Purification of rhHC.

<p>rhHC purified from approximately 0.5 L HEK293 cell culture supernatant. (A) Sequence alignment of native HC and tagged rhHC. Thrombin cleavage site (<b>bold</b>), Myc-tag (<u>underlined</u>) and 6×His-tag (<i>italic</i>) (B) Coomassie stained SDS/PAGE and (C) Western blot analysis of the different purification steps. Lanes: 1, standards, 2, supernatant of HEK293 cells transfected with rhHC, 3, elution of first Ni²⁺ affinity chromatography (tagged rhHC), 4, peak fraction of first size exclusion chromatography (tagged rhHC), 5, flow-through of second Ni²⁺ affinity chromatography after thrombin cleavage, 6, peak fraction of second size exclusion chromatography, 7, human native HC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037421#pone.0037421-Nexo2" target="_blank">[9]</a>. (D) and (E) UV-chromatograms at 280 nm of first and second size exclusion chromatography before and after thrombin cleavage on a Superdex 200 column at a flow rate of 48 ml h⁻¹. Elution volumes of Blue Dextran 2000 (V₀) and albumin (V_alb, 67 kDa) are marked with arrows.