| To improve the affinity and preference to cadmium, four mammalian metallothionein a domain were tandemly fused (4MTa). Here, we report the surface display of 4MTa and enhanced green fluorescent protein (EGFP) fusions by employing the INP-N terminal domain from Xanthomonas campestris pv as anchoring motif. A broad-host-range vector, pIME, coding for INPN-4MTa-EGFP fusion protein was constructed for targeting the fusion protein onto the surface of Pseudomonas putida X4, which is a per se highly robust microorganism able to grow in high concentration of heavy metal contaminated habitats. The surface location of fusion protein was demonstrated by Western blot analysis and immunofluorescence microscopy.The surface-displayed fusion protein retains the metal-binding ability of 4MTa and EGFP fluorescence. The engineered P. putida X4 with surface-expressed 4MTa-EGFP has some major advantages over the same strains cytoplasmic expressing 4MTa-EGFP, including 2-fold higher cadmium binding capacity,1.4-fold stronger fluorescence. Moreover, the tolerance of the surface-engineered bacteria to Cd2+ and pH was enhanced by contrast to the original strains. The OD values comfirms that X4-1 had the strongest tolerance to Cd2+. The result shows that the Cd2+ uptake of surface-engineered strains X4-1 is above 7.263mg/g, which could more effectively bind Cd2+ than the controls over the range of pH 5 to 8. To demonstrate the priority of surface-exposed EGFP-4MTa toward cadmium, whole cell binding of Cd2+ was performed in the presence of up to mass concentration excess of competing heavy metal. The results show that the presence of Cu2+ and Zn2+ with finite concentration did not influence Cd2+ uptake remarkably.In this study, Functional expression of 4MTa-EGFP fusion on the surface of microbes is demonstrated and suggested that it can be a potential whole-cell bio-adsorbent of cadmium for remediation of highly cadmium pollutant. |
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