| Metallothionein(MT) is a group of cysteine-rich, low molecular weight proteins with a combination of heavy metal, scavenging free radicals, radiation resistance, anti-aging and trace mineral balance modulation as well as other important physiological function. Metallothionein was widely applied in environmental protection, pharmaceutical industry, food industry and agriculture. In this research, Lactococcus lactis N-Acetylmuraminidase C-terminal domain(c A) used as expression tag and anchor-domain, successfully improved the stability, expression and purification efficiency of h MT-I protein produced in heterologous expression systems. The surface display system based on protein docking was developed and h MT-I was docked onto the surface of Lactococcus lactis, which absorption capacity of heavy metal has been investigated in detail.C-terminal of N-Acetylmuraminidase gene was amplified from Lactococcus lactis MG1363 genome and fused with h MT-I gene. Restriction-free cloning was employed to construct plasmid p ET21a-c A- h MT-I. Four expression Tag(A、T、H and P) were introduced into 5’-terminal of target gene in expression vector of E.coli BL21(DE3) respectively. The recombinant protein with H tag showed the best productivity among all various tags while most of them were expressed as inclusion bodies. Five molecular chaperone plasmids(p G-KJE8、p Gro7、p KJE7、p G-Tf2 and p Tf16) were transformed to E. coli BL21(DE3) containing H tagged recombinant protein respectively which were co-expressed with molecular chaperone at low temperature. Results showed that the expression level and solubility of c A-h MT-I protein co-expressed with p Tf16 was among the best conditions in which c A-h MT-I produced in soluble form was about 39% in supernatant fraction as well as the expression level was up to 134mg/L.The Lactococcus lactis surface display system was constructed using Green fluorescent protein(GFP) as a reporter. Recombinant plasmid p ET28a-c A-GFP was transformed into E.coli BL21(DE3) to induce expression of report gene. The report protein could be detected as a prominent band with an apparent molecular mass of 53 k D after separation by SDS-PAGE. Supernatant component of engineering bacteria was incubated with Lactococcus lactis after ultrasonic treatment at room temperature. c A-GFP protein was docked and displayed onto cell surface of Lactococcus lactis successfully by SDS-PAGE detection and fluorescence microscopy analysis with loading amount up to 121 mg/g of dry bacteria. c A-GFP displayed on cell surface of Lactococcus lactis was stable which fluorescent intensity stayed 82.2% when stored at 4℃ for 6 days. It demonstrated that Lactococcus lactis surface display system based on c A domain docking was successfully constructed. The identical protocols were applied to display protein h MT-I onto cell surface of Lactococcus lactis. Positive results were confirmed by Protein electrophoresis and ELISA analysis. Loading amount of c A-h MT-I protein was up to 54.16 mg/g of dry bacteria and stored with good stability in buffer at 4℃ or freeze-dried.h MT-I protein displaying on cell surface of Lactococcus lactis showed high adsorption capacity of Cd2+, Pb2+, and Zn2+(for binding metal ions was Cd2+>Pb2+>Zn2+), while with low capacity for Cu2+. The novel method for expressing recombinant human Metallothionein-I in Escherichia coli and displaying on cell surface of Lactococcus lactis efficiently was characterized with high efficient, nontoxic, low cost and purification independent, which can be used for ongoing research and applied in food and cosmetic industry as well as the field of environmental protection. |
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