title: Purification of a recombinant human growth hormone by an integrated IMAC procedure.
abstract: 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. The chelating ligands used were 1,4,7-triaza-cyclononane (tacn) and bis(1,4,7-triazacyclononyl)-propane (dtnp) with copper(II) as the immobilised metal ion. The N-terminal tags were specifically selected for their potential to bind to these immobilised complexes and also for their ease of removal from the tagged protein by the dipeptidyl peptidase, DAP-1. Low levels of detergents in the binding buffer did not dramatically affect the purification, but increased concentrations of NaCl in the loading buffer improved the binding performance. The same IMAC systems, operated in the 'negative' adsorption chromatographic mode, could be used to obtain the purified mature human growth hormone variant, as assessed by MALDI-TOF and N-terminal sequencing studies, following removal of the affinity tag by the dipeptidyl peptidase 1. Western immunoblot analysis of the eluted fractions of both the tagged and de-tagged vhGH demonstrated significant clearance of E. coli host cell proteins (HCPs). Further, these IMAC resins can be used multiple times without the need for metal ion re-charging between runs. This study thus documents an integrated approach for the purification of specifically tagged recombinant proteins expressed in genetically modified E. coli.
authors.affiliation: Centre for Green Chemistry, Building 75, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Jane T
authors.initials: JT
authors.lastname: Mooney
authors.affiliation: Centre for Green Chemistry, Building 75, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Dale P
authors.initials: DP
authors.lastname: Fredericks
authors.affiliation: Centre for Green Chemistry, Building 75, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Chunfang
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Thorkild
authors.initials: T
authors.lastname: Christensen
authors.affiliation: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Christina
authors.initials: C
authors.lastname: Jespergaard
authors.affiliation: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Christine Bruun
authors.initials: CB
authors.lastname: Schiødt
authors.affiliation: Centre for Green Chemistry, Building 75, Monash University, Clayton, Victoria 3800, Australia. Electronic address: Milton.hearn@monash.edu.
authors.forename: Milton T W
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D002614:Chelating Agents; D011994:Recombinant Proteins; D019382:Human Growth Hormone
keywords: 1,4,7-Triazacyclononane (tacn); Bis(1,4,7-triazacyclononyl)propane; Human growth hormone; IMAC adsorbents; N-terminally tagged proteins; Novel affinity tags; Tag removal