA with a corresponding anticodon, wherein the bacterial host contains a constructed DNA sequence encoding the modified protein in which a pair of cysteine codons has been replaced with the unassigned codon, wherein the culturing is under conditions in which the modified protein is expressed.
5. The bacterial host of claim 3 , wherein the modified insulin protein has an A chain comprising at least two selenocysteine residues with at least one diselenide bond between the at least two selenocysteine residues, wherein the protein comprises a sequence selected from the group consisting of: SEQ ID NO: 1142, 1144, 1146, 1148, 1150, 1152, 1154 and residues 90-110 of SEQ ID NO: 1630 and sequences that are at least 80% identical to said sequence.
6. The bacterial host of claim 5 , wherein the modified insulin protein further comprises a B chain having a sequence of residues 25-54 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence; wherein the protein comprises at least one diselenide bond within the A chain and at least one diselenide bond between the A chain and the B chain.
7. The bacterial host of claim 3 , wherein the modified insulin protein comprises an A chain having a sequence of residues 90-110 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence, and a B chain having a sequence of residues 25-54 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence; wherein the protein comprises at least one diselenide bond between the A chain and the B chain.
8. The method of claim 4 , wherein the modified insulin protein has an A chain comprising at least two selenocysteine residues with at least one diselenide bond between the at least two selenocysteine residues, wherein the protein comprises a sequence selected from the group consisting of: SEQ ID NO: 1142, 1144, 1146, 1148, 1150, 1152, 1154 and residues 90-110 of SEQ ID NO: 1630 and sequences that are at least 80% identical to said sequence.
9. The method of claim 8 , wherein the modified insulin protein further comprises a B chain having a sequence of residues 25-54 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence; wherein the protein comprises at least one diselenide bond within the A chain and at least one diselenide bond between the A chain and the B chain.
10. The method of claim 4 , wherein the modified insulin protein comprises an A chain having a sequence of residues 90-110 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence, and a B chain having a sequence of residues 25-54 of SEQ ID NO: 1630 with at least one cysteine to selenocysteine substitution and sequences that are at least 80% identical to said sequence; wherein the protein comprises at least one diselenide bond between the A chain and the B chain.