[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 poly-cloning sites of fusion expression vector pALEX, and fused downstream to its glutathione S-transferase (GST) fusion partner. Fusion protein GST-Cd2+-hMT-3 was expressed after isopropyl-beta-D-thiogalactopyranoside (IPTG) induction and addition of 0.1 mmol/L CdSO4 into the culture medium, and mainly existed in cellular soluble fraction as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Recombinant MT was purified by two purification procedures: "proteinase digestion after purification" method, e. g. by elution of GST-Cd2+-hMT-3 from GST affinity chromatography first, then proteinase digestion and GST affinity chromatographic purification again subsequently; and "proteinase digestion in situ" method, e. g. digestion of GST-Cd2+-hMT-3 directly on the column while its binding to the GST affinity chromatographic resin and collection of Cd2+-MT directly from the flow through fraction after the digestion. It was confirmed that the later procedure exhibited more effective and more convenient by avoiding the 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 column, finally 6-7 mg of Cd2+-hMT-3 was obtained from 3 L of flask culture with the recovery of about 1.8%. SDS-PAGE, amino acid composition and inductively coupled plasma atomic emission spectrometer (ICP-AES) analysis showed that the relative molecular mass of Cd2+-hMT-3 is about 7 000, with a purity above 90%. Its amino acid composition is consistent with the expected value of natural hMT-3, particularly no aromatic amino acid and histidine, and the atomic ratio of 21: (7.5 +/- 0.1) for S: Cd, is also consistent with the theoretical value of 21: 7.
Zheng
Weijuan
W
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, China. wjzheng@nju.edu.cn
Yang
Feng
F
Wu
Fang
F
Lu
Chun
C
Hua
Zichun
Z
D002104:Cadmium / Q000378:metabolism; D019187:Cadmium Compounds / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D013440:Sulfides / Q000494:pharmacology
D019187:Cadmium Compounds; D010455:Peptides; D013440:Sulfides; D002104:Cadmium; C034939:cadmium sulfide; D008668:Metallothionein; D005982:Glutathione Transferase; D010447:Peptide Hydrolases