CHAPTER 4 The Plasmodium spp. putative copper transport protein: Native protein
4.3 Discussion
4.3.3 Recombinant expression of the amino terminus of a putative P. berghei copper
The coding domain of the P. berghei copper transport protein's amino terminus was amplified by RT-PCR, cloned and used for recombinant protein expression. The ability of this protein to bind copper would suggest a role for the whole protein in copper transport. This assumption is
based on the finding that the amino terminus of other copper transport proteins studied, is involved in the initial acquisition and transport of the cuprous ion (De Feo et al., 2009; Larson et al., 2010; Nose et al., 2006). Initial attempts to express the recombinant P. berghei amino terminus (PbCtrNt) made use of the pET23a vector. The expressed protein was, however, found to be targeted to insoluble inclusion bodies (Table 4.1), which is undesirable given the inherent difficulties faced with protein refolding in vitro. Fusion of the amino terminal domain to a soluble carrier protein was therefore explored. Soluble fusion partners, such as glutathione S- transferase (GST) and maltose binding protein (MBP), can improve folding of inherently insoluble, unstable or unfolded proteins (Kapust and Waugh, 1999; Makrides, 1996;
Nallamsetty and Waugh, 2007). The carrier protein is thought to rapidly adopt a native conformation thereby promoting the correct folding of downstream units (Baneyx, 1999).
As with the expression of His6-PbCtrNt (pET23a), expression of GST-PbCtrNt produced an insoluble product (Table 4.1). On the other hand, fusion to the MBP carrier protein produced a soluble product targeted to the cell cytoplasm (pMal-c2), although some of the protein was still found as an insoluble sample (Figure 4.10). Additionally, the expressed product appeared largely degraded or truncated, as evidenced by multiple protein bands below the desired 61kDa product. The addition of protease inhibitors, during lysate preparation, suggested degradation did not occur following cell lysis (data not shown). This indicated that product truncation and/or degradation may have occurred during protein synthesis in the host cell cytoplasm. A strategy used to avoid this problem is expression of a target protein in a host strain bearing mutations to native protease genes (Baneyx, 1999). E. coli BL21 cells lack the lon and ompT genes, which encode proteases catalysing the endoproteolytic cleavage of damaged and recombinant proteins (GST handbook). Expression using this strain made no observable improvement (Figure 4.11). The presence of high molecular weight multimers in all purified samples was another concern. These protein multimers are formed from MBP-PbCtrNt, as indicated by anti- MBP (Figure 4.11) and specific antibody blots (data not shown). Expressing problematic recombinant proteins by fusion to a soluble carrier partner, such as MBP, can be accompanied by large molecular weight aggregates as a result of carrier protein-induced solubilisation (Kapust and Waugh, 1999; Nominé et al., 2001a; Sachdev and Chirgwin, 1999; Zanier et al., 2007).
In an effort to reduce MBP-PbCtrNt degradation and multimer formation various alterations to culture and purification conditions were examined (Table 4.2). Variations to temperature, pH and cell density can affect proteolytic activity, protein secretion and the levels of protein production in E. coli expression systems (Makrides, 1996). Reducing the growth temperature is known to help limit the in vivo aggregation of recombinant proteins and improve production of
soluble protein (Gräslund et al., 2008; Vasina and Baneyx, 1997). This is presumed to be due to slower rates of protein production, thereby allowing newly transcribed recombinant protein time to fold correctly (Vera et al., 2007). In addition, the composition of the cell growth medium should be carefully formulated and monitored, since it too can have significant metabolic effects on the cells and protein production (Makrides, 1996). In this study, changes to nutrient broth composition made no improvement to the MBP-PbCtrNt banding pattern (Table 4.2). The recombinant expression of other malarial parasite proteins at reduced growth temperatures has proven beneficial (Flick et al., 2004; Jalah et al., 2005). For example, expression of soluble P.
falciparum LDH at 15°C increased expression levels (Berwal et al., 2008). Growth at 15°C made little improvement to MBP-PbCtrNt expression (Table 4.2). Recombinant protein production is also affected by cell density. Induction of expression is usually initiated in mid-to- late log phase, which can be identified by a culture having an optical density between 0.4 and 0.6, at 600 nm. At post-log phase, recombinant protein production can be affected by a limited availability of dissolved oxygen and increased carbon dioxide levels (Makrides, 1996).
Expression of GST-PfEMP1 domains post-log phase was, however, found to improve protein production (Flick et al., 2004). A similar observation was not made for MBP-PbCtrNt (Table 4.2).
A critical step in recombinant protein purification is the preparation of the bacterial lysate.
Optimal conditions maximise cell lysis and affect the cell fraction to which the recombinant protein is extracted, while minimizing oxidation and proteolysis (Gräslund et al., 2008). The lysis buffer typically consists of a strong buffering species to overcome the effects of the bacterial lysate, high ionic strength to enhance protein solubility and stability plus a reducing agent to prevent protein oxidation (Gräslund et al., 2008). The conditions used for cell lysis and protein purification were systematically tested for their effects on the quality of purified MBP- PbCtrNt. Different buffers, pH's and the addition of detergents, redox reagents and chaotropic agents were tested (Table 4.2), with protein aggregates only disrupted under reducing conditions in the presence of 8 M urea (Figure 4.12). Previous studies with human papillomavirus (HPV) E6 oncoprotein, as a MBP-fusion (MBP+E6), identified that protein aggregation most likely occurred in vivo during protein expression (Nominé et al., 2001b).
These aggregates were only solubilised using harsh denaturing conditions suggesting that the interactions maintaining MBP+E6 aggregates were probably similar to those found in precipitates of misfolded proteins and bacterial inclusion bodies (Nominé et al., 2001b).
The amino acid residues likely to contribute to aggregate formation are the cysteine residues, of which PbCtrNt has eight (Figure 4.13a). This is likely to have affected heterologous expression since the bacterial cytoplasm has a strong reducing potential and thus few cytoplasmic proteins with disulfide bonds (Fahey et al., 1977). This property generally
translates into the misfolding of heterologous proteins whose tertiary structure depends, in part, on disulfide bond formation (Makrides, 1996). The problem can be overcome by exporting the target protein from the cytoplasm to the oxidising environment of the bacterial cell periplasm (Baneyx, 1999). Attempts at this approach, by fusing PbCtrNt to MBP having a periplasmic malE leader sequence, proved unsuccessful (data not shown), which may be due to the presence of a predicted amino-terminal signal sequence in the inserted gene.
Molecular exclusion chromatography (MEC) was used to fractionate the multiple MBP-PbCtrNt products. MBP-PbCtrNt was, however, found to elute in the void volume of Sephacryl S-100 and S300 resins (Figure 4.14), thereby suggesting a significant number of MBP-PbCtrNt monomers aggregate in the cell cytoplasm to form “soluble” inclusion bodies. A similar observation was made for MBP-E6 expression (Nominé et al., 2001b; Zanier et al., 2007) and although the protein aggregated due to misfolding, it was still found to be able to bind zinc ions with a similar stoichiomery to that of the native protein. This suggested the recombinant protein was synthesised with a native-like fold, thus enabling correct coordination of the metal centre (Zanier et al., 2007). MBP-PbCtrNt was therefore assessed for copper binding in vitro and in vivo with bound copper detected by the copper-specific BCA release assay (Brenner and Harris, 1995). MBP-PbCtrNt bound copper under both examined conditions (Figure 4.20) with an apparent binding preference for the cuprous ion (Cu+). However, considering the misfolded nature of MBP-PbCtrNt, these results were cautiously interpreted since other structural studies were not carried out.
4.3.4 Recombinant expression of the amino termini of two P. falciparum copper