Method for producing circular or multimeric protein species in vivo or in vitro and related methods

摘要

A method is disclosed for the in vitro or in vivo cyclization of protein or peptide sequences. Also disclosed is a method of fusing polypeptide sequences while bound to a solid support These protein manipulation techniques relied on the trans-splicing activity of a split intein such as the naturally occuring split intein form the DnaE gene of Synechocysil sp. PCC6803 (Ssp DnaE intein). The cyclization procedures required the fusion of C- and N-terminal intern splicing domains to the N- and C-termini, respectively, of a target protein (Ineinc-target protein-InteinN). Cyclization in vivo occurred post-translationally when the two complementary intein splicing domains ligated the N- and C-terminus of the target protein. In vitro cyclization also utilized and InteinC-target protein-InteinN precursor protein, in which the intein domains were fused to a chitin binding domain (CBD). Protein expression was conducted under conditions that favored the accumulation of precursor protein. which was imnmobilized on a chitin resin. The circular protein species were eluted from the chitin resin following incubation under conditions that favored protein splicing. Trams-splicing was used to ligate polypeptides on a solid support by generating a protein composed of a CBD fused to a C-terminal intern splicing domain and target protein (1). This was incubated with a protein composed of target protein (2) fused to an N-terminal intein splicing domain and a CBD. The precursor proteins were immobilized on a chitin resin where trans-splicing resulted in the ligation of target protein (1) to target protein (2). These techniques greatly expand the procedures available for protein engineering and modification.