Niklas Holmberg

Immunomodulation of enzyme function in plants by single-domain antibody fragments

Immunomodulation involves the use of antibodies to alter the function of molecules and is an emerging tool for manipulating both plant and animal systems. To realize the full potential of this technology, two major obstacles must be overcome. First, most antibodies do not function well intracellularly because critical disulfide bonds cannot form in the reducing environment of the cytoplasm or because of difficulties in targeting to subcellular organelles. Second, few antibodies bind to the active sites of enzymes and thus they generally do not neutralize enzyme function. Here we show that the unique properties of single-domain antibodies from camelids (camels and llamas) can circumvent both these obstacles. We demonstrate that these antibodies can be correctly targeted to subcellular organelles and inhibit enzyme function in plants more efficiently than antisense approaches. The use of these single-domain antibody fragments may greatly facilitate the successful immunomodulation of metabolic pathways in many organisms.

Aqueous two-phase system formed by thermoreactive vinyl imidazole/vinyl caprolactam copolymer and dextran for partitioning of a protein with a polyhistidine tail

A new type of aqueous two-phase system (ATPS) has been developed for application combining two attractive concepts in downstream processing: the immobilised metal affinity partitioning and the use of thermoprecipitating polymers. ATPS consisting of the thermoprecipitating copolymer of N-vinyl caprolactam/1-vinyl imidazole loaded with Cu ions (Cu-poly-VI-VCL) in the top phase and dextran T70 in the bottom phase was used for purification of recombinant lactate dehydrogenase carrying an affinity tag of 6 histidine residues (His-LDH ) from a crude E. coli extract. The enzyme partitioned preferentially into the top Cu-poly-VI-VCL-rich phase. After phase separation, the latter was mixed with EDTA. Temperature increase to 45°C resulted in thermoprecipitation of VCL/VI-polymer, which could subsequently be recycled. His-LDH remained solubilized in the aqueous phase resulting in 8-fold purification and 80 % recovery in a single step.

Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco

A synthetic gene based on the primary sequence of the mature spruce budworm antifreeze protein (sbwAFP) was constructed by primer overlap extension. The amino acid codons were chosen to mimic those of a highly expressed tobacco nuclear gene. A DNA sequence encoding the amino-terminal leader sequence from the tobacco pathogen related protein 1b (PR), which targets the protein to the apoplastic space, was fused in frame to the synthetic sbwAFP gene. This fusion was placed downstream of the cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator in a T-DNA binary vector. Transgenic tobacco lines transcribing PR-sbwAFP were selected by RT-PCR. The apoplastic protein fractions of sbwAFP expressing tobacco lines exhibited enhanced antifreeze activity as demonstrated by the ability to inhibit ice re-crystallization and increased thermal hysteresis.

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.