The first vector we will consider is the pUC family of vectors

Figure 3.4.1: pUC vector

Although it is not typically used for the expression of recombinant proteins, it has all the necessary elements of an expression vector:

1.      An origin of replication. The pUC family of vectors are high copy vectors. They have a ColE1 origin of replication, but a deletion of the rop replication regulatory region.

2.      A drug resistance marker. The pUC vectors contain a gene for ampicillin resistance (b-lactamase).

3.      An inducible promoter. This family of vectors contains the lac promoter (P_lac) along with the associated lac Operator region (O_lac)

These vectors contain a couple of other elements which add utility to the vector:

1.      The lacI gene. This gene codes for the lac repressor protein. Being a high copy plasmid the host lacrepressor levels may not be sufficient to efficiently repress the lac operator on the plasmids. The plasmid produces lac repressor to augment the host levels.

2.      The lacZ’ gene. This gene product is transcribed from the lac promoter and produces an amino-terminal fragment of the b-galactosidase protein.

3.      A polylinker (multiple cloning site). This short stretch of DNA is located just downstream from the lacpromoter and after the first few codons of the lacZ’ gene. It is a short stretch of nucleotides which contains a variety of restriction endonuclease sites.

a-complementation

The lac promoter of the pUC plasmid can be induced using the lactose analogue isopropylthiol-b-D-galactoside. (“IPTG”).

·         This will result in the expression of the lacZ’ gene.

·         This gene codes for an amino-terminal fragment of the b-galactosidase protein

·         By itself, this fragment is non-functional (it will not hydrolyze lactose or other b-galactosides)

However, b-galactosidase is an interesting protein:

·         If we express the carboxy-terminal fragement of the protein it is also non-functional

·         But, if we combine both peptide fragments they can combine to yield a functional b-galactosidase protein

·         This is termed a-complementation

The lacZDM15 mutation in a bacteria means that its genome has suffered a deletion of the amino-terminal region of the lacZ gene.

·         Such a bacteria has a non-functional b-galactosidase protein

·         If this type of bacteria contains an extra-chromosomal element which expresses the lacZ’ peptide, it can complement the lacZDM15 protein to produce functional b-galactosidase protein (a-complementation)

The galactoside, 5-bromo-4-chloro-3-indoyl-b-D-galactoside (“X-gal”), can be hydrolyzed by b-galactosidase and produces a dark blue color.

·         lacZDM15 bacteria in the presence of lactose (or IPTG) and X-gal will appear a normal white-yellow color.

·         The same bacteria which harbor the above pUC plasmid will be dark blue in color when IPTG and X-gal are present in the media (due to a-complementation and a functional b-galactosidase protein)

The pUC polylinker region

The pUC polylinker region is a short stretch of DNA which is actually inserted within the lacZ’ gene, just downstream from the start codon:

Figure 3.4.2: pUC Polylinker region

·         The different available plink regions are all a multiple of 3 nucleotides in length, therefore the lacZ’ gene remains in-frame.

·         The resulting lacZ’/plink peptide can still function in a-complementation.

What would happen to the lacZ’ peptide if we inserted a DNA fragment into the plink region?

·         The insertion of a random piece of DNA into the plink region will result in three possible different reading frames after the insert (depending on the number of nucleotides in the inserted fragment)

·         There is a 33% (one in three) possibility that the downstream lacZ’ gene will be translated in the correct reading frame

·         There is also the possibility that even if the reading frame is correct that the insertion of a large peptide at the start of the lacZ’ protein will prevent a-complementation.

·         Therefore, most of the time, a DNA fragment inserted into the plink region will abolish b-galactosidase function

·         DNA inserts in the plink region can therefore be identified by growing the host bacteria on media containing IPTG and X-gal and looking for white-yellow colonies (with the caveat that some blue colonies may in fact contain an insert)

DNA coding for a gene of interest can be inserted in the plink region in-frame with the lacZ’ reading frame.

·         The protein coded for by this gene can be expressed by induction with IPTG (i.e. using the P_lac)

·         The expressed protein will have as its amino-terminal sequence the first few amino acids of the b-galactosidase gene (in the absence of any other manipulations/mutagenesis). This is also known as a (short) fusion protein.

·         Although the lac promoter is considered to be relatively weak, the high copy number plasmid may result in a useful level of expression of the protein of interest

The pET vector system (Novagen, Inc.)

The pET vector looks like this:

Figure 3.4.3: pET Vector

It has the following important elements:

·         Ampicillin resistance marker

·         ColE1 origin of replication

·         f1 origin of replication (allows single stranded vector to be produced when co-infected with M13 helper phage)

·         lacI gene (lac repressor protein)

·         T7 transcription promoter (specfic for phage T7 RNA polymerase)

·         lac operator region 3′ to the T7 promoter

·         multiple cloning site (polylinker region) downstream of the T7 promoter

The pET vector is a little different from the pUC vector: pUC uses the lac promoter and pET uses a promoter from phage T7

·         The phage T7 promoter is stronger than the lac promoter

·         Phage T7 RNA polymerase will specifically recognize the T7 promoter region and will not efficiently transcribe from other promoters

·         The T7 promoter will not be efficiently transcribed by E. coli RNA polymerase

Where will the phage T7 RNA polymerase come from?

The pET system involves not only an expression vector, but also a genetically engineered host bacteria. The host bacteria for the pET vector is typically E. coli strain BL(DE3)

·         This strain has integrated into its chromosome the gene for T7 RNA polymerase

·         The T7 RNA polymerase in the host genome is constructed such that it is under the control of a lac promoter and operator

·         Thus, induction by the lactose analogue, IPTG, causes the host to produce T7 RNA polymerase

·         The E. coli host genome also carries the lacI (repressor) gene

Figure 3.4.4: E.Coli BL(DE3) chromosome

Thus, induction by IPTG results in:

·         Derepression of T7 RNA Pol gene on host chromosome with subsequent production of this polymerase

·         Derepression of target gene under lac O regulation

·         Transcription of target gene by T7 RNA Pol

The pET vector itself is available with several different polylinker sequences. They contain the same restriction sites, but differ in the reading frame leading into the pLink region:

Figure 3.4.5: Polylinker sequences

·         The ggatcc site (BamH I restriction endonuclease site) is the first restriction site in the polylinker.

·         The polylinker region is thus available in the three different possible reading frames

·         A gene of interest can thus be cloned to be in-frame with the transcribed region downstream from the T7 promoter. Again, like the pUC vector the expressed protein will be a fusion protein (with 12-13 amino acids at the amino-terminal end of the polypeptide)

The pTrcHis vector system (Invitrogen, Inc.)

The pTrcHis vector looks like this:

Figure 3.4.6: pTrcHis Vector

The important elements are:

·         Ampicillin resistance marker

·         ColE1 origin of replication

·         lacI^q gene. This is a lac repressor mutation which is upregulated (produces more lac repressor than normal)

·         Trc promoter. This is a hybrid of the lac and trp promoters which is a stronger promoter in comparison to the lac promoter

·         The lac operator region downstream of P_trc. This allows regulation by IPTG.

·         An initial stretch of 6 histidine residues in the amino terminal region of the translated protein, also known as a “His tag” region.

·         An enterokinase (EK) cleavage recognition sequence (asp asp asp asp lys , with cleavage after the lys residue)

·         A polylinker region downstream from the EK site

Purpose of the His tag

·         Histidine residues can coordinate to form a metal (Ni²⁺) binding site in a protein.

·         The stretch of six His residues forms such a binding site.

·         Proteins with a His tag have affinity for metal chelating resins, and this characteristic can be used to selectively purify such proteins.

Purpose of the EK cleavage site

·         Although the His tag can allow rapid and selective purifiction of a cloned protein, the presence of these his residues may prevent normal function of the protein

·         The His tag can be cleaved away from the protein by introducing a specific recognition sequence for an endopeptidase

·         The sequence “asp asp asp asp lys” is recognized and cleaved by enterokinase. This sequence is not common and it is doubtful that the protein of interest contains another such sequence

Figure 3.4.7: Enterokinase cleavage

·         His tags/EK sites are typically introduced at the amino terminus of proteins, but can be introduced at the carboxyl terminus as well

Expression hosts

One of the important characteristics of expression hosts (bacteria) is that they allow the expressed protein to accumulate.

·         Some expressed proteins may not be folded correctly. Although they may be refolded later to yield functional protein, misfolded proteins may be rapidly proteolyzed in the host bacterium

·         Host proteases may selectively cleave precursor forms of expressed proteins to produce mature active forms, which may not be desirable.

·         OmpT and lon genes code for proteases which can degrade expressed proteins.

·         Two common mutations in E. coli to eliminate the action of host proteases includes the OmpT^– and lon^– strains.

·         E. coli strain BL(DE3) is both OmpT^– and lon^–