Methods, compositions and systems for production of recombinant spider silk polypeptides
View Patent ↗Disclosed are methods, compositions, and systems for transforming silkworms to produce spider silk and analogs of spider silk. In certain embodiments, the method may include inserting a DNA sequence coding for at least a portion of a spider silk fibroin polypeptide, or an analog of a spider silk fibroin polypeptide, positioned between at least a portion of the 5′ and 3′ ends of a silkworm fibroin gene to generate a fusion gene construct having a sequence that encodes for a polypeptide comprising both spider silk fibroin and silkworm silk fibroin sequences. In certain embodiments, the fused gene is able to replace a native gene present in the silkworm such that the transformed silkworm expresses a polypeptide comprising a spider silk fibroin polypeptide, or an analog thereof, and expresses significantly less of the native silkworm silk.
1. An isolated analog of a spider silk fibroin polypeptide, wherein the analog of the spider silk fibroin polypeptide is encoded by at least 2800 nucleotides encoding alternating beta-sheet domains and alpha-helix domains operably linked to a silkworm silk fibroin amino acid sequence, wherein the amino acid sequence of the beta-sheet domains are each at least 60% identical to at least one of the beta-sheet domains as set forth in SEQ ID NO: 2, and wherein the analog of the spider silk fibroin polypeptide is at least 50% stronger than native silkworm silk.
2. The isolated analog of the spider silk fibroin polypeptide of claim 1 , wherein the spider silk analog of the spider silk fibroin polypeptide comprises about 30 to 1000 spider silk beta-sheet domains alternating with about 30 to 1000 spider silk alpha helix domains.
3. The isolated analog of the spider silk fibroin polypeptide of claim 1 , wherein the analog of the spider silk fibroin polypeptide comprises an amino acid sequence as set forth in at least one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.
4. The isolated analog of the spider silk fibroin polypeptide of claim 1 , wherein the analog of the spider silk fibroin polypeptide further comprises a reporter polypeptide.
5. The isolated analog of the spider silk fibroin polypeptide of claim 1 , wherein the analog of the spider silk fibroin polypeptide is at least 40% more elastic than native silkworm silk.
6. An isolated nucleic acid that encodes an analog of a spider silk fibroin polypeptide, wherein the nucleic acid comprises at least 2800 nucleotides encoding alternating beta-sheet domains and alpha-helix domains operably linked to a silkworm silk fibroin nucleic acid sequence, wherein the amino acid sequence of the beta-sheet domains are each at least 60% identical to at least one of the beta-sheet domains as set forth in SEQ ID NO: 2, and wherein the analog of the spider silk fibroin polypeptide is at least 50% stronger than native silkworm silk.
7. The isolated nucleic acid of claim 6 , wherein the amino acid sequence of the beta-sheet domains each have at least 60% identity to at least one of the beta-sheet domains encoded by the DNA sequence of SEQ ID NO: 34.
8. The isolated nucleic acid of claim 6 , having the sequence as set forth in SEQ ID NO: 34.
9. An isolated nucleic acid that encodes a spider silk fibroin polypeptide or an analog of a spider silk fibroin polypeptide, operably linked to at least one silkworm fibroin nucleic acid sequence.
10. The isolated nucleic acid of claim 9 , wherein the at least one silkworm nucleic acid sequence comprises a 5′ end of a native silkworm fibroin gene, a 3′ end of a native silkworm fibroin gene, or both a 5′ end of a native silkworm fibroin gene and a 3′ end of a native silkworm fibroin gene.
11. The isolated nucleic acid of claim 9 , wherein the analog of the spider silk fibroin polypeptide is encoded by a nucleic acid sequence comprising at least 2800 nucleotides encoding alternating beta-sheet domains and alpha-helix domains, wherein the amino acid sequence of the beta-sheet domains are each at least 60% identical to at least one of the beta-sheet domains as set forth in SEQ ID NO: 2, and wherein the analog of the spider silk fibroin polypeptide is at least 50% stronger than native silkworm silk.
12. The isolated nucleic acid of claim 10 , wherein the silkworm fibroin gene 5′ end comprises a nucleic acid sequence as set forth in at least one of SEQ ID NOs: 5, 10, 15, or 20.
13. The isolated nucleic acid of claim 10 , wherein the silkworm fibroin gene 3′ end comprises a nucleic acid sequence as set forth in at least one of SEQ ID NOs: 8, 13, 18, or 22.
14. A transgenic silkworm transformed with an isolated nucleic acid that encodes a spider silk fibroin polypeptide or an analog of a spider silk fibroin polypeptide, wherein the analog of the spider silk fibroin polypeptide is encoded by at least 2800 nucleotides encoding alternating beta-sheet domains and alpha-helix domains, wherein the amino acid sequence of the beta-sheet domains are each at least 60% identical to at least one of the beta-sheet domains as set forth in SEQ ID NO: 2, and wherein the transgenic silkworm produces a silk that is at least 50% stronger than native silkworm silk.
15. A silk produced by the transgenic silkworm of claim 14 , wherein the silk is at least 50% stronger than native silkworm silk.