A cost-effective enzyme kinetics and inhibition model for biochemistry education and research.

Enzyme kinetics and inhibition studies are crucial in biochemistry education and research. Conventional methods often require expensive equipment and reagents, potentially limiting their accessibility in limited resource settings. Our approach sought to develop a cost-effective experimental design for studying enzyme kinetics and inhibition. Lactase was chosen as a protein model and its activity was investigated by measuring glucose production from lactose hydrolysis. In the study, commercially available lactase pills were used as an enzyme source, while milk was used as a substrate. Instead of scientific equipment, glucometers were used to measure lactase activity. Enzyme kinetics were evaluated using Michaelis-Menten and Lineweaver-Burk plots. In the study, the effects of temperature, pH, and inhibitors were also investigated. The results of our study aligned with established enzyme kinetics theories and previous studies. Lactase showed temperature and pH-dependent activity, with decreased activity observed at both low and high extremes. Results also showed that galactose acts as a competitive inhibitor of lactase. The approach presented here offers a cost-effective procedure for studying enzyme kinetics and inhibition. It can act as a valuable tool for educational purposes and for preliminary research in settings with limited resources.

A Simple Purification Method for Heat-Stable Recombinant Low Molecular Weight Proteins and Peptides Via GST-Fusion Products.

Here, we describe a simple, rapid, cost-effective, and efficient novel one-step purification method for GST-tagged peptides and small proteins. This novel technique applies to proteins and peptides that are known to be thermally stable at 60 °C and do not have elaborate structure(s) and whose heat-induced unfolding is reversible. This method takes advantage of glutathione S-transferase from Schistosoma japonicum (sj26GST) precipitating when heated at 60 °C. Purified GST-fusion products are subjected to enzymatic cleavage to separate the GST tag from the target peptide or small proteins. In our proposed method, the cleavage products are heated at 60 °C for 20 min which results in the precipitation of the GST tag. Subsequently, the GST tag is separated from the target peptide or small protein by high-speed centrifugation. Biophysical experiments such as SDS-PAGE, circular dichroism, isothermal titration calorimetry, mass spectroscopy, and multidimensional NMR spectroscopy confirm that the target peptides and small proteins are purified to more than 95% homogeneity, intact native conformation, and no significant change in the binding affinity of heat-treated purified product to the interacting partners.

Targeting K-Ras Mutations Show Promise Towards Ending Ras's "Undruggable" Era.

It has almost been 40 years since the Ras proteins were discovered as the first human oncogenes. They remain among the most important genes for regulating mammalian cell growth and are involved in more than a quarter of human cancers. Out of 167 members of the Ras superfamily, KRas mutations are the most abundant in human cancers. Particularly, the K-Ras G12C mutations are known to be involved in pancreatic, colon and lung cancers as well as leukemias. Though progress has been made, approaches targeting Ras proteins for therapeutic purposes remain challenging. No drugs treating Ras-related cancers are currently on the market. However, there is now renewed interest in the Ras area, and newer approaches have highlighted the targeting of several types of tumors and treating cancer patients. This review will summarize recent K-Ras drug candidates and approaches in the preclinical, clinical and post-clinical pipelines that show promise for targeting and reducing Ras-related tumors. Macromolecules such as mRNA vaccines, siRNA, and T-cell receptors that target Ras will also be discussed. The newer molecules and the recent approaches to be discussed suggest that the "undruggable" era of Ras proteins could be coming to an end.

Simplification of the purification of heat stable recombinant low molecular weight proteins and peptides from GST-fusion products.

The synthesis and purification of peptides of importance in the fields of research and medicine continue to be a challenging task. Chemical synthesis of oligopeptides, especially those greater than 25 amino acids, is cost prohibitive. On the other hand, several bottlenecks exist in the production of recombinant short peptides in heterologous expression hosts such as Escherichia coli (E. coli). In this study, a rapid, cost-effective, and reliable method for the production and single-step-purification of peptides and small proteins was developed. Five peptides and small proteins were overexpressed in E. coli as GST-fusion products in high yields. The recombinant peptides or proteins were successfully purified after enzymatic cleavage with selective heat-induced precipitation of the GST-affinity tag. Qualitative and quantitative analysis using SDS-PAGE and mass spectrometric methods suggest that the recombinant peptides/ proteins were purified to greater than 95% homogeneity. Results of biophysical experiments, including multi-dimensional NMR spectroscopy, show that the purified proteins/ peptides retain their native conformation. Isothermal titration calorimetry studies indicate no significant change in the binding affinity of the heat-treated purified product to their interacting partner(s) compared to the recombinant peptides purified by conventional chromatographic procedures without subjecting to heat treatment. In our opinion, the results reported render the purification of recombinant proteins/ peptides of biomedical relevance using our proposed method easy and reliable.

Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury.

We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase-3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.