Abstract

High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein's intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His(6)-tag and His(6)- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases.