Fig 1.
Features of the expression casette in the pONE vector set.
The expression cassette begins with an N-terminal secretion signal, followed by a sequence of MBP affinity tag, a protease cleavable linker, the multiple cloning site, and finally a C-terminal His-tag. All of these elements are exchangeable among the vectors, provided the restriction sites are compatible. Many, but not all, restriction sites are compatible among the vectors. There is at least one appropriate restriction enzyme combination for each possible pair of vectors. Minimal requirements that make a vector “pONE” compatible: NcoI and flanking NotI and XhoI sites. At least one of AgeI/KpnI/BamHI sites at the affinity tag/cleavage tag boundary. One of the NheI/AvrII pair. One of the EcoRI/MfeI pair. The pairs generate compatible overhangs, therefore they may be ligated together. (A) Schematic of the multiple cloning site. (B) Sequence details of the intracellular MBP-TEV variant of vectors. (C) Sequence details of the secreted MBP-WELQ variant of vectors.
Fig 2.
Promoters (green) and the MBP gene (red) in each vector are represented by arrows showing the direction of transcription and translation. Other important elements, depicted as rectangles are: 6X histidine tag (blue), resistance markers (orange) and replication origins (black). Secretion vectors also have genes encoding secretion signals (brown) in fusion with MBP. Important, unique restriction sites of the expression cassette are also marked. (A) The only E. coli vector of the set, pONE-10K carries the lacI and lacO elements of the lac operon (yellow). (B) Pichia pastoris vectors: pONE-23A (intracellular) and pONE-24A (secretion) have the AOX1 promoter, while pONE-25A (intracellular) utilizes the GAP promoter. (C) Insect cell vectors: pONE-30A (intracellular) and pONE-31A (secretion) are based on the baculovirus expression system, while pONE-32A (intracellular) and pONE-33A (secretion) can be used for transient transfection. (D) Mammalian vectors, fit for transient transfection: pONE-40A (intracellular) and pONE-41A (secretion). Vector maps were produced using BioEdit [19].
Table 1.
The list of unified vectors of the pONE series.
Fig 3.
Expression patterns of MBP using pONE vectors demonstrate that proteins of interest are expressed and directed to the relevant compartment.
Recombinant MBP (~45 kDa) was expressed in various hosts and pONE vectors, as described in Materials and Methods. Three parallel experiments were performed for each vector (marked by numbers 1–3). Samples were taken from the expression media (‘Extracellular’) and cell lysates (‘Intracellular’), then analysed via SDS PAGE followed by Western blotting. Relative dilutions (each valid within a given blot) are marked below the corresponding lanes. An anti-MBP antibody was used for detection of the recombinant protein. Blots were developed in a bioluminescent reaction (cf. Materials and Methods). Band intensities were determined by densitometry, and converted to total protein mass using internal calibration standards on each blot, with known amounts of MBP. Distribution of MBP between the extra- and intracellular compartments is presented in Table 2. (A) pONE-10K is a vector for intracellular protein expression in E. coli. Considering the sample dilutions, protein leakage is negligible. An MBP form of smaller molecular mass is also present, especially in the extracellular samples. This is likely the form natively expressed by E. coli, which has a molar weight of ~43 kDa (our recombinant variant is ~45 kDa). (B) MBP expression in P. pastoris using the pONE-23A (intracellular) and pONE-24A (secretion) vectors. Some inhomogeneities are detectable in the intracellular samples. These are most probably degradation products, resulting from protease activity in the cytoplasm. (C) pONE-30A (intracellular) and pONE-31A (secretion) are baculovirus based vectors for S. frugiperda cells, while pONE-32A (intracellular) and pONE-33A (secretion) are used for transient transfection. No degradation is observed. MBP targeting works well, with the probable exception of pONE-31A, where approximately half of the product is held back in the cell. (D) MBP expression in HEK293 cells, using the pLEXm based pONE-40A (intracellular) and pONE-41A (secretion) vectors. Intracellular retention of some MBP with the pONE-41A vector can be observed, this is probably misfolded material.
Table 2.
Expression yields and localization of recombinant MBP in various hosts.
Western blots (Fig 3) were subjected to densitometric analysis using ImageJ software [26]. Results were quantified using calibration standards on each Western blot. From these data relative distributions of MBP between the extracellular and intracellular compartments were calculated. “nd” means “not detected”. Expression patterns of MBP using pONE vectors demonstrate that proteins of interest are expressed and directed to the relevant compartment.
Fig 4.
ROCK2 expression and purification in various hosts.
To test the pONE vector set we have used it for the expression screening of a large, multi-domain human protein, the ROCK2 kinase. ROCK2 was expressed in the bacterium E. coli, the yeast P. pastoris and the insect cell S. frugiperda using the intracellular vectors pONE-10K, pONE25-A and pONE30-A, respectively. Cells were lysed and ROCK2 purified from the lysate by amylose affinity chromatography. Samples were taken during the process, and analysed by SDS PAGE and Western blotting using anti-MBP antibody (upper and lower images, respectively). As for the evaluation of the results, it is notable that impurities are generally more visible on Western blots due to its greater sensitivity and non-linearity compared to Coomassie staining. Each image presents the total protein content after expression (‘Total’) and the purified protein after the affinity chromatography (‘Purified’). Bands corresponding to full-length ROCK2 (‘FL’, Mw ~206 kDa with the fusion MBP tag) and the main degradation product (‘DEG’) are marked. E. coli is not suitable for ROCK2 expression, as the amount of full-length protein expressed is negligible compared to the degradation products, likely due to folding problems. In this host, the ROCK2 segment (Mw ~161 kDa) of the fusion protein degrades almost completely, while MBP (Mw ~45 kDa) remains intact, probably protected by its correct fold. In eukaryotic hosts, however, the bulk of ROCK2 is expressed as its full-length form. ROCK2 expressed in P. pastoris proved to be at least as pure as that expressed in S. frugiperda, the most commonly used host for ROCK2 expression. In fact, the presence of degradation products appears to be lower than in insect cells. However, both in P. pastoris and S. frugiperda a small amount of impurities appear along with the purified protein. Based on the Western blots, these are host proteins in the case of P. pastoris, and degradation products of ROCK2 in the case of S. frugiperda. In insect cells the degradation product is probably a fragmented MBP-ROCK2 fusion protein, where the C-terminal domain(s) of ROCK2 are cleaved off. It is demonstrated that yeast can effectively substitute insect cell culture for the expression of large, complex, multi-domain proteins.
Table 3.
Yields and activities for proteins expressed using pONE vectors.
Purification yields and functionality of full-length human ROCK2, Aurora A and LIMK1 kinases as well as a RASSF1A fragment expressed by different hosts (as illustrated by Figs 4 and 5).
Fig 5.
Purity of various protein constructs expressed using pONE vectors.
The pONE vector set was also applied to express the following proteins. Full-length LIMK1 (Mw ~117 kDa) was expressed in the insect cells S. frugiperda, using the vector pONE-30A. The kinase domain of Aurora kinase A (Mw ~79 kDa) as well as a RASSF1A fragment (residues 121–340, Mw ~70 kDa) were both expressed in the bacterium E. coli using pONE-10K. All molecular masses correspond to the recombinant proteins in fusion with an MBP tag. Samples were taken before harvesting the cells (‘Total’) and from the final, purified protein product (‘Purified’). The samples were analysed by SDS-PAGE and subsequent staining by Coomassie brilliant blue. All proteins were expressed in substantial quantities (Table 3), and could be purified. Using pONE vectors, pure recombinant proteins (LIMK1, Aurora A kinase domain and a RASSF1A fragment) were obtained with high yield.