A New Strategy to Produce a Defensin: Stable Production of Mutated NP-1 in Nitrate Reductase-Deficient Chlorella ellipsoidea
Figure 2
Characterisation of the purified mNP-1.
(A) Tricine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) of purified mNP-1. Extracted proteins from transgenic nrm-4 cells were dialysed with a molecular weight cut-off of 3,500 Da and chromatographed using a G-25 column (Pharmacia & Upjohn, NJ, USA). The fractions with the activity against E. coli were collected and continuously chromatographed 4 times. Purified peptides (15 µg per lane) from the G-25 chromatography were analysed using Tricine-SDS-PAGE (10–20% gel gradient). The mNP-1 migrates faster than a similarly sized molecular weight marker (M) due to its greater charge. mNP-1 denoted by 1 and 2. (B) Reversed-phase high-pressure liquid chromatography (RP-HPLC) of purified mNP-1. A mixture containing 20 µg each of mNP-1 was applied to a C-18 column and developed using a linear water-acetonitrile gradient at a flow rate of 1 ml/min and monitored at 280 nm on a UltiMate 3000 (Dionex, Sunnyvale, USA). (C) MALDI-TOF-MS analysis of purified mNP-1. The mNP-1 isolated from RP-HPLC was analysed by MALDI-TOF-MS using a Bruker Autoflex (Bruker, Germany). The peak at the molecular weight, 4023.3 Da, is the mNP-1 with a single charge; another peak of molecular weight 2013.7 Da is the mNP-1 containing two charges.