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1.
PLoS One ; 12(2): e0171926, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28207780

RESUMO

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.


Assuntos
Proteínas Alimentares/química , Inocuidade dos Alimentos , Pepsina A/química , Proteínas Alimentares/imunologia , Concentração de Íons de Hidrogênio , Fatores de Tempo
3.
Methods Mol Biol ; 1286: 35-42, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25749943

RESUMO

Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency.


Assuntos
Acetiltransferases/isolamento & purificação , Cromatografia de Afinidade/métodos , Corantes/química , Triazinas/química , Cromatografia de Afinidade/economia , Escherichia coli/enzimologia , Plantas Geneticamente Modificadas , Sefarose/química , Fatores de Tempo
4.
Protein Expr Purif ; 90(2): 129-34, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23748142

RESUMO

The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 µg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed.


Assuntos
Acetiltransferases/isolamento & purificação , Cromatografia/métodos , Triazinas , Acetiltransferases/química , Corantes , Resinas de Troca Iônica
5.
Bioorg Med Chem Lett ; 13(9): 1565-70, 2003 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-12699756

RESUMO

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.


Assuntos
Compostos de Boro , Corantes Fluorescentes , Nylons/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Fluorescência , Humanos , Espaço Intracelular/metabolismo , Verapamil/farmacologia
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