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pET System

Advantages Powerful Highest expression levels, tightest control over basal expression Versatile Choice of N-terminal and C-terminal peptide tags for detection, purification and localization Expanded multiple cloning sites in all three reading frames Choice of expression controls and host strains to optimize expression levels f1 origin of replication for mutagenesis and sequencing Rapid E. coli-based system for rapid results Convenient restriction sites for subcloning from other vectors Choice of methods for one-step purification of target proteins without antibodies SïTagô sequence allows rapid quantification of target proteins in crude extracts Complete Variety of system configurations plus many supporting products pET Vector Backbones The pET vectors shown below are grouped according to the functional elements present on the plasmid backbones. Features of the T7 cloning and expression regions (indicated here as "T7" and "T7lac") are shown below and in the Appendix. Note that the pET vector sequences are numbered by the pBR322 convention, so that the T7 expression region is reversed on these maps and in the published sequences. pET vector backbones: "plain" T7 promoter vectors pET vector backbones: T7lac promoter vectors Cellulose Binding Domains Several pET vectors and pBACô transfer plasmids feature CBDïTag sequences, which encode cellulose binding domains derived from microbial cellulases. These unique domains enable the use of cellulose-based solid supports for highly specific, low cost affinity purification of recombinant fusion proteins. The system is especially useful for protein immobilization applications, such as screening for protein:protein interactions. Three CBDïTag sequences are available for pET constructs; all share high affinity for CBIND cellulose supports and are efficiently eluted with ethylene glycol, but differ slightly in other characteristics. CBDclosïTag (158 aa): active as N-terminus of fusion proteins, designed for cytoplasmic expression CBDcenAïTag (114 aa): active as N-terminus of fusion proteins, includes its native signal sequence for potential export into the periplasm, may be eluted from cellulose with water CBDcexïTag (107 aa): active as C-terminus of fusion proteins, includes signal sequence for export, high levels of fusion proteins may be found in media, may be eluted from cellulose with water CBDclosïTag and CBDcexïTag fusions are also available with pBAC transfer plasmids for baculovirus expression. In addition, two new pBAC transfer plasmids contain the CBDcenDïTag sequence, which enables purification of fusion proteins directly from insect cell culture medium.

The pET System* is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Based on the T7 promoter-driven system originally developed by Studier and colleagues (1ó3), the pET System has been greatly expanded and now includes over 35 vector types, 11 different E. coli host strains and many other companion products designed for efficient detection and purification of target proteins (see Choosing a pET Vector and How to Order below). Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that almost all of the cell's resources are converted to target gene expression. The desired product can comprise more than 50% of the total cell protein a few hours after induction. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, so they are virtually "off" and cannot cause plasmid instability due to the production of proteins potentially toxic to the host cell. Once established, plasmids are transferred into expression hosts containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, and expression is induced by the addition of IPTG. Many genes that have been difficult to establish in E. coli promoter-based systems (e.g., tac, lac, trc, pL) have been stably cloned and expressed in the pET System. Control Over Basal Expression Levels The pET System also uniquely provides six possible vector-host combinations that enable tuning of basal expression levels to optimize target gene expression (2). These options are necessary because no single strategy or condition is suitable for every target protein. Host Strains After plasmids are established in a non-expression host, they are most often transformed into a host bearing the T7 RNA polymerase gene (DE3 lysogen) for expression of target proteins. Figure 1 illustrates in schematic form the host and vector elements available for control of T7 RNA polymerase levels and the subsequent transcription of a target gene in a pET vector. In DE3 lysogens, the T7 RNA polymerase gene is under the control of the lacUV5 promoter. This allows some degree of transcription in the uninduced state and in the absence of further controls is suitable for expression of many genes whose products have innocuous effects on host cell growth. For more stringent control, hosts carrying either pLysS or pLysE are available. The pLys plasmids encode T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase, and thus reduces its ability to transcribe target genes in uninduced cells. pLysS hosts produce low amounts of T7 lysozyme, while pLysE hosts produce much more enzyme and, therefore, represent the most stringent control available in DE3 lysogens (4). Figure 1. Control elements of the pET system Several different host strains are available as DE3 lysogens. The most widely used host is BL21, which has the advantage of being deficient in both lon (5) and ompT proteases. Novagen has introduced two derivatives of BL21 designed for special purposes. The B834 series is methionine deficient and, therefore, enables high specific activity labeling of target proteins with 35S-methionine or selenomethionine (6). The BLR strain is a recA- derivative that improves plasmid monomer yields and may help stabilize target plasmids containing repetitive sequences. The AD494 strains are thioredoxin reductase (trxB) mutants that enable disulfide bond formation in the E.coli cytoplasm. This allows for the potential production of properly folded, active proteins (7). Other available strain backgrounds include the K-12 strains HMS174 and NovaBlue, which are recA-, like BLR. These strains may stabilize certain target genes whose products may cause the loss of the DE3 prophage. NovaBlue is potentially useful as a stringent host due to the presence of the high affinity lacIq repressor encoded by the F episome. In addition, Novagen offers the DE3 Lysogenization Kit for making new expression hosts with other genetic backgrounds. An alternative for expressing extremely toxic genes or preparing a new DE3 lysogen is to provide T7 RNA polymerase by infection with CE6. Although not as convenient as inducing a DE3 lysogen with IPTG, this strategy may be preferred for certain applications. High Stringency T7lac Promoter In addition to the choice of three basic expression stringencies at the host level, the pET system provides two different stringency options at the level of the T7 promoter itself: the "plain" T7 promoter and the T7lac promoter (8; also shown in Fig. 1). The T7lac promoter contains a 25 bp lac operator sequence immediately downstream from the 17 bp promoter region. Binding of the lac repressor at this site effectively reduces transcription by T7 RNA polymerase, thus providing a second lacI-based mechanism (besides the repression at lacUV5) to suppress basal expression in DE3 lysogens. Plasmids with the T7lac promoter also carry their own copy of lacI to ensure that enough repressor is made to titrate all available operator sites. In practice, it is usually worthwhile to test several different vector/host combinations to obtain the best possible yield of protein in its desired form (9). Figure 2 illustrates dramatic differences in the expression of two target proteins with various combinations. Figure 2. Effect of vector/host combination on expression levels of two proteins The indicated cell cultures were grown at 37 °C to OD600 of approximately 0.8 and expression induced with 1 mM IPTG for 2.5 h. Total cell protein samples were run along with Novagen's Perfect Proteinô Markers on a 4ó20% SDS polyacrylamide gradient gel followed by staining with Coomassie blue. Vectors used were pET-20b(+) and pET-22b(+) for the recombinant antibody and pET-23b(+) and pET-21b(+) for p53. pET Vector Characteristics A wide variety of pET vectors is available. All except the specialized pSCREENô-1b(+) vector are derived from pBR322 and vary in leader sequences, expression signals, relevant restriction sites, and other features (see pET Vector classification table following the section, How to Order). There are two major categories of pET plasmids known as transcription vectors and translation vectors: The transcription vectors (including pET-21, pET-23, and pET-24) express target RNA but do not provide translation signals. They are useful for proteins from target genes that carry their own bacterial translation signals. Note that the transcription vectors can be identified by lack of a letter suffix after the name. The translation vectors contain efficient translation initiation signals and are designed for protein expression. Most contain cloning sites in reading frames a, b, or c that correspond to the GGA, GAT, or ATC triplet of the BamH I site, respectively. Refer to Choosing a pET Vector for further details. References Moffatt, B.A. and Studier, F.W. (1986) J. Mol. Biol. 189, 113ó130. Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and Studier, F.W. (1987) Gene 56, 125ó135. Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60ó89. Studier, F.W. (1991) J. Mol. Biol. 219, 37ó44. Phillips, T.A., Van Bogelen, R.A., and Neidhardt, F.C. (1984) J. Bacteriol. 159, 283<ó287. Leahy et al. (1992) Science 258, 987ó991. Derman, A.I., Prinz, W.A., Belin, D., and Beckwith, J. (1993) Science 262, 1744ó1747. Dubendorff, J.W. and Studier, F.W. (1991) J. Mol. Biol. 219, 45ó59. Mierendorf, R., Yaeger, K., and Novy, R. (1994) inNovations 1, 1ó3. Choosing a pET Vector Use the following section in conjunction with the Fusion Tag Table and the pET Vector Characteristics Table to decide which vector is best for your application. Ordering information for the vectors and kits follows the tables. The Appendix contains sequences of cloning and expression regions for all of the pET vectors. Primary Considerations Choosing a pET vector for expression usually involves a combination of factors. Consider the following three primary factors: The application intended for the expressed protein Specific information known about the expressed protein Cloning strategy Applications for proteins expressed in pET vectors vary widely. For example, analytical amounts of a target protein may be needed for activity studies, screening and characterizing mutants, screening for ligand interactions, and antigen preparation. Large amounts of active protein may be required for structural studies, use as a reagent, or affinity matrix preparation. Any number of vectors may be suitable for expression of analytical amounts of protein for screening or antigen preparation, yet only one combination of vector, host strain, and culture conditions may work best for large scale purification. If a high yield of active protein is needed on a continual basis, it is worth testing a matrix of vector, host, and culture combinations to find the optimal result. Any known information available about the target protein may help determine the choice of vector. For example, some proteins require no extraneous sequence on one or both termini for activity. Most pET vectors enable cloning of unfused sequences; however, expression levels may be affected if a particular translation initiation sequence is not efficiently utilized in E. coli. In these cases, an alternative is to construct a fusion protein with efficiently expressed amino terminal sequences (available with many pET vectors; indicated on the Characteristics Table with an N) and then remove the fusion partner following purification by digestion with a site-specific protease. The LIC (ligation independent cloning) vectors are especially useful for this strategy, because the cloning procedure enables the removal of all amino terminal vector-encoded sequences with either enterokinase or Factor Xa (in the Characteristics Table, look for vectors containing both LIC and a protease cleavage site). Cloning strategies can affect the choice of vector due to the need for restriction site and reading frame compatibilities. Because many of the pET vectors share common restriction site configurations, it is usually possible to clone a target gene into several vectors with a single preparation of the insert. Different considerations apply when using PCR cloning strategies. The LIC vector kits are recommended for this purpose, and enable the preparation of inserts by PCR and eliminate the need for restriction digestion of vector or insert. The Appendix contains sequences of cloning and expression regions for all of the pET vectors. Consider Solubility and Cellular Localization Once you have considered your application and cloning strategy, a good starting point for any expression project is to determine the cellular localization and solubility of the target protein. Protein solubility can be greatly influenced by the vector, host cell, and culture conditions. In many applications, it is desirable to express target proteins in their soluble, active form. Solubility of a particular target protein is determined by a variety of factors, including the individual protein sequence. In most cases, solubility is not an all-or-none phenomenon; the vector, host, and culture conditions can be used to increase or decrease the proportion of soluble and insoluble forms obtained. The pET-32 vector series enables the fusion of target sequences with thioredoxin (TrxïTag), which usually increases the fraction of soluble protein. In addition, the trxB mutant strain AD494 can be used to allow the formation of disulfide bonds, which are required for proper folding and activity of many eukaryotic proteins in the cytoplasm. Induction at lower temperatures (18ó25 °C) can also increase the proportion of soluble target proteins. See also Host Strains described earlier in this section. An alternative strategy to obtain active, soluble proteins is to use vectors that enable export into the periplasm, which is a more favorable environment for folding and disulfide bond formation. For this purpose vectors carrying signal peptides are used, such as the new pET-39b(+) and pET-40b(+) vectors carrying the DsbA and DsbC fusion sequences, respectively. Some purification strategies optimize production of insoluble inclusion bodies in the cytoplasm. Inclusion bodies are extracted, solubilized; then the target protein is refolded in vitro. This procedure usually produces the highest yields of initial protein mass and protects against proteolytic degradation in the host cell. However, the efficiency of refolding into active protein varies significantly with the individual protein and can be quite low. Therefore, this approach is often used for producing antigens or in other applications for which proper folding is not required. One pET vector, pET-31b(+), is specifically designed for the generation of insoluble fusion proteins. Fusion Tags for Different Needs If a fusion sequence is tolerated by the application you are using, it is useful to produce fusion proteins carrying the SïTagô, T7ïTag®, or HSVïTag® peptides for easy detection on Western blots. These peptides (fusion sequences) are small in size and the detection reagents for them are extremely specific and sensitive. The SïTag and T7ïTag sequences can also be used for affinity purification using the corresponding resins and buffer kits. The HisïTagô sequence is very useful as a fusion partner for purification of proteins in general. It is especially useful for those proteins initially expressed as inclusion bodies, because affinity purification can be accomplished under totally denaturing conditions that solubilize the protein. The CBDïTagô sequences are also generally useful for low cost affinity purification (see sidebar). They are also uniquely suited to refolding protocols (especially pET-34b(+) and -35b(+), which contain the CBDclosïTag sequence); because only properly refolded CBDs bind to the cellulose matrix, the CBIND affinity purification step can remove improperly folded molecules from the preparation. While any of the tags can be used to immobilize target proteins, the CBDïTag sequences are ideally suited for this purpose due to the inherent low non-specific binding and biocompatibility of the cellulose matrix. Refer also to Novagen's inNovations Newsletter No. 7 for more details about cellulose binding domains. The various fusion tags available and their corresponding pET vectors are listed in the following table. A number of pET vectors carry several of the fusion tags in tandem as 5' fusion partners. In addition, many vectors enable expression of fusion proteins carrying a different peptide tag on each end by allowing in-frame read-through of the target gene sequence. Using vectors with protease cleavage sites (thrombin, Factor Xa, enterokinase) between the 5' tag and the target sequence enables optional removal of one or more tags following purification. Vectors that represent a good selection for cellular localization and affinity tag configurations are pET-30 Ek/LIC, pET-32 Ek/LIC, pET-34 Ek/LIC and pET-36 Ek/LIC. The pET Ek/LIC Vector Combo Kit includes all 4 of these vectors in ready-to-use form to allow convenient construction of several target gene configurations at once. How to Order After reading through Choosing a pET Vector and the previous information, refer to the Vector Characteristics Table to select a pET vector that fits your needs. Note that the pET Vectors are available as purified plasmid DNA as well as configured into Expression Systems. For pET Vectors 31b(+) through 40b(+), the individual vectors and supplemental kits are described in greater detail beginning on page 48. The list of pET Host Strain Glycerol Stocks, Competent Cells, and genotypes complete the pET Vector section. Detection and Purification Reagents are listed at the end of the pET section.