Person: ALBAYRAK, CEM
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Publication Digital Quantification of Proteins and mRNA in Single Mammalian Cells(2016-03-01T00:00:00Z) Albayrak, CEM; Jordi, Christian A.; Zechner, Christoph; Lin, Jing; Bichsel, Colette A.; Khammash, Mustafa; Tay, Savas; ALBAYRAK, CEMAbsolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells.Publication Neovascularization of engineered tissues for clinical translation: Where we are, where we should be?(2021-06-01T00:00:00Z) Nazeer, Muhammad Anwaar; Karaoglu, Ismail Can; Ozer, Onur; Albayrak, Cem; Kizilel, Seda; ALBAYRAK, CEMOne of the key challenges in engineering three-dimensional tissue constructs is the development of a mature microvascular network capable of supplying sufficient oxygen and nutrients to the tissue. Recent angiogenic therapeutic strategies have focused on vascularization of the constructed tissue, and its integration in vitro; these strategies typically combine regenerative cells, growth factors (GFs) with custom-designed biomaterials. However, the field needs to progress in the clinical translation of tissue engineering strategies. The article first presents a detailed description of the steps in neovascularization and the roles of extracellular matrix elements such as GFs in angiogenesis. It then delves into decellularization, cell, and GF-based strategies employed thus far for therapeutic angiogenesis, with a particularly detailed examination of different methods by which GFs are delivered in biomaterial scaffolds. Finally, interdisciplinary approaches involving advancement in biomaterials science and current state of technological development in fabrication techniques are critically evaluated, and a list of remaining challenges is presented that need to be solved for successful translation to the clinics.Publication Pluripotency transcription factor Sox2 is strongly adsorbed by heparin but requires a protein transduction domain for cell internalization(2013-02-01T00:00:00Z) Albayrak, CEM; Yang, William C.; Swartz, James R.; ALBAYRAK, CEMThe binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins. (C) 2012 Elsevier Inc. All rights reserved.Publication Broadening Horizons and Teaching Basic Biology Through Cell-Free Synthesis of Green Fluorescent Protein in a High School Laboratory Course(2013-12-01T00:00:00Z) Albayrak, CEM; Jones, K. C.; Swartz, James R.; ALBAYRAK, CEMCell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school laboratory course. Ninety students in a biotechnology class used CFPS to study the effects of the concentrations of amino acids, cell extract, DNA, and the energy source on accumulation of active super-folder green fluorescent protein. Students estimated product concentrations simply by comparing solution colors to a printed green color gradient. This simple and inexpensive method allows for immediate measurements, and 26 of the 30 groups observed measurable product concentrations within 60 min. These student-generated data were then discussed to illustrate concepts of data analysis such as outliers and standard deviation. We also combined the laboratory experience with a visit to a university campus that included a laboratory tour and a college-style lecture. Our overall objective was to excite the students about the scientific enterprise and to instill a sense of personal relevance and attainability so that these students could realistically consider technical careers.Publication Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair(2013-02-01T00:00:00Z) Albayrak, CEM; Swartz, James R.; ALBAYRAK, CEMEven though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated kat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4 x 10(-5) s(-1). Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450 +/- 20 mu g/ml CAT109pAzF and 428 +/- 2 mu g/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe. (C) 2012 Elsevier Inc. All rights reserved.Publication High-Content Quantification of Single-Cell Immune Dynamics(2016-04-01T00:00:00Z) Junkin, Michael; Kaestli, Alicia J.; Cheng, Zhang; Jordi, Christian; Albayrak, CEM; Hoffmann, Alexander; Tay, Savas; ALBAYRAK, CEMCells receive time-varying signals from the environment and generate functional responses by secreting their own signaling molecules. Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems. We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically. Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs. Employing this system to analyze macrophage signal processing under pathogen inputs, we found that the dynamics of TNF secretion are highly heterogeneous and surprisingly uncorrelated with the dynamics of NF-kappa B, the transcription factor controlling TNF production. Computational modeling of the LPS/TLR4 pathway shows that post-transcriptional regulation by TRIF is a key determinant of noisy and uncorrelated TNF secretion dynamics in single macrophages.Publication Protein Scaffold-Based Multimerization of Soluble ACE2 Efficiently Blocks SARS-CoV-2 Infection In Vitro and In Vivo(2022-07-01T00:00:00Z) Kayabolen, Alisan; Akcan, Ugur; Ozturan, Dogancan; Ulbegi-Polat, Hivda; Sahin, Gizem Nur; Pinarbasi-Degirmenci, Nareg; Bayraktar, Canan; Soyler, Gizem; Sarayloo, Ehsan; Nurtop, Elif; Ozer, Berna; Guney-Esken, Gulen; Barlas, Tayfun; Yildirim, Ismail Selim; Dogan, Ozlem; Karahuseyinoglu, Sercin; Lack, Nathan A.; Kaya, Mehmet; Albayrak, Cem; Can, Fusun; Solaroglu, Ihsan; Bagci-Onder, Tugba; ALBAYRAK, CEMSoluble ACE2 (sACE2) decoys are promising agents to inhibit SARS-CoV-2, as their efficiency is unlikely to be affected by escape mutations. However, their success is limited by their relatively poor potency. To address this challenge, multimeric sACE2 consisting of SunTag or MoonTag systems is developed. These systems are extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates, perform better than the dimeric or trimeric sACE2, and exhibit greater than 100-fold neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag fused to a more potent sACE2 (v1) achieves a sub-nanomolar IC50, comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutations for variants of concern, including delta and omicron, are also neutralized efficiently with multimeric sACE2. Finally, therapeutic treatment of sACE2(v1)-MoonTag provides protection against SARS-CoV-2 infection in an in vivo mouse model. Therefore, highly potent multimeric sACE2 may offer a promising treatment approach against SARS-CoV-2 infections.Publication Investigating the Antioxidant Capacity of Lunasin Expressed in Aspergillus oryzae(2024-01-01) KARAMAN E.; Albayrak C.; UYSAL S.; KARAMAN, ELİF; ALBAYRAK, CEM; UYSAL, SERDARObjective: Lunasin is a bioactive protein that possesses anti-carcinogenic, anti-inflammatory, and antioxidant properties. Traditional isolation methods are resource-intensive, and chemical synthesis faces cost and environmental issues. This study aims to achieve cost-effective lunasin expression in Aspergillus oryzae with a focus on exploring its antioxidant properties in vitro. Materials and Methods: The expression vector carrying four lunasin sequences fused with amylase and an 8xHis-tag was introduced into pyrG auxotrophic A. oryzae. Subsequently, the recombinant protein was purified using metal affinity chromatography. The study uses sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analyses, and size-exclusion chromatography to evaluate the composition and purity of the protein, a linoleic acid assay to demonstrate the inhibitory effect on lipid peroxidation, and the 2,2\"-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid] ABTS) assay to evaluate the radical scavenging activity. Results: SDS-PAGE and western blot analyses confirmed sustained lunasin expression in A. oryzae, appearing in both fusion and non-fusion forms. Yields were 5.8 mg/L for non-fusion and 4 mg/L for fusion lunasin expression. Moreover, 0.1 μM non-fusion lunasin surpassed α-tocopherol and butylated hydroxyanisole (BHA; p < 0.05) in reducing lipid peroxidation at 4 and 72 h. Unlike the fusion lunasin, the non-fusion lunasin displayed concentration- and time-independent inhibitory effects on linoleic acid peroxidation as well as significant ABTS scavenging activity (p < 0.05). Conclusion: The study has shown for the first time A. oryzae to efficiently express and secrete both fusion and non-fusion lunasin proteins in a soluble form, with the non-fusion lunasin exhibiting superior antioxidant effectiveness compared to the fusion lunasin. The findings underscore A. oryzae\"s potential as a promising host for producing functional lunasin with antioxidant properties, opening avenues for broader applications in biotechnology and bioactive peptides.Publication Real-time tracking, retrieval and gene expression analysis of migrating human T cells(2015-01-01T00:00:00Z) Mehling, Matthias; Frank, Tino; Albayrak, CEM; Tay, Savas; ALBAYRAK, CEMDynamical analysis of single-cells allows assessment of the extent and role of cell-to-cell variability, however traditional dish-and-pipette techniques have hindered single-cell analysis in quantitative biology. We developed an automated microfluidic cell culture system that generates stable diffusion-based chemokine gradients, where cells can be placed in predetermined positions, monitored via single-cell time-lapse microscopy, and subsequently be retrieved based on their migration speed and directionality for further off-chip gene expression analysis, constituting a powerful platform for multiparameter quantitative studies of single-cell chemotaxis. Using this system we studied CXCL12-directed migration of individual human primary T cells. Spatiotemporally deterministic retrieval of T cell subsets in relation to their migration speed, and subsequent analysis with microfluidic droplet digital-PCR showed that the expression level of CXCR4 - the receptor of CXCL12 - underlies enhanced human T cell chemotaxis.Publication Direct Polymerization of Proteins(2014-06-01T00:00:00Z) Albayrak, CEM; Swartz, James R.; ALBAYRAK, CEMWe report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step decamer using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-L-phenylalanine (pAzF) or pp-ropargyloxy-L-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were monomer coupled by copper-catalyzed azide alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained >= 63% of their specific activity (i.e., fluorescence) after coupling. Polymerization of a concentrated solution of triply substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or copolymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.