Compositions and methods for fabricating synthetic dragline spider silk

Abstract

Compositions comprising a mixture of proteins derived from MaSP, nucleic acids encoding same and method for the preparation of synthetic dragline spider silk are provided. The compositions of the invention comprise a mixture of proteins of differing molecular weight, wherein each protein of said mixture comprises, independently, multiple repeats of a repetitive region of a MaSP (major ampullate spidroin) protein or a functional homolog, variant, derivative or fragment thereof.

1 . A non-natural proteinous fiber, wherein said proteinous fiber comprise: an amino acid sequence as set forth in SEQ ID NO: 1: (X 1 ) Z X 2 GPGGYGPX 3 X 4 X 5 GPX 6 GX 7 GGX 8 GPGGPGX 9 X 10 , wherein X 1  is, independently, at each instance A or G wherein at least 50% of (X 1 ) Z  is A, Z is an integer between 5 to 30; X 2  is S or G; X 3  is G or E; X 4  is G, S or N; X 5  is Q or Y; X 6  is G or S; X 7  is P or R; X 8  is Y or Q; X 9  is G or S; and X 10  is S or G, said proteinous fiber is characterized by a DSC peak at 250° C. to 330° C. 
     
     
         2 . The non-natural proteinous fiber of  claim 1 , wherein said proteinous fiber comprises 4 to 32 repeats of said SEQ ID NO: 1. 
     
     
         3 . The non-natural proteinous fiber of  claim 1 , wherein said proteinous fiber comprises a first moiety and contiguous thereto a second moiety, the first moiety is an amino acid sequence of 5-30 amino acids comprising at least 50% alanine residues, the second moiety is an amino acid sequence of 20-60 amino acids comprising at least 80% residues selected from the group consisting of glycine, serine, proline and tyrosine, optionally wherein the second comprises at most two glutamine residues. 
     
     
         4 . The non-natural proteinous fiber of  claim 1 , wherein said said proteinous fiber comprises the amino acid sequence as set forth in SEQ ID NO: 33. 
     
     
         5 . The non-natural proteinous fiber of  claim 1 , wherein said proteinous fiber comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS) and SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS). 
     
     
         6 . The non-natural proteinous fiber of  claim 1 , wherein said proteinous fiber comprises a single N-terminal region selected from the group consisting of: 
       
         
           
                 
               
                   SEQ ID NO: 5 
                 
                   (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV), 
                 
                     
                 
                   SEQ ID NO: 6 
                 
                   (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVRPLSNLDN 
                 
                   A), 
                 
                     
                 
                   SEQ ID NO: 7 
                 
                   MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVDPPGCRNSA 
                 
                   RAGSS), 
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       or any functional homolog, variant, derivative, or fragment thereof, wherein said homolog of said N-terminal region shares at least 70% homology with any one of SEQ ID NOs: 5-7. 
     
     
         7 . The non-natural proteinous fiber of  claim 1 , wherein said proteinous fiber comprises comprises a single C-terminal region as set forth in SEQ ID NO: 9 (GPSGPGAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNS LVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS), or any functional homolog, variant, derivative, fragment or mutant thereof wherein said homolog of said C-terminal region shares at least 70% homology with SEQ ID NO: 9. 
     
     
         8 . The non-natural proteinous fiber of  claim 1 , wherein said proteinous fiber is further characterized by a DSC pattern with an endothermic peak in the range of 220° C. to 250° C. 
     
     
         9 . An article comprising the non-natural proteinous fiber of  claim 1 . 
     
     
         10 . A nucleic acid molecule encoding the fiber of  claim 1 . 
     
     
         11 . An expression vector comprising the nucleic acid molecule of  claim 10 , wherein said nucleic acid molecule is under expression control of an operably linked promoter and, optionally, regulatory sequences. 
     
     
         12 . A host cell transformed with the expression vector of  claim 11 .