Rapid assembly of synthetic genes encoding protein polymers

A general method is described for the rapid assembly of synthetic genes enc oding protein polymers based on the Seamless cloning technique. Genes encod ing repeats of the elastin-mimetic polypeptides [(Val-Pro-Gly-Val-Gly)(4)(V al-Pro-Gly-Xaa-Gly)] (Xaa = Lys 1; Ile, 2) were constructed using this tech nique. The use of this method eliminates the dependence of the concatameriz ation on a limited pool of nonpalindromic restriction endonucleases and red uces the number of subcloning steps. A synthetic gene of approximately 3000 base pairs in length was isolated that encoded a protein polymer based on repeat sequence 1. An inducible expression of this gene in bacterial cultur es of recombinant E. coli afforded a 90 kDa protein polymer of 1 in high yi eld (64 mg/L of bacterial culture). The protein was purified to homogeneity using immobilized metal affinity chromatography. The sequence of the prote in polymer was confirmed by N-terminal amino acid sequence analysis and MAL DI-TOF mass spectrometry of site-specific proteolytic cleavage fragments.