Trans inteins for protein domain shuffling and biopolymerization

A method for making a polymeric protein comprising repeating protein polymer motifs, the method comprising the steps of:

• i) ligating a plurality of n genes for n monomeric components of the polymeric protein between a C-intein and an N-intein of two different trans-inteins, wherein the C-intein of the nth gene specifically interacts with the N-intein of the (n−1)th monomeric component to covalently link the nth gene product with the (n−1)th gene product;
• ii) ligating each of a plurality of the gene-intein ligation products of step (i) to an expression vector;
• iii) introducing a plurality of said vectors containing the gene-intein ligation products into a suitable host cell;
• iv) expressing the plurality of gene-intein ligation products in the host cell;
• and
• v) isolating the harvesting the polymeric protein therefrom.

A method for generating a trans-intein from a cis-intein comprising the steps of:

• i) inserting into a first nucleic acid that encodes a protein a second nucleic acid comprising a nucleotide sequence encoding a cis-intein comprising an amino terminal portion (N-intein) and a carboxyl-terminal portion (C-intein) separated by a linker domain;
• ii) breaking the cis-intein into two overlapping fragments, wherein the first fragment comprises a portion of the intein extending from the 5′ end of the intein through the 3′ end of the linker domain and wherein the second fragment comprises a portion extending from the 5′ end of the linker domain through the 3′ end of the intein;
• iii) performing incremental truncation of each of the N-intein and the C-intein to produce every combination of deletion within the linker domain;
• iv) performing intramolecular blunt-ended ligation to produce an N-intein truncation library and a C-intein truncation library wherein each truncation fragment comprising the library terminates translation of protein fragments encoded thereby on stop codons in all reading frames in N-inteins and initiates translation with a start codon from C-inteins;
• v) introducing both libraries into a suitable host cell and
• vi) selecting said host cells for trans-intein activity by detecting production of the protein encoded by the first nucleic acid.