Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASL^tag)

Figures & data

Figure 1. Fluorescence microscopy of E. coli BL21(DE3) cells transformed with pET-22b/INPN-SspCA (left) or with pET-ASL^tag-SspCA (right). The cells were incubated with BG-FL and then analysed by fluorescence microscopy. Images show bright field (BHF) and AlexaFluor488 (green). As expected, the fluorescence is only evidenced for the bacterial cell transformed with the ASL^tag system.

Figure 2. Western Blot performed on the outer membrane purified from the whole bacterial cells. The anti His-tag antibody was raised against the C-terminus of His-tagged SspCA. Legend: Lane Std, molecular markers, M.W. starting from the top: 75.0, 50.0, and 37.0 kDa; Lane 1, anchored SspCA; Lane 2, anchored H⁵-SspCA.

Figure 3. Model representation of an outer membrane fraction (OM; pdb from Tieleman and Berendsen⁶⁵) describing the in vivo immobilisation of SspCA (in blue; PDB ID: 4G7A; panel A) and in fusion with H⁵ (in green; PDB ID: 6GA0; panel B). The INPN domain is omitted because inserted in the OM. The catalytic reaction of SspCA (the hydration/dehydration of CO₂) and H⁵ (the conversion of BG-FL in the free guanine and the fluorescent benzyl-guanine derivative, B-FL, covalently linked to the active site of H⁵) are also shown.

Figure 4. Protonography (Panel A), fluorescence gel-imaging (Panel B) and Coomassie staining (Panel C) of SspCA and H⁵-SspCA carried out with different amounts of the whole E. coli cells (see Materials and Methods). Filled green, white and black arrows represent the ASL^tag-SspCA, INPN-SspCA and the free SspCA, respectively.

Figure 5. The thermostability of immobilised SspCA and H⁵-SspCA on the bacterial surface. Measures were carried out at indicated temperatures, by using aliquots of the whole cells incubated up to 24 h. Legend: continuous line, membrane-bound H⁵-SspCA; dashed line, membrane-bound SspCA. Each point is the mean of three independent determinations.

Figure 6. The long-term stability of immobilised SspCA and H⁵-SspCA on the bacterial surface. Measures were carried out at indicated temperatures up to 10 d, using aliquots of whole bacterial cells. Continuous line: free SspCA; Dashed line: membrane-bound SspCA. Each point is the mean of three independent determinations.