RESUMO
Hydrogenases, ferredoxins, and ferredoxin-NADP+ reductases (FNR) are redox proteins that mediate electron metabolism inâ vivo, and are also potential components for biological H2 production technologies. A high-throughput H2 production assay device (H2 PAD) is presented that enables simultaneous evaluation of 96 individual H2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H2 PAD senses the chemo-optical response of Pd/WO3 thin films to the H2 produced. H2 PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H2 production rates. H2 PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems.
Assuntos
Ferredoxina-NADP Redutase/metabolismo , Ensaios de Triagem em Larga Escala , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Biocatálise , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Hidrogênio/química , Hidrogenase/química , MutaçãoRESUMO
HGP is a 24-amino acid peptide derived from HIV gp41 that increases vesicular escape when incorporated into gene delivery vehicles. The typical yield of HGP from solid phase peptide synthesis is low due to its length and hydrophobicity. The goal of this work was to investigate truncated sequences that maintained activity in order to improve the ease and yield of synthesis. A shortened, 15-amino acid sequence retained comparable lytic activity and the ability to interact with lipids when compared to the full length peptide. A scrambled peptide showed poor lytic activity, confirming that the activity of these endosomal escape peptides is sequence specific. Peptides were covalently attached to polyethylenimine (PEI) and used to condense plasmid DNA to form nanoparticulate carriers. When delivery efficiencies of PEI-peptide conjugates were compared in vitro, PEI modified with the truncated HGP sequence increased transgene expression over unmodified PEI and full length HGP.