Purification and Analysis of the Three isoforms of Mitogen Activated Protein Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MKNK1)

Cellular responses to stimuli are essential to the proper functioning of all organisms. The specific protein interactions that pass along a message that induce cellular changes are numerus and include the extensive p38 MAPK pathway known to be activated by environmental stressors such as inflammatory cytokines, DNA damage, or oxidative stress. Dysregulation and improper responses to these conditions have been linked to various cancers and immune disorders, making members of the pathway highly desirable drug targets. We are focused on Mitogen Activated Protein Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MKNK1) which has three isoforms, long, primary and short; long has never been studied but primary and short are known to be involved in translational regulation (via binding to eIF4G and phosphorylation of eIF4E), though the overall function and role of MKNK 1 is not entirely known. Thus far, we have successfully expressed and purified all isoforms using plasmids that enabled creation of gst-tagged fusion proteins. Further purification via tag removal was conducted to prepare the proteins for metal analysis, predicted in the cysteine containing loop of MKNK1s. Kinase assay was also performed to detect MKNK1 phosphorylation by p38, using a coupled assay with kemptide as the final substrate. Further analysis is underway to explore the functional differences of the isoforms

An Assessment of biological Potency and Molecular Characteristics of Different Innovator and Noninnovator Interferon-Beta Products

Approved innovator products and their noninnovator ‘‘copy’’ versions are likely to vary in their quality, eg, physicochemical characteristics and biological activity, with important implications for clinical efficacy and safety. Therefore, it is important to study and thoroughly evaluate the noninnovator products in comparison with approved products at the preclinical and clinical stages. We have obtained 4 noninnovator interferon (IFN)- b-1a products currently marketed in Latin America and Iran and compared these with approved IFN-b-1a products (Avonex and Rebif) obtained from the same geographical regions with respect to biological potency, estimated by in vitro bioassays, and molecular characteristics, assessed by immunoblotting and high-performance liquid chromatography. In this article, we present our data showing that the noninnovator IFN-b-1a products can vary considerably in their biological potency. In addition, we showed that all IFN-b-1a products formulated with human serum albumin contained variable amounts of higher-molecular-weight aggregates of IFN-b-1a and adducts with human serum albumin, these being more prevalent in 2 noninnovator IFN-b-1a products where biological potency was reduced compared with approved IFN-b-1a products. Additionally, significant lot-to-lot variability was observed for one of the noninnovator products. Taken together, the results of this study highlight the need for not only thorough in vitro characterization, but also preclinical and clinical assessment to ensure patient safety and efficacy