N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E.

Removal of cleavage slow points from affinity tags used in the IMAC purification of recombinant proteins.

The complete enzymatic removal of affinity tags from tagged recombinant proteins is often required but can be challenging when slow points for cleavage exist. This study documents a general approach to remove N-terminal tags from recombinant proteins specifically designed to be efficiently captured by IMAC resins. In particular, site-directed mutagenesis procedures have been used to modify the amino acid sequence of metal binding tags useful in IMAC purifications of recombinant proteins with the objective to increase cleavage efficiency with the exopeptidase, dipeptidyl aminopeptidase 1.

Purification of a recombinant human growth hormone by an integrated IMAC procedure.

In this study, integration of three discrete process aspects of the IMAC purification of Escherichia coli expressed recombinant proteins has been investigated. To this end, novel N-terminally tagged human growth hormone variants (tagged-vhGHs) have been expressed in E. coli by tank fermentation and captured directly from the cell lysate by a new IMAC approach.

NMR studies of the backbone flexibility and structure of human growth hormone: a comparison of high and low pH conformations.

(15)N NMR relaxation parameters and amide (1)H/(2)H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.