pET Vector Characteristics
A wide variety of pET vectors is available. All except the specialized pSCREEN˘-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,
Refer to Choosing a pET Vector for further details.
- Moffatt, B.A. and Studier, F.W. (1986) J. Mol. Biol. 189,
- Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and
Studier, F.W. (1987) Gene 56, 125ˇ135.
- Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W.
(1990) Meth. Enzymol. 185, 60ˇ89.
- Studier, F.W. (1991) J. Mol. Biol. 219, 37ˇ44.
- Phillips, T.A., Van Bogelen, R.A., and Neidhardt, F.C. (1984)
J. Bacteriol. 159, 283<ˇ287.
- Leahy et al. (1992) Science 258, 987ˇ991.
- Derman, A.I., Prinz, W.A., Belin, D., and Beckwith, J. (1993)
Science 262, 1744ˇ1747.
- Dubendorff, J.W. and Studier, F.W. (1991) J. Mol. Biol.
- Mierendorf, R., Yaeger, K., and Novy, R. (1994) inNovations 1, 1ˇ3.
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.
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 (Trx´Tag), 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 (18ˇ25 °C) can also increase the proportion of soluble
target proteins. See also Host Strains described earlier in this
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 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 S´Tag˘, T7´Tag®, or HSV´Tag®
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 S´Tag and T7´Tag sequences can also be used for affinity purification using the
corresponding resins and buffer kits.
The His´Tag˘ 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 CBD´Tag˘ 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 CBDclos´Tag 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 CBD´Tag 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.