| The choice of vector and expression host can significantly increase the activity and amount of target protein present in the soluble fraction. A vector can enhance solubility and/or folding in one of three ways– 1) provide for fusion to a polypeptide that itself is highly soluble (e.g. GST, Trx, NusA)
2) provide for fusion to an enzyme that catalyzes disulfide bond formation (e.g. thioredoxin, DsbA, DsbC)
3) provide a signal sequence for translocation into the periplasmic space When using vectors designed for cytoplasmic expression, folding can be improved in hosts that are permissive for the formation of disulfide bonds in the cytoplasm. The thioredoxin reductase (trxB) mutation has been shown to allow the formation of disulfide bonds in the E. coli cytoplasm, which is further enhanced by the additional mutation in the glutathione reductase (gor) gene in Origami™ and Rosetta-gami™ hosts.
Fusion Tags in pET Vectors
Cytoplasmic Localization
The pET-44, pET-43.1 and pET-32 vectors incorporate fusion tags specifically designed to enhance the solubility of target proteins in the E. coli cytoplasm. These vectors are also compatible with trxB mutant hosts AD494 and BL21trxB, and with the trxB/gor mutant Origami, Origami B, and Rosetta-gami strains. Nus•Tag™ sequence: pET-43.1 and pET-44 series
The pET-43.1 and pET-44 vectors incorporate the 495 aa solubility-promoting NusA (Nus•Tag) sequence, which was discovered through a systematic search for E. coli proteins that have the highest potential for solubility when overexpressed. [Harrison, R. (2000) inNovations 11, 4–7; Novy, R. and Drott, D. (2002) inNovations 14, 12-13] Trx•Tag™ sequence: pET-32 series
Many proteins that are normally produced in an insoluble form in E. coli tend to become more soluble when fused with the 109 aa N-terminal thioredoxin (Trx•Tag) sequence. The Trx•Tag expressed from pET-32 vectors not only enhances the solubility of many target proteins, but appears to catalyze the formation of disulfide bonds in the cytoplasm of trxB mutants [Stewart et al. (1998) EMBO J. 17, 5543–5550]. GST•Tag™ sequence: pET-41 and 42 series
Schistosomal glutathione-S-transferase (GST) is commonly used as an N-terminal fusion partner when expressing proteins in E. coli. Although not specifically designed for this purpose, the 220 aa GST•Tag sequence has been reported to enhance the solubility of its fusion partners. The pET-41 and -42 series of vectors encode the GST•Tag sequence driven by the powerful T7lac promoter. Note that these vectors carry kanamycin resistance, so are not recommended for use with trxB mutant hosts. Periplasmic Localization
pET Dsb Fusion Systems 39b and 40b
DsbA and DsbC are periplasmic enzymes that catalyze the formation and isomerization of disulfide bonds, respectively. The 208 aa DsbA•Tag™ [pET-39b(+)] and 236 aa DsbC•Tag™ [pET-40b(+)] vectors enable fusion of target polypeptides to these enzymes, which include their N-terminal secretion signals. If the fusion protein is exported to the periplasm, the Dsb partner can assist in proper disulfide bond formation. Note that other pET vectors that carry signal sequences without the additional DsbA or DsbC coding regions are also available (pET-12 with ompT signal sequence; and pET-20, pET-22, pET-25, pET-26, and pET-27 with pelB signal sequence).
Features Common to
Above Vectors
| • | Tightly controlled T7lac promoter and optimal bacterial ribosome binding site | | • | Internal 6 aa His•Tag® (N-terminal in pET-44) and 15 aa S•Tag™ sequences for efficient affinity purification and detection by homogeneous FRET assay, Western blot, and immunofluorescence | | • | Thrombin and Enterokinase cleavage sites for complete N-terminal tag removal (pET-42 has thrombin and enterokinase or factor Xa sites) | | • | Multiple cloning sites in all three reading frames [except for pET-39b(+), 40b(+)] | | • | Optional C-terminal HSV•Tag® epitope and His•Tag sequences | | • | Most available as linearized vectors prepared for efficient, directional ligation-independent cloning (LIC) of PCR products |
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E. coli Host Strains
Tuner
Tuner™ strains are lacYZ deletion mutants of the popular protease-deficient BL21 strain, and enable adjustable levels of protein expression throughout all cells in a culture. The lac permease (lacY) mutation allows uniform entry of IPTG into all cells in the population, which produces a concentration-dependent, homogeneous level of induction. By adjusting the concentration of IPTG, expression can be regulated from very low level expression up to the robust, fully induced expression levels commonly associated with pET vectors. Lower level expression may enhance the solubility and activity of difficult target proteins. Rosetta
Rosetta™ strains are derived from Tuner strains, hence they possess the same advantages afforded by the lacYZ mutation and BL21 background. However, Rosetta strains carry an additional feature, the pRARE plasmid, which encodes tRNAs for mammalian codons that rarely occur in E. coli, and therefore enhances the expression levels of proteins otherwise limited by codon usage [Novy et al. (2001) inNovations 12, 1–4]. The pRARE plasmid is selectable on chloramphenicol and is compatible with all pET vectors. Origami
Origami host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, the combination of which greatly enhances disulfide bond formation in the cytoplasm [Prinz et al. (1997) J. Biol. Chem. 272, 15661–15667]. Studies have shown that expression in Origami(DE3) yielded 10-fold more active protein than in a standard host even though overall expression levels were similar. Origami hosts are compatible with ampicillin-resistant plasmids and are ideal for use with pET-43.1, pET-44 and pET-32 series vectors.
Origami B
Origami B host strains combine the protein-folding benefits of Origami E. coli with precise control of expression levels in Tuner strains (see above). These strains have the protease-deficient genetic background of BL21 and are compatible with ampicillin-resistant plasmids. Rosetta-gami
Rosetta-gami strains are derived from Origami strains, and carry the pRARE plasmid for rare tRNAs in a K-12 background with the trxB/gor mutations for enhanced disulfide bond formation in the cytoplasm. These strains are compatible with ampicillin-resistant vectors, such as the pET-43.1, pET-44 and pET-32 series. AD494
AD494 strains are K-12 derived thioredoxin reductase (trxB) mutants that enable disulfide bond formation in the cytoplasm. The trxB mutation is selectable on kanamycin; therefore, this strain is recommended for use with plasmids carrying the ampicillin resistance marker bla. BL21trxB
BL21trxB strains possess the same thioredoxin reductase mutation (trxB) as the AD494 strains in the protease deficient BL21 background, this strain is recommended for use with plasmids carrying the ampicillin resistance marker bla.
And for Protein Extraction...
Novagen has developed a family of reagents designed for simple, efficient extraction of soluble proteins from E. coli without mechanical disruption. BugBuster™ Protein Extraction Reagent is a proprietary mixture of detergents that perforates the cell wall without denaturing proteins. BugBuster comes as a ready-to-use reagent for direct resuspension of bacterial pellets, and is also available as a 10X concentrate to enable user control of pH, concentration, and buffer additives. The addition of a small amount of lysozyme along with BugBuster increases the extraction efficiency and cell lysis, which can increase the yield of larger target proteins. BugBuster is compatible with Benzonase® Nuclease, a broad spectrum nuclease that degrades all forms of DNA and RNA, for efficient viscosity reduction and nucleic acid removal of protein samples. Protease inhibitors are also available for use with BugBuster reagents. BugBuster HT combines BugBuster Protein Extraction Reagent and Benzonase in one convenient solution, and is well-suited for processing multiple samples. For automated procedures and other high-throughput applications, Novagen has developed the novel PopCulture™ Reagent. This detergent-based concentrate is added directly to cell cultures for extraction of proteins without the need for centrifugation or other cell collection methods [Grabski et al. (2001) inNovations 13, 1–4]. |
