Ozgur Gul

Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis

ABSTRACT The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997–1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.

Effect of codon optimization on the expression of Trichoderma reesei endoglucanase 1 in Pichia pastoris

Trichoderma reesei cellulases are important biocatalysts for a wide range of industrial applications that include the paper, feed, and textile industries. T. reesei endoglucanase 1 (egl1) was successfully expressed as an active and stable catalyst in Pichia pastoris for the first time. Codon optimization was applied to egl1 of T. reesei to enhance its expression levels in P. pastoris. When compared with the originally cloned egl1 gene of T. reesei, the synthetic codon optimized egl1 gene (egl1s) was expressed at a higher level in P. pastoris. Batch fermentations of both clones with the same copy number under controlled conditions indicated that codon optimized EGI enzyme activity increased to 1.24 fold after 72 h of methanol induction. Our research indicated that P. pastoris is a suitable host for cellulase production.

Apoptotic blocks and chemotherapy resistance: strategies to identify Bcl-2 protein signatures

Acquired or innate resistance to chemotherapy is a major drawback of cancer therapeutics, which is frequently seen in epithelial cancers. However, the molecular mechanisms underlying chemotherapy resistance remain poorly understood. The mitochondrial pathway is a critical death pathway common to many different types of chemotherapy. Aberrations in this pathway can result in resistance to chemotherapy. The Bcl-2 family of proteins control commitment to programmed cell death by mitochondrial apoptosis. In this review, we will summarize the strategies in determining the components of apoptotic defects responsible for chemotherapy resistance, mainly focused on Bcl-2 protein network.

Antibody array-based immunosensor for detecting cardiovascular disease risk markers.

Quantitative detection of proteins in multiplexed platforms presents technical advantages at clinical and laboratory settings compared to the monoplex ELISA method. With this purpose, we implemented a pilot study using in-house–designed sandwich-type antibody array for multiplexed detection of seven cardiovascular disease (CVD) risk markers and compared the performance of our immunosensor to conventional ELISA kits. Results indicated that our immunosensor can determine serum amyloid A (SAA), vascular cell adhesion molecule (VCAM), and myoglobin (MYO) concentrations accurately, precisely, and above all very much similar to ELISA. Hence, multiplexed detection and quantification of SAA, VCAM, and MYO with our immunosensor can be considered as a potential multiplexed alternative to the ELISA method.

Label-free, capacitive immunosensor for protein detection

This paper presents a biosensor implementation for the detection of protein molecules using specific antibodies. Affinity sensors allow the detection and quantification of target molecules in complex mixtures by affinity-based interactions. Immobilized antibody molecules are the probes that bind to specific protein molecules (targets) in biological fluids. In this study, interdigitated electrodes in the form of capacitance on glass slide were designed/simulated and used to measure the changes in the dielectric properties of the interdigated capacitances.

Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells

Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells.

Biosensors for the detection of cardiovascular risk markers in human serum

This paper presents two affinity-based biosensor implementations for the detection and quantification of cardiovascular risk proteins/markers, such as C-reactive protein, myoglobin, TNF-alpha, serum amyloid, using specific antibodies. The first method is based on interdigitated microelectrodes while the other is based microarrays. The signal transduction in micro-IDEs from biological-signal to the electronic/electrical signal occurs via fringe fields and dielectric change, induced by the capture of antigens by already immobilized antibody between the microelectrodes. Being process/integration-compatible with microelectronic/fabrication technologies, this method provides many advantages for the utilization in portable and/or remote sensing applications, such as smaller size, lower power consumption, small sample consumption, disposability, etc. The other method also analyzes the capability and reliability of sandwich-type antibody microarrays for the detection quantification of cardiovascular risk markers, using optical scanner system for the detection of fluorescent-labeled, sandwiched-structured antibodies on the chemically modified glass substrates. Our findings in micro-array study provides the advantages for lab-based detection and quantification applications such as being able to detect and quantify multiple markers at once and also better dynamic ranges over some of the commercial ELISA test-kits.

Biosensor implementation for cardiovascular risk marker analysis

In this paper we presented the detection of cardiovascular marker protein set in the form of sandwiched- antibody microarrays. The antibody set consist of C-reactive protein, fatty acid binding protein, myoglobin, D-dimer, troponin T protein, known cardiovascular risk markers in human serum. Chemically-modified glass substrate surfaces were used as solid support for immobilization of sandwiched, antibody-antigen-antibody structures. An optical scanner system was used for the detection of fluorescent-labeled, captured antibody set. We have shown that the proposed structure/process is capable of detecting 1mug/ml concentration of antigens, used in this study