Biomedical subjects

Javier F Chaparro-Riggers

Publications and source records attributed to Javier F Chaparro-Riggers.

4 recordsLinked to original sources

PubMed2007

Better library design: data-driven protein engineering.

Data-driven protein engineering is increasingly used as an alternative to rational design and combinatorial engineering because it uses available knowledge to limit library size, while still allowing for the identification of unpredictable substitutions that lead to large effects. Recent advances in computational modeling and bioinformatics, as well as an increasing databank of experiments on functional variants, have led to new strategies to choose particular amino acid residues to vary in order to increase the chances of obtaining a variant protein with the desired property. Strategies for limiting diversity at each position, design of small sub-libraries, and the performance of scouting experiments, have also been developed or even automated, further reducing the library size.

Computational Biology↗

PubMed2006

High-throughput screening for enhanced protein stability.

High thermostability of proteins is a prerequisite for their implementation in biocatalytic processes and in the evolution of new functions. Various protein engineering methods have been applied to the evolution of increased thermostability, including the use of combinatorial design where a diverse library of proteins is generated and screened for variants with increased stability. Current trends are toward the use of data-driven methods that reduce the library size by using available data to choose areas of the protein to target, without specifying the precise changes. For example, the half-lives of subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold, respectively, using a crystal structure to guide mutagenesis choices. Sequence homology based methods have also produced libraries where 50% of the variants have improved thermostability. Moreover, advances in the high-throughput measurement of denaturation curves and the application of selection methods to thermostability evolution have enabled the screening of larger libraries. The combination of these methods will lead to the rapid improvement of protein stability for biotechnological purposes.

Computational Biology↗

PubMed2006

Structure-guided consensus approach to create a more thermostable penicillin G acylase.

The thermostabilization of penicillin G acylase (PGA) is a difficult problem due to the large size of the protein and its complex maturation process. We developed a data-driven protein design method that requires fewer homologous sequences than the traditional consensus approach and utilizes structural information to limit the number of variants created. Approximately 50% of our 21 single-point mutants were found experimentally to be more thermostable than the wild-type PGA, two had almost threefold longer half-life at 50 degrees C, with very little effect on activity. An analysis of four programs that predict the thermostability conferred by point mutations shows little agreement between the programs and with the experimental data, emphasizing that the chosen stabilizing mutations are very difficult to predict, but that our data-driven design method should prove useful.

Bacteria↗

PubMed2006

Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases.

Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

Peptide Hydrolases↗