Browsing by Author "Madlambayan, Gerard"
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Item Optimization Protocol for Extraction and Purification of Recombinant Protein Modeled with Taq PolymeraseTir, Lily; Madlambayan, Gerard; Lal, ShaileshThis experiment explores recombinant protein drug protocols and how to better optimize and purify large scale recombinant proteins expressed in bacterial cells. Using Escherichia coli bacteria, the gene Taq will be cloned into the bacterial plasmid, grown, induced, and incubated . Taq proteins will then be transcribed, lysed out of cells, and purified using an ion exchange chromatography column. By altering elements of this current process — varying the column flow rate and using different salt gradient concentrations for column purification — more concentrated yields were achieved with greater purity. It was found that lower flow rates of 100 mL/hour at normal salt gradient concentration of 25 to 700 mM produced the most optimal protein fractions. While Taq polymerase is not a recombinant protein drug, it can serve as a model to represent therapeutic protein drugs and purification optimizations that would work well for both. Utilizing these results, they can potentially provide the pharmaceutical industry novel ways to increase their production while lowering costs. Drug manufacturers would be able to produce these drugs at higher rates and more affordable prices, while saving on resources and time. Patients purchasing recombinant protein drugs would benefit from this as drugs become more widely accessible and reasonably priced. Optimization of protein purification can overall ease economic burdens, and initiate change from typical protocols.Item PHOSPHORYLATION PATTERNS, AGGREGATION PROPENSITIES, AND MORPHOLOGICAL STUDIES OF THE VARIOUS TAU PROTEIN ISOFORMS(2022-03-25) Laryea, Erving Torgbor; Wu, Colin; Avery, Adam; Madlambayan, GerardTau protein is a microtubule-binding protein as well as a biomarker of neurodegeneration. Its core function is to stabilize microtubules for proper neuronal communication. When hyperphosphorylated, it detaches itself from microtubules and self-assembles into cytotoxic structures. However, little is known about how phosphorylation, the commonest posttranslational modification process found in eukaryotic cells regulate Tau protein structure, conformation, and function. Herein, the role of specific kinases or kinase combinations from the three main classes of proteins kinases that phosphorylate Tau: Proline-directing protein kinase (Glycogen synthase kinase (GSK-3β)) and non-proline directing protein kinase (Microtubule associated regulating kinase (MARK4)) and Tyrosine kinase (Fyn) were systematically evaluated in vitro. After the expression and purification of all the six Tau isoforms from E. Coli cells, Tau 441, also known as full-length Tau, which comprises of all the domains found in the other isoforms was extensively investigated. Phosphorylation of Tau 441 by GSK-3β, MARK4 and Fyn was detected by immunostaining using phosphospecific antibodies. With Tau protein been identified to co-localize with sulfated aminoglycans such as heparin and heparin sulfates, single and multi-kinase phosphorylated aggregation studies of Tau 441 were conducted in the presence or absence of heparin. Functional assays including proteostat assays, turbidity assays and SDS-PAGE were used to evaluate the aggregation properties of Tau 441 after phosphorylation.The phosphosites on all the single and multi-kinase phosphorylated Tau 441 samples were characterized by Tandem mass spectrometry. Tau 441 protein structure and conformational changes after phosphorylation was also determined by Hydrogen Deuterium Exchange mass spectrometry (HDX-MS). The flexibility and accessibility to the first and second hexapeptide repeats (H1 and H2), a repeat motif found in the MTBR of Tau protein identified to increase the aggregation tendencies of Tau protein were used to describe the single kinase or multi-kinase combinations evaluated ability to promote Tau fibrillization.Item President's Report to the Board of Trustees, April 12, 2024(2024-04-12) Oakland University. Board of Trustees; Pescovitz, Ora Hirsch; Carey, Elaine; Chamra, Louay; Harper, Graeme; Barry, Meaghan; Madlambayan, GerardItem THE ROLE OF CD49f - INTEGRIN α6- IN HUMAN STEM CELL BIOLOGY(2021-11-15) Timilsina, Suraj; Villa-Diaz, Luis; K. Lal, Shailesh; Madlambayan, GerardOwing to the intrinsic capability for unlimited self-renewal and the ability to make all the cells in the body, pluripotent stem cells (PSC) are an ideal candidate to be used as starting material for cell therapies. The development of a standard human PSC (hPSC), which includes embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC), culture methods using completely defined and xeno-free culture environment will advance our knowledge of hPSC biology, and also increase the effectiveness of hPSC expansion on defined conditions for potential human applications. It has been shown that stem cell fates are controlled by their specialized microenvironment, the stem cell niche, via direct cell-cell interactions, cell-extracellular matrix (ECM) contact- largely by surface proteins known as integrins- and the molecular signals emitting from the niche. Activation of integrins by binding to their ligands triggers signal transduction mechanisms involved in cell fate determination. In addition, because of their cell surface localization, integrins are used as biological markers to identify cell populations, and in this regard, integrin α6 (ITGA6), also known as CD49f, is a key biomarker identifying stem cells as it is commonly expressed in all identified stem cell types. Although, numerous findings strongly suggest that CD49f plays important functions in stem cell biology, the underlying molecular mechanisms by which CD49f sustain stem cell’s self-renewal have been only partially described. In this project I have established a chemically defined and xeno-free culture condition for long-term maintenance and derivation of hPSC using chemically defined and xeno-free culture conditions. I also described a novel molecular mechanism involved in the maintenance of self-renewal and proliferation of hPSC using simulated microgravity (sμg). Moreover, my results highlighted CD49f as a reliable biomarker in identifying and characterizing functional status of human mesenchymal stem cells (hMSC).