Single-step trypsin cleavage of a fusion protein to obtain human insulin and its C peptide.

The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG-binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ-proinsulin fusion protein were generated, having different trypsin-sensitive cleavage sites, Arg, Lys-Arg or Lys. The ZZ-proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography.

Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner.

We have previously shown that human insulin-like growth factor I (IGF-I), fused to ZZ (two domains derived from staphylococcal protein A), can be refolded at relatively high concentrations, without the use of solubilizing agents [Samuelsson, E., Wadensten, H., Hartmanis, M.

Single-step recovery of a secreted recombinant protein by expanded bed adsorption.

We have used an expanded bed adsorption procedure for efficient recovery of a recombinant fusion protein, directly from a crude fermentor broth without prior cell removal. The fusion protein was designed to have a relatively low isoelectric point (pI) to allow anionic exchange adsorption at pH 5.5 where most Escherichia coli host proteins are not adsorbed.

Fusion proteins in biotechnology.

Gene fusion techniques allow the production of recombinant proteins featuring the combined characteristics of the parental products. Originally, these techniques were used to probe transcriptional and translational activity, to translocate proteins across cell membranes, and to facilitate the recovery of proteins. Recently, new applications have emerged in areas such as protein refolding, immunology, drug targeting and protein display.

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.