An expression vector tailored for large-scale, high-throughput purification of recombinant proteins.

@article{citeulike:1631228, abstract = {Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his(6)-tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his(6)-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his(6)-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his(6)-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his(6)-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his(6)-tag.}, address = {Biosciences Division, Argonne National Laboratory, IL 60439, USA. mdonnelly@anl.gov}, author = {Donnelly, M. I. and Zhou, M. and Millard, C. S. and Clancy, S. and Stols, L. and Eschenfeldt, W. H. and Collart, F. R. and Joachimiak, A. }, citeulike-article-id = {1631228}, doi = {http://dx.doi.org/10.1016/j.pep.2005.12.011}, issn = {1046-5928}, journal = {Protein Expr Purif}, keywords = {protein-expression}, month = {June}, number = {2}, pages = {446--454}, posted-at = {2007-09-07 13:05:09}, priority = {2}, title = {An expression vector tailored for large-scale, high-throughput purification of recombinant proteins.}, url = {http://dx.doi.org/10.1016/j.pep.2005.12.011}, volume = {47}, year = {2006} }

TY - JOUR ID - citeulike:1631228 L3 - citeulike-article-id:1631228 TI - An expression vector tailored for large-scale, high-throughput purification of recombinant proteins. JF - Protein Expr Purif VL - 47 IS - 2 SP - 446 EP - 454 AD - Biosciences Division, Argonne National Laboratory, IL 60439, USA. mdonnelly@anl.gov SN - 1046-5928 N2 - Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his(6)-tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his(6)-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his(6)-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his(6)-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his(6)-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his(6)-tag. KW - protein-expression AU - Donnelly, MI AU - Zhou, M AU - Millard, CS AU - Clancy, S AU - Stols, L AU - Eschenfeldt, WH AU - Collart, FR AU - Joachimiak, A PY - 2006/06// UR - http://dx.doi.org/10.1016/j.pep.2005.12.011 ER -