Proteinase inhibitor optimization

Affinity and specificity of proteinase inhibitors can be optimized by phage display... 

Proteinases in soil extracts have been assayed using casein and substituted amides and peptides as assay substrates. Mayaudon et al. found that the casein hydrolysing activities of soil extracts were directly related to soil organic matter contents and inversely related to clay contents. Specific activities doubled when associated coloured humic compounds were removed from the extracts. The partially-purified proteinases appeared to be of a serine protease type as judged from their responses to inhibitors. Optimal activity occurred at pH 8.5 and at 50 0. The estimated Km value (22mg casein ml was considerably higher than those (0.15 and 0.54mg casein ml reported by Nannipieri et for proteinases of crude extracts from two soils. 

Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube 1.0 pL RNasin (RNase inhibitor), 11.5 pL UV-irradiated dH20, 37.5 U DNase (RQl RNase free DNase, Promega Inc., Madison, Wl)/(1 U/pL) Total (per slide) 50.0 pL. 

Cicero DO, Barbato G, Koch U, Ingallinella P, Bianchi E, Nandi MC, Steinkuhler C, Cortese R, Matassa V, De Francesco R, Pessi A, Bazzo R, Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the Hepatitis C Virus (HCV) NS3 protein by NMR and modelling studies, J. Mol. Biol., 289 385-396, 1999. 

Optimization of the peptide backbone of these aldehydes to take advantage of the binding interactions in the 8,-84 subsites afforded potent inhibitors of HLE, for example, aldehyde (6-6) [124]. Concurrent with the increases in potency, the selectivity of these compounds also improved. For example, aldehyde (6-5) was inactive at 100 //M against other enzymes, including the serine proteinases trypsin, chymotrypsin, and cathepsin G [125]. Aldehyde (6-5) was compared with a,-PI and was shown to be more potent in vitro (on a weight basis) and more stable towards oxidative inactivation by cigarette smoke [128]. 

The achievement of a successful analysis depends not only on optimized analytical procedures but also on (1) the ability of the extraction procedure to recover DNA from the sample and to remove potential assay inhibitors, and (2) the quality and purity of the DNA extracted. The analytical technique is useless if the target cannot be extracted from the sample. At this point in time there are no standardized procedures for the extraction of DNA from food samples. The development of DNA extraction procedures is matrix dependent. High fat content and low dry matter seem to explain the decreased extraction efficiency of full-fat soybean flour compared to its defatted counterpart (Gryson et al., 2008). Traditionally, the extraction of DNA has been carried out by treating the sample with detergents such as sodium dodecyl sulfate and proteinases such as proteinase K, followed by the removal of proteins and polysaccharides with phenol-chloroform and precipitation of DNA with ethanol. 

Although there have been numerous and comprehensive studies on intracellular proteinases, they have been carried out predominantly with the purified enzymes. This work has provided information on catalytic mechanisms, susceptibility of proteinases to different classes of inhibitors, specificities of the enzymes toward a variety of artificial and natural substrates, and details on optimal conditions for each proteinase (Barrett, 1969). Unfortunately, these experiments provide little if any direct evidence about whether a particular enzyme is involved in the breakdown of intracellular enzymes or other proteins. 

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