Gösta Lilius

The metabolic effects of native and transgenic hemoglobins on plants

The strictly aerobic bacterium Vitreoscilla expresses a hemoglobin-like protein, VHb, when subjected to oxygen stress. When expressed in plants, this has several intriguing physiological effects, such as improving the overall growth rate, speeding germination and flowering, and increasing the productivity of certain oxygen-requiring metabolic pathways. Although the mechanisms behind the effects of VHb in heterologous hosts are not yet fully characterized, it has been suggested that VHb facilitates oxygen transport and/or storage. This hypothesis is supported by the kinetic properties of VHb, which allow very rapid dissociation of oxygen from the protein.

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

Abstract 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, 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 α-helical His-X3-His-X3-His showed a preference for Co2+ over Zn2+. 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 Zn2+, Co2+ or Ni2+. LDHHis6 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 Zn2+.

Artificial antifreeze proteins can improve NaCl tolerance when expressed in E. coli

A chemically synthesized DNA fragment encoding an artificial antifreeze protein was expressed in E. coli as a translational fusion with a truncated protein A. Two constructions were made, with two and four antifreeze domains, respectively. The fusion proteins stimulated the growth of their bacterial host cells at inhibitory NaCl concentrations. The fusion protein carrying four antifreeze domains also conferred improved tolerance towards freezing.

An artificial bifunctional enzyme, γ-glutamyl kinase/γ-glutamyl phosphate reductase, improves NaCl tolerance when expressed in Escherichia coli

SummaryAn artificial bifunctional enzyme, γ-glutamyl kinase/γ-glutamyl phosphate reductase, was obtained by fusing the Escherichia coli genes proA and proB. The proB gene was fused to the 5′-end of the proA gene with a linker encoding five amino acids. When expressed in E. coli enhanced intracellular concentrations of proline were observed. At 0.6 M NaCl the growth rates for the strain carrying the fusion enzyme and a control harbouring a plasmid encoding the wild-type enzymes were 320 and 530 min, respectively.