Prokaryotic expression systems have been used successfully for the expression of many proteins including PDE enzymes . In most cases, the foreign proteins have been expressed either as fusion proteins, or directly but containing a polyhistidine tag. In both cases, the fusion partner or the polyhistidine tag has been used to aid purification. Table 4.1 gives an overview of some o f the proteins expressed using E. coli as the host cell.
Table 4.1 Foreign genes expressed using E. coli as the host.
F o r e ig n p ro tein F u s io n p a r t n e r V e c t o r R e f e r e n c e
P. gingivalis cysteine protease His,-tag pET Margetts e t a l , 2000
Mouse phosphofructokinase His,-tag pE T2 0b Gunasekera and Kemp, 1999
Hu m an leptin His,-tag pET Vame rin et a l , 1998
Rat connexin 32 Thioredoxin pTrxFus Mambet isaeva et a/., 1997
GluR2 receptor His,-tag pETGQ Chen and Gouaux, 1997
Bovine mitochondrial translation His,-tag pQ E M a and Spremulli, 1996
initiation factor 2
Thyroid hormone reeeptor GST pG EX-KG Ball e r a /., 1995
Human glutathione S-transferase His,-tag pET- 15b Chang et a l , 1999
Plasmodium falc ip aru m Ag63, Ag361 GS T p GE X-3X Smith and Johnson, 1988
P D F s :
PD E7 A C-terminal His-tagged p E T2 I-C Richter et a l , 2002
HSPDE4A C-termi nal His-tagged pET Richter et a l , 2000
RNPDE4D1 GS T pGE X-KG Kovala et a/., 1997
PDE3 N-terminal His-tagged pQ E-30 Tang et a l , 1997
Despite the success of using prokaryotic expression systems, there are some well-known problems associated with the use of bacterial expression systems especially when the protein of interest is a eukaryotic protein requiring complex post-translational modifications, such as glycosylation, which cannot be carried out by the E. coli.
Additionally, overproduction o f heterologous proteins in E. coli often results in their misfolding and segregation into insoluble aggregates called inclusion bodies (Prouty et a l,
1975; Williams et a l, 1982). A large range o f recombinant proteins have been shown to
be accumulated in inclusion bodies and includes proteins that were expressed as fusion proteins, such as myoglobin, bovine and human growth hormones, a,-antitrypsin, as well as proteins that were expressed directly (interleukin-2, bovine and human growth hormones, calf prochymosin) (Marston, 1986). It should be noted that it is not just the foreign proteins that the host segregates into inclusion bodies but also normal E. coli proteins synthesised to high levels using recombinant DNA technology (Marston, 1986).
There have been many strategies employed to overcome the accumulation of recombinant proteins in inclusion bodies. This has included the co-expression of molecular chaperones
which aid the proper folding of proteins but are not themselves part o f the final product (Baneyx, 1999; Hannig and Makrides, 1998). The expression of foreign proteins at room temperature (Kovala et al., 1997; Ball et a l, 1995) and 30°C (Gunasekera and Kemp, 1999) as opposed to 37°C has also been used successfully. The supplementation of the bacterial medium with agents such as sorbitol which do not permeate through the cell membrane have also been used successfully (Blackwell and Morgan, 1991; Gunasekera and Kemp,
1999).
The redox state of the E. coli cytoplasm has also been said to play a role in the aggregation o f recombinant proteins. The cytoplasmic environment is reducing with at least five bacterial proteins involved in the reduction of disulphide bridges. This is also thought to be a factor in the misfolding of proteins (Baneyx, 1999). E. coli mutants lacking genes for
the thioredoxins and glutaredoxins {trxA., trxC, grxA, grxB or grxC genes) have been used
for the expression o f foreign proteins which do not form aggregates (Schneider et a l,
1997).
The fusion protein systems developed for the purpose of purification and detection of the expressed fusion protein have been shown to facilitate protein folding. The presence of the fusion partner is thought to help in the correct folding of the foreign protein, improving solubility as well as providing protection from proteolytic cleavage. Smith and Johnson (1988) developed the pGEX vector system which expressed foreign proteins with a
Schistosoma japonicum glutathione S-transferase (GST) protein as a fusion partner. These
were successful in generating soluble fusion proteins (Smith and Johnson, 1988). Other fusion partners used have included maltose-binding protein which is thought to directly interact with the recombinant protein thereby acting as an ‘intramolecular’ chaperone but the MBP has to be synthesised first because if the fusion order is reversed, proteins become
insoluble (Sachdev and Chirgwin, 1998; Baneyx, 1999; Kapust et a l, 1999).
Recombinant proteins are also prone to proteolytic degradation especially if they are misfolded. This is carried out by at least five ATP-dependent proteases which includes the protease La, a Ion gene product and HflB, a hflB gene product (Baneyx, 1999; Gottesman, 1996). The protease La, a serine protease, has been shown to catalyse the degradation o f
proteolysis occurring (Mizusawa and Gottesman, 1983). The concentration of La protease also increases when cells accumulate large amount of foreign proteins, especially at high temperatures, which is followed by proteolysis o f the foreign proteins by La (Goff et a l,
1984). The protease HflB has a zinc protease motif and the in vivo activity can be modulated by zinc ions. This protease is thought to be an essential E. coli protease involved in the proteolysis o f both cytoplasmic and membrane protein. Protease-deficient strains of E. coli have been developed to overcome proteolytic degradation of recombinant proteins but these mutant strains show reduced cell growth rates and also compromised strain fitness (Chin et a l, 1988; Baneyx, 1999).
Recombinant proteins destined fbrE. coli periplasm has also been explored. The periplasm
provides an oxidizing environment that contains enzymes catalyzing the formation of disulphide bonds so is an attractive target of eukaryotic protein disulphide bond formation. This has been successfully used to express the cysteine-rich recombinant protein, human tissue plasminogen activator (Baneyx, 1999; Qiu et a l, 1998), and also the glutamate receptor (Arvola and Keinanen, 1996). Unfortunately, only small amounts (lOOpg/L) of the proteins have been produced by this method. Also, although there is no periplasmic ATP pool, proteolytic degradation does occur but via energy-independent proteases. Secretion of polypeptides to the extracellular medium is another desirable strategy but information regarding the secretion o f proteins from the host cell remains scant and also may not be possible for large proteins (Baneyx, 1999).
Codon usage between prokaryotes and eukaryotes can also have an impact on foreign gene expression in E. coli (Hannig and Makrides, 1998; Baneyz, 1999). Heterologous genes containing codons that are rarely used in E. coli may be poorly expressed. Moreover, the occurrence of rare codons (e.g. AGA, AGG) in the heterologous gene is associated with a low level of their tRNA species. However, this can be overcome by using site-directed mutagenesis to replace the rare codons with E. co//-preferred codons (e.g CGC) (Zahn, 1996; Baneyx, 1999)
Although many strategies have been developed to overcome inclusion body formation when recombinant genes are expressed in E. coli, the fact that foreign proteins are sequestered into inclusion bodies can be used to advantage in the purification of the foreign proteins.
Inclusion bodies generally contain large amounts of the desired protein and they can be relatively easily isolated from the remaining cellular components. The foreign proteins can then be retrieved from these inclusion bodies using the dénaturants, urea (8M) or guanidine (6M), and then allowed to gently refold into their native state following dilution or dialysis o f the dénaturant. The refolding process can be very slow and the conditions have to be empirically determined. Many proteins have been extracted from inclusion bodies and refolded to yield biologically active proteins including PDE enzymes (Richter et a l, 2000, 2002). In most cases, the conditions for the refolding of the recombinant proteins are
individually optimised. Richter and co-workers (2000) expressed PDE4A in E. coli as a
histidine tagged protein and purified the recombinant protein from inclusion bodies to yield
a biologically active protein. Patra and co-workers (2000) successfully extracted
recombinant human growth hormone from E. coli inclusion bodies using a range of pH
buffers (pH 3-13), with and without urea, for the solubilisation o f the inclusion bodies. Chen and Gouaux (1997) used an extensive folding screen involving changes in twelve different factors ranging from different protein concentrations for the starting material, buffer pH, divalent ions, temperature, presence and absence of chaotrope as well addition of additives such as arginine and ethylene glycol. These conditions were investigated for the refolding of the glutamate receptor (GluR2) ligand binding domain (-40 kD A) extracted
from E. coli inclusion bodies (Chen and Gouaux, 1997). Their experiments revealed that
the pH (pH 8.5), temperature (4°C) and presence o f divalent metal ions (Mg^^, Ca^^) were the most important factors for the optimal refolding of GluR2 receptor protein. It is therefore apparent that purification from inclusion bodies requires lengthy investigation into finding the optimum refolding conditions and this is generally made more difficult if the target protein is very large. Additionally, the inclusion bodies themselves are not free from certain proteases which can adsorb onto the surface of the inclusion bodies and may actually degrade the desired protein while it is being refolded. This includes the protease ompT protease, which adsorbs to the inclusion bodies, as well as an inner membrane protease FtsH which is also active under denaturing conditions (Gottesman, 1996; Baneyx,
4.2 Background and aims of the present study using the bacterial expression