Mathias Uhlen

Structure of the IgG-binding regions of streptococcal protein G.

The gene encoding the IgG‐binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5‐kb insert gave a functional product in Escherichia coli. Protein analysis of affinity‐purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG‐binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N‐terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C‐terminal part of streptococcal protein G consists of three IgG‐binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one‐step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature‐inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram‐positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta‐galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG‐Sepharose in high yield (95‐100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta‐galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline‐phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N‐terminal sequencing of the affinity‐purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG‐binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N‐terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N‐terminal IgG‐binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.

Production of specific antibodies against protein A fusion proteins.

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta‐galactosidase, alkaline phosphatase and human insulin‐like growth factor I (IGF‐I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG‐Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF‐I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF‐I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.

Development of Non-Pathogenic Staphylococci as Vaccine Delivery Vehicles

Among the bacteria being considered as live recombinant vaccine vehicles, the most well studied during the past decade are attenuated Salmonella species1 and mycobacterial bacille Calmette-Guerin (BCG) due to their capacity to colonize mucosal surfaces and invade macrophages in the liver, spleen and lymph nodes of the host.2,3 Surface-display of the foreign antigens to be delivered, has in both these systems proven to be beneficial in eliciting an immune response.4–7 The risk of reversion to a virulent phenotype and the potential side-effects in immunocompromised individuals and infants have, however, raised concern of the use of Salmonella or BCG-based recombinant vaccines in humans.8

Characterization of Diaphanous-related formin FMNL2 in human tissues

BackgroundDiaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2) in human tissues.ResultsAn FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2s proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration.ConclusionFMNL2 is expressed in multiple human tissues, not only in the central nervous system. The expression is especially strong in gastrointestinal and mammary epithelia, lymphatic tissues, placenta, and in the reproductive tract. In cultured melanoma cells, FMNL2 co-localizes with F-actin dots at the tips of cellular protrusions.

Application of chemiluminescent probes in investigating lysosomal sensitivity to superoxide verus suspected radical scavengers

The role of the superoxide anion radical (O2(-).) relative to catalytic/inhibitory substances in lysosomes is poorly understood. Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to radical activities. The Sepharose-4B-isoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-). and was inhibited or catalysed by various radical scavengers, transition metals and other substances which may affect lysosomal metabolism and lipofuscin formation. We conclude that lysosomal radical activities may be inhibited by butylated hydroxytoluene, hydrocortisone, ACF, RNA, alpha-tocopherol, and, in special circumstances, with fully metabolized iron. Choline and oxidized copper and iron cations in overload concentrations in incubated freshly in lysosomes may catalyse radical activities, and they may be important factors in lipofuscin formation and its role in aging.