Protein expression and purification trials using the pCri System.

<p>(<b>A</b>) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in <i>E. coli</i> BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. (<b>B</b>) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in <i>E. coli</i> Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. (<b>C</b>) Partially purified MBP-fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of fragilysin (white) and the MBP (black) after TEV proteinase cleavage. (<b>D</b>) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in <i>E. coli</i> cells, and extracellularly (lanes 5 and 6) in <i>P. pastoris</i> cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. (<b>E</b>) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in <i>E. coli</i> BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. (<b>F</b>) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). (<b>G</b>) MecR1 was expressed in <i>E. coli</i> BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112643#s2" target="_blank">Materials and Methods</a>”. A black arrow indicates the detected MecR1. (<b>H</b>) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. (<b>I</b>) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.