Cleavable C-terminal His-tag vectors for structure determination |
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Authors: | William H Eschenfeldt Natalia Maltseva Lucy Stols Mark I Donnelly Minyi Gu Boguslaw Nocek Kemin Tan Youngchang Kim Andrzej Joachimiak |
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Institution: | (1) Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Bldg. 202/Rm. BE111, 9700 South Cass Avenue, Argonne, IL 60439, USA;(2) Center for Structural Genomics of Infectious Diseases, Computational Institute, University of Chicago, Chicago, IL 60667, USA; |
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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 His6-tag and His6- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases. |
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