Barry Perry

Olfactory receptor-encoding genes and pseudogenes are expressed in humans

A putative olfactory receptor-encoding gene was cloned from human genomic DNA and shown to be expressed by isolation of a full-length cDNA from olfactory tissue. A second cDNA clone was found to encode an olfactory receptor pseudogene. The expression of a pseudogene from the olfactory gene repertoire, in neurons which express only a single receptor type, implies that many neurons will be non-functional.

Translational regulation of the plant S-adenosylmethionine decarboxylase

It is becoming apparent that control of protein synthesis by metabolites is more common than previously thought. Much of that control is exerted at the level of initiation of mRNA translation, orchestrated by upstream open reading frames (uORFs) and RNA secondary structure. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis and both mammalian and plant AdoMetDCs are translationally regulated by uORFs in response to polyamine levels by distinct mechanisms.

GOlf Complements A Gpa1 Null Mutation in Olf Saccharomyces Cerevisiae and Functionally Couples to the Ste2 Pheromone Receptor

Abstract We have produced a plasmid designed for the expression of heterologous G protein α subunits in the yeast Saccharomyces cerevisiae Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein α subunit, to the yeast pheromone response pathway. The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal- null mutation. A similar response is obtained when an alternative G protein a subunit, Golf, is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.

Modification of the stability of phospholipase A2 by charge engineering.

Electrostatic interactions play an important role in stabilizing the folded conformation of globular proteins. Here we predict the change in stability of charge engineered mutants, construct these mutants and compare the predicted change in stability with that observed. The change in stability was correctly predicted for two of the three mutants and the factors responsible for the discrepancy between observation and prediction for the third mutant are discussed.

Expression proteomics identifies biochemical adaptations and defense responses in transgenic plants with perturbed polyamine metabolism.

Soluble proteins from leaves of transgenic tobacco plants with perturbed polyamine metabolism, caused by S‐adenosylmethionine decarboxylase overexpression, were analysed by comparative proteomics. A group of proteins was found to be increasingly repressed, in parallel with the degree of polyamine perturbation, in each of the three independent transgenic lines. These were identified as isoforms of chloroplast ribonucleoproteins, known to be involved in chloroplast mRNA stability, processing and translation. Another group of eight proteins strongly induced in the most metabolically perturbed line was identified as multiple, uncharacterised isoforms of the defense protein PR‐1, a known marker for systemic acquired resistance.

A novel method for the purification of porcine phospholipase A2 expressed in E. coli.

Porcine phospholipaseA2 expressed in E. coli as a fusion protein was isolated, renatured and specifically cleaved by trypsin as described in (1). Active phospholipaseA2, was purified to homogeneity on a column of PBE-94 over a pH region 7.4-4.5. Using this method, several phospholipase A2 mutant enzymes have now been purified in a single step and all behaved identically during chromatofocusing. The method will therefore be extremely useful not only for those interested in understanding the structure-function relationships of phospholipaseA2 but also for preparing the enzyme in large quantities for industrial and pharmaceutical purposes.

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan.

Making a small enzyme smaller; removing the conserved loop structure of hen lysozyme

Engineering a smaller lysozyme is a challenge for both random and site‐directed mutagenesis. This paper illustrates the power of knowledge‐based protein engineering in the design of a smaller lysozyme that folds correctly and has activity against bacterial cell walls. In this smaller lysozyme the conserved disulphide bridged loop is replaced by a short loop. The long loop was selected because it buries a predominantly hydrophilic surface. The short loop was discovered by searching for appropriate fragments in the protein databank. This approach is important in the design of small enzymes useful to the food industry.