Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (11), e112643

The pCri System: A Vector Collection for Recombinant Protein Expression and Purification


The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

Theodoros Goulas et al. PLoS One.


A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
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.
Figure 2
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.

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles


    1. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16: 23–52. - PubMed
    1. Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115: 113–128. - PubMed
    1. Kost T, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23: 567–575. - PMC - PubMed
    1. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, et al. (2012) The Pfam protein families database. Nucleic Acids Res 40: D290–D301. - PMC - PubMed
    1. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786. - PubMed

Publication types

MeSH terms


Grant support

This study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; CSD2006-00015; and 2014SGR9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.