Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA

Abstract

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.

Structural Characterization and In Vitro Antioxidant Activity of Metallothionein from Oratosquilla oratoria

“…At present, research hotspots concerning MTs are mainly focused on the stress response mechanisms and biological functions of MTs. To date, the reported preparation methods for MTs mostly involve cloning the coding sequence of the MT, inducing an overexpression in bacteria or fungi, and then isolating the protein through a series of purification steps [ 18 , 29 , 30 , 31 ]. Only a few studies have centered on the direct extraction of high-purity MTs [ 32 , 33 , 34 ], and there are no reports on the extraction of MTs from marine crustaceans (including O. oratoria ).…”