Protocol

High Throughput Protein Expression and Purification

Volume 498 of the series Methods in Molecular Biology pp 105-115

A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins

  • William H. EschenfeldtAffiliated withBiosciences Division, Argonne National Laboratory
  • , Stols LucyAffiliated withBiosciences Division, Argonne National Laboratory
  • , Cynthia Sanville MillardAffiliated withBiosciences Division, Argonne National Laboratory
  • , Andrzej JoachimiakAffiliated withBiosciences Division, Argonne National Laboratory
  • , I. Donnelly MarkAffiliated withBiosciences Division, Argonne National Laboratory

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Summary

Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate pro tein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here.

Key words:

Structural genomics High throughput Protein purification Ligation-independ ent cloning Coexpression In vivo proteolysis Maltose-binding protein TEV protease TVMV protease