Table 1.
pCri-a vectors.
Table 2.
pCri-b vectors.
Table 3.
pCri-a-Strep vectors.
Figure 1.
Vector overview of the pCri System.
(A) Vectors for cytoplasmic protein expression. (B) Vectors for periplasmic and extracellular protein expression. An N-terminal His6-tag can be fused in all vectors for intracellular expression except of pCri-7. Other tags can also be fused including MBP, TRX, GST, SUMO, MISTIC, and LSL (Table 1–3). In all vectors, a C-terminal His6-tag or Strep-tag is attached if a stop codon is omitted within the target gene. Black arrows indicate the proteinase (i.e. TEV, SENP1 or thrombin) and signal peptide (SP) cleavage sites. Restriction sites allowing directional cloning are also shown. For more details regarding each vector, refer to Fig. S1.
Table 4.
Sequencing primers for the pCri System.
Figure 2.
Protein expression and purification trials using the pCri System.
(A) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli 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) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli 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. (C) 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. (D) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris 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. (E) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. (F) 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). (G) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “Materials and Methods”. A black arrow indicates the detected MecR1. (H) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. (I) 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.