Biology subjects

Bozkurt, E. U.

Publications and source records attributed to Bozkurt, E. U..

4 recordsLinked to original sources

bioRxiv2026

Multiparameter optimization extends the lifetime of cell-free protein synthesis in a high-throughput format

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, yet the factors governing reaction longevity remain poorly understood despite their importance for high-throughput applications. Here, the three principal determinants of CFPS performance--DNA template design, reaction composition, and lysate genotype--were systematically optimized to extend reaction lifetime in a 384-well plate format. Different energy regeneration systems were evaluated through real-time pH monitoring and metabolomic analyses to identify the metabolic constraints limiting prolonged protein synthesis. Lysates prepared from engineered Escherichia coli BL21(DE3) strains were further examined to assess the contributions of DNA, RNA, and amino acid stabilization. Systematic optimization of amino acid, nucleoside triphosphate, polyethylene glycol, and lysate concentrations identified DNA template stability and amino acid preservation as the primary factors sustaining CFPS activity. Combining these improvements yielded reactions that remained productive for >14 h and produced 567 {+/-} 64 g mL-1 active deGFP. These findings establish practical strategies for extending CFPS lifetime and improving high-throughput cell-free platforms.

molecular biology↗

bioRxiv2025

Developing Anti-EGFR/Anti-HER2 Bifunctional Antibody for Solid Tumors by Protein Engineering

Overexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) is common in solid tumors like breast, colorectal, and head and neck cancers, driving oncogenic signaling, therapy resistance, and poor prognosis. Monotherapies often fall short in fully suppressing tumor progression, particularly in cases with EGFR/HER2 co-amplification. Dual-targeting strategies offer enhanced efficacy by mitigating resistance and amplifying antitumor responses. Our study focused on developing and characterizing a bispecific anti-EGFR/HER2 antibody designed to simultaneously block ligand binding and receptor activation while harnessing Fc-mediated immune effector functions. Utilizing knob-into-hole and CrossMab technologies, we engineered a bispecific antibody capable of binding the extracellular domains of both EGFR and HER2. In vitro analyses confirmed its dual-binding capacity. Comparative studies revealed that this bispecific construct outperformed its monospecific counterparts in suppressing proliferation and, notably, in increasing the expression of apoptotic markers in EGFR/HER2-expressing tumor cells. Together, these findings highlight the synergistic therapeutic potential of bispecific antibodies as a promising modality for overcoming the limitations of current monotherapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/696462v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@192972corg.highwire.dtl.DTLVardef@102f9b2org.highwire.dtl.DTLVardef@57377corg.highwire.dtl.DTLVardef@1fbeb9d_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

A Highly Potent SARS-CoV-2 Blocking Lectin Protein

COVID-19 pandemic effected more than 180 million people around the globe causing more than four million deaths as of July 2021. Sars-CoV-2, the new coronavirus, has been identified as the primary cause of the infection. The number of vaccinated people is increasing however prophylactic drugs are highly demanded to ensure a secure social contact. There have been a number of drug molecules repurposed to fight against Sars-CoV-2, however the proofs for the effectiveness of these drug candidates is limited. Here we demonstrated griffithsin (GRFT), a lectin protein, to block the entry of the Sars-CoV2 into the Vero6 cell lines and IFNAR-/-mouse models by attaching to spike protein of the Sars-CoV-2. Given the current mutation frequency of the Sars-CoV-2 we believe that GRFT protein-based drugs will have a high impact in preventing the transmission both on Wuhan strain as well as any other emerging variants including delta variant causing high speed spread of COVID-19.

biochemistry↗

bioRxiv2020

Genetic Circuits Combined with Machine Learning Provides Fast Responding Living Sensors

Whole cell biosensors (WCBs) have become prominent in many fields from environmental analysis to biomedical diagnostics thanks to advanced genetic circuit design principles. Despite increasing demand on cost effective and easy-to-use assessment methods, a considerable amount of WCBs retains certain drawbacks such as long response time, low precision and accuracy. Furthermore, the output signal level does not correspond to a specific analyte concentration value but shows comparative quantification. Here, we utilized a neural network-based architecture to improve the aforementioned features of WCBs and engineered a gold sensing WCB which has a long response time (18 h). Two Long-Short Term-Memory (LSTM)-based networks were integrated to assess both ON/OFF and concentration dependent states of the sensor output, respectively. We demonstrated that binary (ON/OFF) network was able to distinguish between ON/OFF states as early as 30 min with 78% accuracy and over 98% in 3 h. Furthermore, when analyzed in analog manner, we demonstrated that network can classify the raw fluorescence data into pre-defined analyte concentration groups with high precision (82%) in 3 h. This approach can be applied to a wide range of WCBs and improve rapidness, simplicity and accuracy which are the main challenges in synthetic biology enabled biosensing.

synthetic biology↗