Construction and cloning of recombinant rat trypstatin variants and their expression as fusion proteins in E. coli.

A synthetic master gene coding for rat trypstatin was designed, assembled of eight oligonucleotides, ligated into the cloning vector pUC8 and cloned in E. coli. In addition to the expected product DNA sequencing revealed the presence of several gene variants. The gene coding for (-1M F44G) rat trypstatin was mutated to a wild type form (-1M) rat trypstatin by cassette mutagenesis. For the first expression experiments the E. coli pEx31 system was used. After SDS-PAGE analysis fusion proteins were detected consisting of the N-terminal part of MS2-polymerase, a linker region and of the appropriate rat trypstatin variant. These fusion proteins were synthesized in amounts as high as 25% of total E. coli proteins.