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β-galactosidase is a natural cytoplasmic enzyme of E. coli, composed of 116 kDa monomers which form a 465 kDa homotetramer. It is encoded by the lacZ gene from the lacZYA operon (Juers et al., 2012). A simple and quick activity assay is available whereby the amount of o-nitrophenol (yellow) released from the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) by β-galactosidase, can be measured. It does not possess any DSBs and therefore, does not require the use of a DSB enabling technology (section 1.4.1.). With respect to export in E. coli, β-galactosidase was reported to not export via Sec because of multiple regions within the amino acid sequences as well as N-terminal positively charged amino acids (Lee et al., 1989). Furthermore, Tat export has not been reported to date in E. coli. However, successful translocation of β-galactosidase through the Tat pathway has been demonstrated in Bacillus subtilis using the signal peptide of alkaline phosphatase D (Xia et al., 2010, Ren et al., 2016). Due to the large size of the complex, it was believed that the monomeric components are primarily exported prior to assembly into active complexes after crossing the membrane. It is important to note here that the reason why this enzyme was secreted by Xia et al. (2010) and Ren et al. (2016) might lie in the different Tat mechanism of Bacillus subtilis as detailed in section 1.3.2.6., the different sequence of their engineered β-galactosidase, the use of the signal peptide from PhoD, the requirement of TatAd-Cd co-expression and that the alkaline phosphatase D is not found in Gram-negative bacteria. Thus, it is plausible to speculate that β-galactosidase at least in monomeric form, can be exported by the Tat machinery of E. coli and therefore would make it a suitable reporter partner to test.

A preliminary experiment was performed to confirm the absence of β-galactosidase export via the Sec pathway. To this end, the E. coli lacZ gene was fused to a C-terminal His tag for detection (coding for LacZ-His) and fused to the N-terminal OmpASP for export. A cytoplasmic control with no signal

Chapter 6 – Reporter proteins

95 peptide was also used. During this experiment, Tat export was also investigated by N-terminal fusion of the five selected NTS signal peptides (AmiA, HyaA, PaoA, TorA and YcbK) described in Chapter 5. All proteins were expressed in the MC4100 WT and Tat-null strains in LB at 30°C with induction of 2 h at 30°C (Table 2-3, condition 1) and fractionated using the PureFrac method (Appendix 4). As expected, cytoplasmic LacZ-His expressed well and was localised in the cytoplasm and insoluble fraction, presumably as inclusion bodies (Figure 6-1A). Moreover, the insoluble materials showed greater activity than the cytoplasmic proteins (Figure 6-1B). But, the latter observation is most likely to be the result of a higher concentration of enzyme in the insoluble fraction than in the cytoplasmic fraction as the activity presented is not specific to the amount of protein. The activity in the insoluble fraction is suspected to come from the inclusion bodies as they have been shown to be capable of retaining the proteins activities (García-Fruitós et al., 2005, de Groot and Ventura, 2006). Nevertheless, the fact that the enzyme is active reflected the stability of the monomers and the correct assembly into an active tetrameric form.

Similarly, when fused to OmpASP and in support of published data (Lee et al., 1989), LacZ-His did not

export via the Sec pathway. Surprisingly, no export via Tat was evident with respect to the five NTS signal peptides tested, in terms of periplasmic localisation or periplasmic activity. However, cytoplasmic stability appears to be signal peptide dependent where AmiASP, PaoASP and YcbKSP enable

polypeptides to form stable β-galactosidase complexes in the cytoplasm whereas HyaASP and TorASP

do not, irrespectively of the strain (Figure 6-1B). This difference showed that signal peptides do not perform in the same way and can lead to various extent of instability of the recombinant protein. The differences highlighted in the set of signal peptides in terms of net charge, hydrophobicity or length (Appendix 2), do not correlate to any behaviour given to β-galactosidase. The Western-blots revealed no signs of double bands which is a usual indication of signal peptide cleavage. But the western-blot resolution prevents from identifying them as premature or mature proteins. Since β- galactosidase presented activity in the cytoplasm, N-terminal signal peptides do not interfere with the folding of the monomers nor with the assembly of the active tetramers. With the signal peptides, activity was also demonstrated in the insoluble fractions (Figure 6-1B). Since a similar activity of

Chapter 6 – Reporter proteins

96 about 50 kU/L was observed in the absence of a signal peptide, it is suspected to come from inclusion bodies rather than membrane-associated proteins due to their signal peptide.

Figure 6-1: Cellular localisation and activity profiles of β-galactosidase expressed in E. coli

His-tagged LacZ with no signal peptide (No SP) or fused with OmpASP, AmiASP, HyaASP, PaoASP, TorASP or YcbKSP

were expressed in the MC4100 WT and Tat-null strains. The cells were harvested after 2h induction at 30°C and fractionated using the PureFrac method into the cytoplasmic (C), insoluble (I) and periplasmic (P) fractions. A. Western-blots represent the detection of LacZ-His, LacI and MBP using anti-His, anti-LacI and anti-MBP antibodies respectively. Molecular weight markers are indicated in kilodalton (kDa). The figure is a composite image where the marker lanes reflects the approximate position of the molecular weights. B. β-galactosidase activity assays were performed in triplicate and measured against a titration curve of a commercially available β-galactosidase as a standard. The background signals were eliminated by subtracting the activity value from the empty vector’s fractions to each corresponding fraction. The bars indicate the cytoplasmic (white), insoluble (grey) and periplasmic (black) fractions.

Chapter 6 – Reporter proteins

97 The localisation results are strengthened by the LacI and MBP controls which proved the purity of the periplasmic fraction while the periplasmic proteins are still extracted (Figure 6-1A). Overall, β- galactosidase was not a suitable reporter protein because it showed no translocation via Tat likely because it folded into its tetrameric structure in the cytoplasm before reaching the Tat translocon preventing export. To better understand the expression, secretion and stability behaviours of β- galactosidase with the different signal peptides, analysing the specific activity of the sample would have been more informative. However, since the reporter protein is only a tool in this study and β- galactosidase presented no sign of Tat export, no further work was performed on this recombinant protein.