Method for producing proteins tagged at the N- or C-terminus

A method for producing one or more proteins in which one or more domains are full length and correctly folded and wherein each protein is tagged at either the N- or C-terminus with one or more marker moieties, said method comprising:

• (a) providing one or more DNA molecules having an open reading frame encoding said proteins together with 5′ and/or 3′ untranslated regions;
• (b) amplifying said DNA molecules under conditions that incorporate α-S-dNTPs as well as dNTPs into the daughter DNA molecules;
• (c) specifically protecting the 5′ or 3′ end of said daughter DNA molecules from nuclease digestion;
• (d) treating said DNA molecules first with a 3′ to 5′-nuclease to generate a set of nested deletions followed by treating with a single-strand nuclease under conditions that allow removal of said 5′ or 3′ untranslated regions including a start codon or a stop codon of said open reading frame;
• (e) cloning the fragments generated in step (d) into an expression vector containing a coding sequence for one or more 5′ or 3′ marker moieties;
• and
• (f) expressing said encoded proteins in which one or more domains are full length and correctly folded.