Purification of Cadmium-Ion Binding Metallothionein-3 by Proteinase Digestion on Affinity Chromatographic Column

The gene encoding human metallothionein-3 (hMT-3) was synthesized and inserted into the polycloning sites of fusion expression vector pALEX, and then fused downstream to its glutathione S-transferase (GST) fusion partner. Fusion protein, GST-Cd2+-hMT-3, was expressed after isopropyl-β-D-thiogalactopyranoside (IPTG) induction and addition of 0.1 mmol/L CdSO4 into the culture medium. GST-Cd2+-hMT-3 mainly existed in cellular soluble fraction as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Recombinant Cd2+-hMT-3MT was purified by two alternative strategies: (1) “protease digestion after purification” method, performed by elution of GST-Cd2+-hMT-3 from GST affinity chromatography, then protease digestion, and GST affinity chromatographic purification subsequently, and (2) “protease digestion in situ” method, performed by digestion of GST-Cd2+-hMT-3 directly on the column when it is bound to the GST affinity chromatographic resins, and collection of Cd2+-hMT-3 directly from the eluate after the digestion. It was confirmed that the latter procedure is more effective and convenient, avoiding the need for conventional elution, dialysis, and lyophilization processes, and increasing the purity, recovery, or yield of the final product. After further purification by a Superdex™ 75 HR 10/30 precolumn, 6–7 mg of Cd2+-hMT-3 was finally obtained from 3 L of flask culture with a recovery of about 1.8% and a protein yield of 2 mg/L. SDS-PAGE, amino acid composition, and inductively coupled plasma atomic emission spectrometer (ICP-AES) analyses showed that the relative molecular mass of Cd2+-hMT-3 is about 7000, with a purity of above 90%. Its amino acid composition is consistent with the expected value of natural hMT-3, particularly no aromatic amino acid and histidine, and moreover, the atomic ratio of 21:(7.5±0.1) for S:Cd is consistent with the theoretical value of 21:7.