Lactate dehydrogenase (LDH) from 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, His6 and His-X3-His-X3-His, were compared regarding their effect on LDH gene expression and metal ion specificity. His6 exhibited strong binding to all of the tested transition metals (Zn2+, Co2+, Ni2+ and Cu2+) while the alpha-helical His-X3-His-X3-His showed a preference for Co2+ over Zn2+.This alpha-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 Zn2+,Co2+ or Ni2+. LDHHis6 could also be precipitated from a crude tobacco protein extract using ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) charged with Zn2+.
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