As many bacterial promoter regions are relatively weak, plasmids have been engineered that possess stronger promoters and greater controls have been introduced in the initiation of transcription. Promoters such as the lacUVS (lac), trp, tacitrc {pp-lac hybrid promoters), the X phage promoter and the T7 phage promoter are now used to control and initiate recombinant gene expression in E. coli. These promoters are essential as eukaryotic promoters have very poor function in E. coli. Many of these promoters are chosen because they can be regulated. If the recombinant gene is toxic to the host upon expression, coupling to a strong, unregulated promoter is not recommended. High levels of constitutive transcription have been shown to interfere with plasmid DNA replication and lead to plasmid instability (Remaut et al., 1981).
3.2.1.1. The Pl promoter
By using the phage X (P J promoter sequence, tight control of transcription can be controlled. This promoter region is regulated by the X repressor protein, cl, and by using host cells that can be induced to either turn off production of this protein, or produce a cl protein that can be selectively degraded, transcription can be selectively initiated.
Most examples of this system are pBR322 derived plasmids that contain the P l promoter region. Upon insertion into temperature sensitive mutants that express thermolabile cl repressor protein, a temperature shift from 28°C to 42°C degrades the cl and enables transcription of the plasmid encoded sequence. Examples of the tight control achieved by this system for the expression of toxic genes have been demonstrated (Shimatake & Rosenberg, 198l;D erom e/a/., 1982; Remaut a/., 1987). A problem associated with the promoter is that the temperature shift increases heat-shock gene expression, of which some products are proteases. A more recent derivative of the promoter based expression system involves the host cell expression of Xcl being under the tight regulation of a, trp promoter that has being inserted upstream of the Xcl repressor gene. Production of Xcl is inhibited by the addition of free tryptophan which binds to the trp repressor and the activated complex binds to the trp promoter (LaVallie et aL, 1992).
3.2.1.2. The T7 promoter
Insertion of the bacteriophage promoter region into an expression vector is another way of tightly controlling transcription. This system involves the bacteriophage T7 promoter region which is only recognised by bacteriophage T7 RNA polymerase. Again the pBR322 vector backbone has been used, and the T7 gene 10 promoter, the T7 gene 10 translation start and the T7 transcription terminator have been cloned into it. To initiate transcription, T7 RNA polymerase needs to be introduced to the system. This can be done by fusing the T7 gene 1 that encodes this complex into the E. coli genome or by introduction of the coding sequence by direct infection with a bacteriophage carrying this information on a ^ vector (Studier & Moffat, 1986). When fused into the E. coli genome, the transcription of the T7 gene 1 can be brought under control of the lacUW5 promoter which is IPTG inducible. The hybrid vectors that work via this system are known as the pET series of vectors or the pRSET vectors (Studier & Moffat, 1986; Schoepfer, 1993). Many products from these systems are expressed to high levels very quickly but the occurrence of inclusion bodies is higher as the expression is not so tightly regulated (Taylor et al., 1992; Schoepfer, 1993).
3.2.1.3. trpUac Hybrid promoters
Previously, the trp and lac promoters had been used to direct expression of foreign gene products within E. coli. By themselves these promoters are relatively weak, but by fusing the -35 region of the trp promoter and the -10 region of the lacUWS promoter along with the lac operator, this trp-lac hybrid (known as the tac or trc promoter) is relatively strong and also controllable by addition of the gratuitous inducer, IPTG. What distinguishes the two types of hybrid promoter from each other is that the nucleotide spacing in between the -35 and -10 regions is 17 for the trc promoter and 16 for the tac promoter (Amann, 1988; Amann, 1985; DeBoer, 1983). Both promoters are the same relative strength being up to 11 times more efficient than /acUV5 and three times more efiBcient than trp promoters alone (DeBoer, 1983). Many expression vectors are based on this promoter hybrid including the pGEX and pTrcHis expression systems which will be discussed in detail later.
In addition to the strong, regulated promoter to produce large amounts of mRNA, another major factor for efficient expression of recombinant genes in E. coli is a ribosome-binding site to ensure efficient mRNA translation. In E. coli the ribosome- binding site consists of an initiation codon and a sequence 3-9 bp long which is located 3-11 bp upstream fi’om the initiation site for the gene. This sequence is called the Shine- Delgamo (SD) sequence and is complementary to the 3' end of E. coli 16S rRNA (Shine & Delgamo, 1975; Steitz, 1979). Strong termination signals at the end of the sequence to be expressed are also needed to enhance plasmid stability (Gentz et aL, 1981), as having strong promoters and ribosomal binding sites without efficient termination will produce potentially high amounts of unstable RNA due to read-through. This unstable RNA could enter the RNA metabolism pathway leading to low levels of mRNA translation.
3.3. Expression systems for fusion proteins
Direct expression of foreign polypeptides within E. coli can be limited as the protein may be degraded by the host cell. This is most apparent with small peptides (Marston, 1986). Another problem associated with direct expression is solubility and subsequent purification of the protein product. In an attempt to circumvent these problems, fusion of the sequence with a highly expressed non-toxic protein has been used. A number of fusion systems exist for E. coli which use such proteins (e.g. P-galactosidase, chloramphenicol acetyltransferase, glutathione transferase, maltose binding protein, phosphate binding protein, protein A, protein G, streptavidin and thioredoxin) or synthetic peptides (e.g. poly-Arg, -Glu or -His residues). These fusion partners may be placed either -NH2 terminal or -COOH terminal to the recombinant gene sequence (Uhlén & Moks, 1990).
Commonly, the fusion partner is selected in part, due to its ability to be adsorbed specifically and reversibly by a complementary binding substance (or ligand). If this ligand is immobilised on an insoluble support, then selective separations can be performed on complex mixtures. This technique is known as affinity chromatography which can be carried out in a batch or column format. This forms a major step in the isolation process of the foreign gene product (Figure 3.1). Recovery of the fusion protein from the affinity matrix is facilitated by eluting the protein off of the matrix by using fi*ee ligand or by varying the binding conditions.
Often the product is needed as a separate entity from the fusion partner. This is achieved by engineering cleavage-recognition sequences for site-specific proteases into the polylinker sequence between the fusion partner and the recombinant gene product. This enables selective removal of the fiision partner. Cleavage of the fiision protein can be carried out whilst the fusion protein is still attached to the affinity matrix. This yields the protein of interest whilst leaving the fusion partner still attached to the affinity matrix. If the fiision protein is cleaved in solution, the fiision partner can be removed by passing the protein mixture over the affinity matrix.
INTRODUCE VECTOR INTO SUITABLE HOST STRAIN OF E.COU]
ELUTION OF FUSION PROTEIN
FURTHER PURIFICATION OF PROTEIN ADSORB FUSION PROTEIN
ONTO AFFINITY MATRIX SELECT A POSITIVE COLONY, GROW AND INDUCE EXPRESSION
INSERT FOREIGN GENE INTO FUSED EXPRESSION VECTOR
PROTEOLYTIC CLEAVAGE OF FUSION PROTEIN WHILST STILL ADSORBED
TO MATRIX PROTEOLYTIC CLEAVAGE
IN SOLUTION TO REMOVE AFFINITY TAG
(optional)
GROW E.COU UNDER SELECTIVE PRESSURE, PICK
COLONIES THAT HAVE THE CORRECTLY INSERTED VECTOR
HARVEST AND LYSE CULTURE
(under denaturing or non-denaturing conditions) CLARIFY LYSATE
Figure 3.1.
A flow chart of the stages involved in the expression and purification of a foreign gene within Eschericia coli. This methodology has been utililised in the expression of aggrecan and link protein domains using the pGEX and pTrcHis fused expression systems (Chapter 6).
The following two expression systems are both fused expression systems that enable purification of the resultant fusion protein by affinity chromatography.