Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts

A novel, two-step preparative technique is described for the purification o f authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coil cells. The first step is based on immobili zed metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific Ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exc lusion chromatography is used for further purification to > 99.5% purity. T he methodology is reproducible, leading to 77% recovery. Identity and purit y of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamid e. electrophoresis, isoelectric focusing, mass spectrometry (matrix-assiste d laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC an d high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg. (C) 2001 Elsevier Science B.V. All rights reserved.