Methods, Compositions and Systems for Production of Recombinant Spider Silk Polypeptides

Abstract

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.

That which is claimed is: 
     
         1 . A method to make a silkworm that is capable of producing a silk comprising a spider silk polypeptide or analog thereof comprising the steps of ligating a DNA sequence coding for at least a portion of a spider silk fibroin polypeptide, or an analog of a spider silk fibroin polypeptide, 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, and wherein the portion of the 5′ and 3′ ends of a silkworm fibroin gene are long enough to allow for homologous recombination to occur such that when the fused gene is inserted into a silkworm, it is able to replace the native gene present in the silkworm. 
     
     
         2 . The method of  claim 1 , wherein the spider silk analog may comprise a sequence made up of about 4 to 1000 spider silk beta-sheet domains alternating with about 4 to 1000 spider silk alpha helix domains. 
     
     
         3 . The method of  claim 1 , wherein the fusion gene construct comprises a nucleic acid that encodes for a peptide having the 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 method of  claim 1 , wherein the fusion gene construct comprises a nucleic acid that encodes for a spider silk analog peptide having the amino acid sequence as set forth in SEQ ID NO: 2. 
     
     
         5 . The method of  claim 1 , wherein the fusion gene construct comprises a nucleic acid that encodes for a silkworm silk gene 5′ end having a sequence as set forth in at least one of SEQ ID NO: 5, 10, 15 or 20. 
     
     
         6 . The method of  claim 1 , wherein the fusion gene construct comprises a nucleic acid that encodes for a silkworm silk gene 3′ end having a sequence as set forth in at least one of SEQ ID NO: 8, 13, 18 or 22. 
     
     
         7 . The method of  claim 1 , wherein the fusion gene construct further comprises a reporter gene. 
     
     
         8 . The method of  claim 1 , wherein the fusion gene construct comprises an isolated DNA as set forth in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. 
     
     
         9 . The method of  claim 1 , further comprising transforming a first population of silkworms with a fusion gene construct encoding for a first spider silk fibroin polypeptide, or analog thereof, and transforming a second population of silkworms with a fusion gene construct encoding for a second spider silk fibroin polypeptide. 
     
     
         10 . The method of  claim 1 , further comprising breeding the recombinant silkworms to generate silkworms that are homozygous at a fibroin loci for the fused gene construct. 
     
     
         11 . The method of  claim 1 , wherein at least one of light fibroin loci and one heavy fibroin chain in a recipient silkworm are replaced by a fusion gene construct encoding a spider silk fibroin polypeptide or an analog of a spider silk fibroin polypeptide. 
     
     
         12 . The method of  claim 1 , wherein the silkworm produces a silk that is at least 25% stronger than native silkworm silk. 
     
     
         13 . The method of  claim 1 , wherein the silkworm produces a silk that is at least 40% more elastic than silkworm silk. 
     
     
         14 . A composition comprising a DNA molecule having a nucleic acid sequence encoding both spider silk fibroin and silkworm silk fibroin amino acid sequences, the composition comprising a recombinant DNA construct 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, and wherein the portion of the 5′ and 3′ ends of a silkworm fibroin gene are long enough to allow for homologous recombination to occur such that when the fused gene is inserted into a silkworm, it is able to replace the native gene present in the silkworm. 
     
     
         15 . The composition of  claim 14 , wherein the spider silk analog may comprise a sequence made up of about 4 to 1000 spider silk beta-sheet domains alternating with about 4 to 1000 spider silk alpha helix domains. 
     
     
         16 . The composition of  claim 14 , wherein the fusion gene construct comprises a nucleic acid that encodes for a peptide having the 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: 42. 
     
     
         17 . The composition of  claim 14 , wherein the fusion gene construct comprises a nucleic acid that encodes for a spider silk analog peptide having the amino acid sequence as set forth in SEQ ID NO: 2. 
     
     
         18 . The composition of  claim 14 , wherein the fusion gene construct comprises a nucleic acid that encodes for a silkworm silk gene 5′ end having a sequence as set forth in at least one of SEQ ID NO: 5, 10, 15 or 20. 
     
     
         19 . The composition of  claim 14 , wherein the fusion gene construct comprises a nucleic acid that encodes for a silkworm silk gene 3′ end having a sequence as set forth in at least one of SEQ ID NO: 8, 13, 18 or 22. 
     
     
         20 . The composition of  claim 14 , wherein the fusion gene construct further comprises a reporter gene. 
     
     
         21 . The composition of  claim 14 , wherein the fusion gene construct comprises an isolated DNA as set forth in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. 
     
     
         22 . A system comprising a transformed silkworm or silkworm egg, wherein the transformed silkworm or silkworm egg comprises a recombinant DNA construct encoding 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, and wherein the portion of the 5′ and 3′ ends of a silkworm fibroin gene are long enough to allow for homologous recombination to occur such that when the fused gene is inserted into a silkworm, it is able to replace the native gene present in the silkworm.