Purification of a recombinant human growth hormone by an integrated IMAC procedure.
作者： Jane T. Mooney , Dale P. Fredericks , Chunfang Zhang , Thorkild Christensen , Christina Jespergaard
DOI: 10.1016/J.PEP.2013.11.002
关键词:
摘要: Abstract In this study, integration of three discrete process aspects the IMAC purification Escherichia coli expressed recombinant proteins has been investigated. To end, novel N-terminally tagged human growth hormone variants (tagged-vhGHs) have in E. by tank fermentation and captured directly from cell lysate a new approach. The chelating ligands used were 1,4,7-triaza-cyclononane (tacn) bis(1,4,7-triazacyclononyl)-propane (dtnp) with copper(II) as immobilised metal ion. N-terminal tags specifically selected for their potential to bind these complexes also ease removal protein dipeptidyl peptidase, DAP-1. Low levels detergents binding buffer did not dramatically affect purification, but increased concentrations NaCl loading improved performance. same systems, operated ‘negative’ adsorption chromatographic mode, could be obtain purified mature variant, assessed MALDI-TOF sequencing studies, following affinity tag peptidase 1. Western immunoblot analysis eluted fractions both de-tagged vhGH demonstrated significant clearance host (HCPs). Further, resins can multiple times without need ion re-charging between runs. This study thus documents an integrated approach genetically modified .
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