Evaluation of genetically attached histidine affinity tails for purification of lactate dehydrogenase from transgenic tobacco

• Published in: Plant science (Limerick) Vol. 134; no. 1; pp. 103 - 114
• Main Authors: Mejàre, Malin, Lilius, Gösta, Bülow, Leif
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 11.05.1998; Elsevier Science
• Subjects: amino acid sequences; Analytical, structural and metabolic biochemistry; Bacillus stearothermophilus; biochemical techniques; Biological and medical sciences; Enzymes; Enzymes and enzyme inhibitors; evaluation; fractionation; Fundamental and applied biological sciences. Psychology; gene expression; histidine; Immobilized metal affinity chromatography; Lactate dehydrogenase; Metabolism; Metal affinity precipitation; molecular sequence data; Nicotiana tabacum; nucleotide sequences; Oxidoreductases; plant extracts; Plant physiology and development; Polyhistidine tails; purification; recombinant proteins; Tobacco; transgenic plants; Metal affinity precipitation; Lactate dehydrogenase; Tobacco; Immobilized metal affinity chromatography; Polyhistidine tails; Heterologous system; Purification; Chemical precipitation; Enzyme; Metal ion; Bacillaceae; Gene expression; Nicotiana tabacum; L-Lactate dehydrogenase; C terminal-Sequence; Histidine; Bacillales; Affinity chromatography; Dicotyledones; Angiospermae; Bacillus stearothermophilus; Bacteria; Spermatophyta; Transgenic plant; Oxidoreductases; Solanaceae; Experimental plant
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(98)00050-8

Lactate dehydrogenase ( Bacillus stearothermophilus) has been expressed in transgenic tobacco. To facilitate purification polyhistidine tails were fused to the 5′-end of the gene. Two different tails, His 6 and His-X 3-His-X 3-His, were compared regarding their effect on LDH gene expression and metal ion specificity. His 6 exhibited strong binding to all of the tested transition metals (Zn 2+, Co 2+, Ni 2+ and Cu 2+) while the α-helical His-X 3-His-X 3-His showed a preference for Co 2+ over Zn 2+. This α-helical His tail also increased the level of gene expression compared to the native enzyme construct. The histidine modified proteins could be successfully purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn 2+, Co 2+ or Ni 2+. LDHHis 6 could also be precipitated from a crude tobacco protein extract using ethylene glycol-bis( β-aminoethyl ether) N, N, N′, N′-tetraacetic acid (EGTA) charged with Zn 2+.