ProteinParser:a Community Based Tool for the Generation of a Detailed Protein Consensus and FASTA Output

Comparison of bioinformatic data is a common application in the life sciences and beyond. In this communication, a novel Java based software tool, ProteinParser, is outlined. This soft- ware tool calculates a detailed consensus, or most common, amino acid at a given position in an aligned protein set, whilst also generating a full consensus protein FASTA output. A second application of this software tool, computing a consensus amino acid given a toler- ance threshold, is also demonstrated. The phytase and the common bacterial �-lactamase proteins are analysed as ‘proof of concept’ examples. Consensus proteins, as generated by ProteinParser, are regularly utilised in the selection of residues for protein stabilisation muta- genesis; however, this widely applicable software tool will find many alternative applications in areas such as protein homology modelling

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 °C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes

Site directed mutagenesis studies of horseradish peroxidase

The peroxidases are a ubiquitous subset of enzymes found in both the animal and plant kingdoms. Of all the peroxidases, the majority of research has focussed on the Class III Horseradish Peroxidase (E.C. 1.11.1.7). The basic form, HRP-C, is the most common and is utilised in this study. Recombinant HRP-C was first expressed over fifteen years ago; however, its production has been plagued by the formation of inclusion bodies and low yields. In this present study, the HRP gene and a PelB leader sequence were directionally cloned to form a fusion protein, expressed to the bacterial periplasmic envelope. By manipulating subsequent expression conditions, fully functional HRP was successfully produced, with the inclusion of a poly-Histidine tag permitting single step purification. Site directed mutagenesis was utilised to probe the stability of the recombinant enzyme. Two methodologies were employed to select residues for mutation, initially a rational approach, based on previous knowledge proposed 16 mutations. The second method based on a peroxidase sequence alignment utilising novel bioinformatic software, proposed 6 mutations. These mutants were generated, expressed and purified via optimised conditions. All mutants were characterised based on thermal, solvent and H2O2 stabilities, as well as kinetic analysis. Important stabilising roles for Glutamic Acid 238, Glutamine 106 and the helical secondary structure within the HRP protein were noted. Peroxidase structure evolution could be followed via a novel archetype peroxidase sequence generated. Mutations, which increased hydrogen bonding in the Lysine 232/Lysine 241 axis, improved stability. Genetic engineering was also employed to generate a recombinant HRP-C that permitted simple directed immobilisation (one triple mutant and one pentuple mutant), allowing maximal accessibility to the enzymes’ active site. Improved immobilisation capacity was achieved, however, at the cost of free and immobilised stability. Application o f wildtype recombinant HRP-C to screen-printed and etched platinum electrode biosensors was investigated. Improved direct electron transfer was noted for recombinant HRP over plant HRP for both configurations