Tomas Moks

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. A slightly modified version of this review is also published in Current Opinion in Structural Biology 1992, 2:569-575.

A gene fusion system for generating antibodies against short peptides

A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.

Fusion proteins in biotechnology and structural biology

Abstract 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. A slightly modified version of this review is also published in Current Opinion in Biotechnology 1992, 3 :363–369.

Uniformly spaced banding pattern in DNA sequencing gels by use of field-strength gradient

A method is described for casting and operating ultrathin wedge-shaped field-strength gradient gels for DNA sequencing. One of the gradients results in a uniformly-spaced oligonucleotide banding pattern from 60 to 300 nucleotides in a 53 cm long 4% polyacrylamide gel. From these field-strength gradient gels, more sequencing data can be obtained with greater confidence than from gels with uniform thickness. The gel-casting method is fast and simple. At 2.5 kV two gels can be run simultaneously in 2 h with the same power supply.

Approaches to solid phase DNA sequencing

Abstract A novel approach to enable two-directional solid-phase DNA sequencing, involving immobilization of double stranded plasmid to avidin agarose, is described. A plasmid vector, pRIT28, has been designed to allow enzymatic incorporation of biotinylated dUTP into two alternative sites.

Differential stability of recombinant human insulin-like growth factor II in Escherichia coli and Staphylococcus aureus.

Recombinant human insulin-like growth factor II (IGF-II), produced as a soluble extracellular fusion protein, was shown to be proteolytically degraded in Escherichia coli. In contrast, the fusion protein secreted from Staphylococcus aureus was stable and the full length product could be recovered by affinity chromatography. After site specific cleavage of the fusion protein, soluble IGF-II with biological activity was obtained without refolding procedures. These results demonstrate that a eukaryotic protein unstable in E. coli can be stabilized by expression in a Gram positive host. The full-length fusion protein from S. aureus was used to characterize the protease responsible for the degradation in E. coli. Biochemical and genetic analysis suggests a specific degradation by the outer membrane protease (OmpT).

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.

Utilizing the tubular bioreactor for continuous recovery of secreted fusion protein from recombinant Escherichia coli

In order to improve the cultivation properties of a traditional continuous stirred tank reactor (CSTR), we introduced a circulation unit made of four inorganic membranes in stainless steel tubes in parallel configuration, the so-called Tubular Bioreactor (TBR). Furthermore, the TBR outlet tube, which has a restriction nozzle at the end, was installed on top of the fermentor vessel, thereby creating a strong jet flow into the reactor and thus improving the mixing and the oxygen transfer rate. The kLa could be increased by approximately 50%. This setup was used for cultivations of recombinant Escherichia coli in a minimal medium and high cell density. More than 50 g dry cell mass/dm3 was obtained. Simultaneously, we have produced an elongated form of human insulin-like growth factor II, which was a secreted fusion protein utilizing the E. coli secretion system based on staphylococcus protein A. The product could be recovered continuously through the TBR-membrane.

Refolding of human recombinant insulin-like growth factor II (IGF-II) in vitro, using a solubilizing affinity handle

Publisher Summary Insulin-like growth factor II (IGF-II) consists of 67 amino acid residues in a single chain. In analogy with IGF-I, there are 6 cysteine residues forming 3 disulfide bonds. The recombinant IGF-II was initially produced as a secreted fusion protein, ZZ-IGF-II, in E. coli. However, most full-length fusion protein was found in multimeric aggregates held together with disulfide bonds. A redox system, using the fusion partner as a solubilizer, was developed to transform multimeric, misfolded variants into the monomeric, correctly folded molecule. This chapter presents a comparison between IGF-II refolding as a fusion protein and as a native molecule. A hydrophilic fusion partner may act as a solubilizer of insulin-like growth factor II during refolding. The fusion partner apparently does not interfere with the refolding. Instead it seems to keep the more hydrophobic IGF-II moiety soluble, making denaturating agents or organic solvents unnecessary in the refolding buffers. The fusion partner can then subsequently be removed using site specific cleavage. This concept can be used to refold proteins that normally precipitate during refolding.